PDHA1-Ser293 / 300 common phosphorylated protein marker for inhibiting tumor angiogenesis and application of PDHA1-Ser293 / 300 common phosphorylated protein marker

Abstract

The invention provides a PDHA1-Ser293 / 300 common phosphorylation protein marker for inhibiting tumor angiogenesis and application of the PDHA1-Ser293 / 300 common phosphorylation protein marker, and belongs to the technical field of molecular biology. The biological function of the PDHA1-Ser293 / 300 common phosphorylation participating in glioma angiogenesis is studied, the biological function of the PDHA1-Ser293 / 300 common phosphorylation for inhibiting glioma angiogenesis and further hindering tumor progression is disclosed, and an important theoretical basis is provided for development of a new approach for targeted therapy of glioma angiogenesis.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a PDHA1-Ser293 / 300 common phosphorylation protein marker for inhibiting tumor angiogenesis and an application thereof. Background Art

[0002] Malignant brain tumors are one of the most serious diseases that endanger human health. Glioma is the most common type of primary brain tumor, accounting for 81% of malignant brain tumors, with high morbidity and mortality. Although a variety of chemotherapeutic drugs have been developed, few drugs have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of gliomas due to the presence of the blood-brain barrier (composed of endothelial cells, capillaries, and basement membrane). This unique structure of the central nervous system prevents most anti-tumor drugs from entering the brain, which poses a huge challenge to the development of anti-glioma drugs.

[0003] Angiogenesis is one of the hallmarks of glioma, providing the necessary conditions for the growth and metastasis of malignant cells. In the tumor microenvironment (TME), malignant cells release pro-angiogenic factors, which bind to specific receptors expressed on the membrane of vascular endothelial cells (VECs), triggering a pro-angiogenic cascade reaction and promoting cell behavior to form endothelial tubular structures. Cancer cells triggered by the Warburg effect secrete a large amount of lactic acid to prevent intracellular acidification, and then produce acidity in the extracellular environment. The lactic acid and protons secreted in the TME have specific effects on promoting the proliferation and growth of tumor cells, and one of the main pathways is to promote the angiogenesis of VECs. The indispensable role of VECs in maintaining vascular homeostasis, remodeling and angiogenesis has been widely studied and is considered to be an important mechanism to resist tumor progression. PDHA1 plays an important role in the occurrence and development of various malignant tumors. Studying the relationship between PDHA1 and angiogenesis is of great significance for the development of anti-glioma drugs. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a PDHA1-Ser293 / 300 co-phosphorylated protein marker for inhibiting tumor angiogenesis and its application. The present invention studies the biological function of PDHA1-Ser293 / 300 phosphorylation in glioma angiogenesis, reveals the biological function of PDHA1-Ser293 / 300 phosphorylation in inhibiting glioma angiogenesis and then hindering tumor progression, and provides an important theoretical basis for the development of new approaches for targeted treatment of glioma angiogenesis.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a PDHA1-Ser293 / 300 common phosphorylation protein marker for inhibiting tumor angiogenesis. The sequence of the PDHA1-Ser293 / 300 common phosphorylation protein marker is shown in SEQ ID NO:1.

[0007] Preferably, the PDHA1-Ser293 / 300 co-phosphorylated protein marker is PDHA1-Ser293 / 300 co-hyperphosphorylation, and the PDHA1-Ser293 / 300 co-hyperphosphorylation is obtained by mutating the serine at the 293rd and 300th positions on the PDHA1 protein to glutamic acid.

[0008] The present invention provides the use of the PDHA1-Ser293 / 300 common phosphorylation protein marker as a molecular target in the preparation or screening of drugs for inhibiting tumor angiogenesis.

[0009] Preferably, the PDHA1-Ser293 / 300 co-phosphorylated protein marker is PDHA1-Ser293 / 300 co-hyperphosphorylation, and the PDHA1-Ser293 / 300 co-hyperphosphorylation is obtained by mutating the serine at the 293rd and 300th positions on the PDHA1 protein to glutamic acid.

[0010] Preferably, the drug for inhibiting tumor angiogenesis is a drug for inhibiting glioma angiogenesis.

[0011] The present invention provides an application of a reagent causing the co-phosphorylation of PDHA1-Ser293 / 300 in the preparation of a drug for inhibiting tumor angiogenesis.

[0012] Preferably, the reagent causing co-phosphorylation of PDHA1-Ser293 / 300 comprises one or more of a nucleotide sequence capable of causing PDHA1 protein mutation, a plasmid comprising the nucleotide sequence, and a recombinant cell comprising the nucleotide sequence.

[0013] Preferably, the drug for inhibiting tumor angiogenesis is a drug for inhibiting glioma angiogenesis.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses mIF technology to perform correlation analysis on the phosphorylation of PDHA1-Ser293 / 300 and the expression level of PDK2 protein in high-grade glioma VECs and the content of vascular endothelial marker protein CD31, and clarifies the regulatory function of PDHA1-Ser293 / 300 phosphorylation on glioma angiogenesis. Subsequently, small animal in vivo and cell in vitro research techniques are used to detect the effects of artificial intervention of PDHA1-Ser293 / 300 phosphorylation status on angiogenic phenotypes such as VECs cell proliferation, migration, and tubule formation ability, proving the biological effect of PDHA1-Ser293 / 300 hyperphosphorylation in inhibiting VECs angiogenesis. By analyzing the mitochondrial oxidation rate and combining non-target metabolome and transcriptome high-throughput sequencing technology, it is further revealed that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 regulates cytoplasmic Ca through the mitochondrial energy metabolism pathway. 2+ The molecular mechanism of PDHA1-Ser293 / 300 phosphorylation inhibiting glioma angiogenesis and hindering tumor progression was revealed, which provides an important theoretical basis for the development of new targeted treatments for glioma angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 3 is the relationship between PDHA1 phosphorylation level and vascular density in clinical vascular tissue of glioma detected by mIF, where A is the representative mIF detection result obtained by scanning under a 20x microscope, green fluorescence represents PDHA1-Ser300 phosphorylation, red fluorescence represents CD31, yellow fluorescence represents PDK2, and blue fluorescence represents cell nuclei; B-G are statistical results of protein expression differences in different subgroups; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05;

[0016] Figure 2 Schematic diagram of AAV viral vector for overexpression of PDK2;

[0017] Figure 3 Schematic diagram for constructing mouse intracranial tumor model;

[0018] Figure 4 This is an optical microscopic image of PDK2 expression in mouse intracranial tumor vascular endothelial cells detected by mIF, where the left scale bar in the EGFP / Cd31 / Pdk2 / DAPI result image is 0.050 mm, and the right scale bar is 0.020 mm;

[0019] Figure 5This is the statistical result of mIF detection of PDK2 expression in intracranial tumor vascular endothelial cells of mice, * indicates P < 0.05;

[0020] Figure 6 Analysis of the dimensions of mouse intracranial tumor growth for small animal in vivo imaging;

[0021] Figure 7 This is the survival curve of model mice;

[0022] Figure 8 The panoramic image of the coronal staining of the brain tissue of the model mouse and the statistical results of the tumor infiltration area, where the blue mark is the cell nucleus, the area with dense cell nuclei is the tumor cell, * indicates P < 0.05, and the scale is 2.000mm;

[0023] Fig. 9 CD31 staining and statistical results of brain tumor tissues in model mice, * indicates P < 0.05, the left scale bar is 0.200 mm, and the right scale bar is 0.050 mm;

[0024] Fig.10 is the map of pLK0.1-TRC;

[0025] Fig.11 The structure map of the target shRNA;

[0026] Fig.12 It is the lentiCRISPRv2 map;

[0027] Fig.13 It is the construction of PDHA1 knockout hCMEC / D3 cells, where A is a schematic diagram of PDHA1-sgRNAs target, B is the result of T7 endonuclease detection of PDHA1-sgRNAs target cutting efficiency, C is Western blotting detection of PDHA1 expression changes in CRISPR / Cas9-PDHA1-sgRNA#1 infected cells, D is Western blotting detection of PDHA1 expression changes in CRISPR / Cas9-PDHA1-sgRNA#1 infected cell monoclonal cells, and E is Sanger sequencing results showing PDHA1 genome changes in different CRISPR / Cas9-PDHA1-sgRNA#1 monoclonal cells;

[0028] Fig.14The phenotypic analysis diagram of PDHA1 knockout inhibiting cell angiogenesis, where A and B are Edu results showing that PDHA1 homozygous knockout inhibits hCMEC / D3 cell proliferation, C is CCK8 results showing that PDHA1 homozygous knockout inhibits hCMEC / D3 cell proliferation, D and E are Transwell results showing that PDHA1 homozygous knockout inhibits hCMEC / D3 cell migration ability, F-H are tubule formation analysis results showing that PDHA1 homozygous knockout inhibits hCMEC / D3 cell vascular mimicry formation ability, S represents single cell, sgCON represents the sgRNA of control gene, *** represents P<0.001; ** represents P<0.01; * represents P<0.05; ns represents P≥0.05;

[0029] Fig.15 The phenotype diagram of hCMEC / D3 cell angiogenesis inhibition by simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, wherein A is a display diagram of PDHA1 mutation sites, EE represents PDHA1-S293E / S300E, AA represents PDHA1-S293A / S300A, S293E represents PDHA1-S293E, S300E represents PDHA1-S300E, S293A represents PDHA1-S293A, S300A represents PDHA1-S300A, WT represents wildtype, and E represents Gl utamate, A represents alanine; S represents serine, B represents Western blotting to detect the phosphorylation and protein expression levels of PDHA1 in cells with different genotypes of PDHA1, C and D represent Edu to detect the proliferation of hCMEC / D3 cells with different genotypes, E and F represent Transwell to detect the migration of hCMEC / D3 cells with different genotypes, G and H represent tubule formation experiments to detect the vascular mimicry formation ability of hCMEC / D3 cells with different genotypes, sgCON represents the sgRNA of control gene, *** represents P < 0.001; ** represents P < 0.01; * represents P < 0.05; ns represents P ≥ 0.05;

[0030] Fig.16Figure 1 shows the effect of single site mutations of PDHA1-Ser293 and -Ser300 on angiogenesis of hCMEC / D cells, where A is CCK8 analysis of the difference in cell proliferation when PDHA1-Ser293 and -Ser300 are overphosphorylated or dephosphorylated, B and C are Edu detection of proliferation of hCMEC / D3 cells of different genotypes, D and E are Transwell detection of migration of hCMEC / D3 cells of different genotypes, F to H are tubule formation experiments to detect the vascular mimicry formation ability of hCMEC / D3 cells of different genotypes, S293E represents PDHA1-S293E, S300E represents PDHA1-S300E, S293A represents PDHA1-S293A, S300A represents PDHA1-S300A, WT represents wildtype, E represents Glutamate, A represents alanine, and S represents serine;

[0031] Fig.17 Figure 1 is the result of PDK2 overexpression inhibiting angiogenesis in hCMEC / D3 cells, where A is Western blotting to detect the changes in PDHA1-Ser293 / 300 phosphorylation modification in PDK2 overexpressing cells, B is Western blotting to detect the changes in PDHA1-Ser293 / 300 phosphorylation modification in PDK2 knockdown cells, C and D are Edu to detect the proliferation phenotype of PDK2 overexpressing hCMEC / D3 cells, E and F are Transwell to detect the migration phenotype of PDK2 overexpressing hCMEC / D3 cells, G and H are tubule formation experiments to detect the vascular mimicry formation ability of PDK2 overexpressing hCMEC / D3 cells, I and J are Edu to detect the PDHA1- Ser293 / 300 hyperphosphorylated hCMEC / D3 cell proliferation phenotype, K and L are Transwell assays for PDK2 overexpressed PDHA1-Ser293 / 300 hyperphosphorylated hCMEC / D3 cell migration phenotype, M-O are tubule formation assays for PDK2 overexpressed PDHA1-Ser293 / 300 hyperphosphorylated hCMEC / D3 cell vascular mimicry formation ability, P and Q are Edu assays for PDK2 knockdown hCMEC / D3 cell proliferation phenotype, R-T are tubule formation assays for PDK2 knockdown hCMEC / D3 cell vascular mimicry formation ability, EE represents PDHA1-S293E / S300E, WT represents wildtype, SHC represents shRNA for control gene, *** represents P<0.001; ** represents P<0.01; * represents P<0.05, ns represents P≥0.05;

[0032] Fig.18The results of simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 blocking mitochondrial oxidative phosphorylation and glycolysis in hCMEC / D3 cells are shown in Figure 1. A shows that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits PDHc activity in cells. B shows that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 affects the binding of PDHA1 and PDHB proteins in cells. C shows that the O2K system analysis results show that PDHA1-Ser293 / 3 00 is the graph of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting cell oxygen consumption rate, D is the graph of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting cell basic respiration, E is the graph of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting cell maximum respiration, F is the graph of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting cell respiratory reserve capacity, G is the graph of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting cell complex II oxidative phosphorylation, H is the graph of PDHA1-Ser29 3 / 300 simultaneous hyperphosphorylation inhibits the non-mitochondrial oxidation rate of cells, I is the diagram of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting the basic glycolysis of cells, J is the diagram of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting the maximum glycolysis capacity of cells, K is the diagram of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting the formation of non-mitochondrial lactate in cells, L is the O2K system analysis results showing that PDHA1-Ser293 / 300 Simultaneous hyperphosphorylation inhibits the ability of cell acid production. M is the ability of PDHA1-Ser293 / 300 to inhibit extracellular pH exchange by simultaneous hyperphosphorylation. N and O are the ability of PDHA1-Ser293 / 300 to inhibit ROS accumulation in cells by simultaneous hyperphosphorylation. P and Q are the ability of PDHA1-Ser293 / 300 to inhibit mitochondrial membrane potential in cells by simultaneous hyperphosphorylation. EE indicates PDHA1-S293E / S300E. WT indicates wildtype. WCL indicates whole cell lysate. *** indicates P<0.001; ** indicates P<0.01; * indicates P<0.05; ns indicates P≥0.05.

[0033] Fig.19 Heatmap shows the differential metabolites in cells with different genotypes, where EE represents PDHA1-S293E / S300E; AA represents PDHA1-S293A / S300A, and CON represents control;

[0034] Fig. 20 The trend diagram of 27 differential metabolites in cells of different genotypes;

[0035] Fig.21The results of the signal pathway analysis of 27 differential metabolites enrichment are shown in Figure 1, where EE represents PDHA1-S293E / S300E; AA represents PDHA1-S293A / S300A, and CON represents control;

[0036] Fig. 22 Figure 2 is a diagram showing that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 participates in the regulation of hCMEC / D3 cell angiogenesis by inhibiting the cAMP / PKA / CREB signaling pathway, wherein A is the ELISA result showing that the cAMP content in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 is reduced, B is a schematic diagram of the cAMP / PKA / CREB signaling pathway, C is the Western blotting result showing the changes in the expression of cAMP effector proteins and substrate proteins in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, D-F are tubule formation experiments to detect the angiogenesis phenotype inhibited by simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 treated with 8-Bromo-cAMP, G is Western blotting, Blotting was used to identify the CREB gene knockdown and PDHA1-Ser293 / 300 hyperphosphorylated cell lines. H-J are tubule formation experiments to detect the angiogenesis phenotype of 8-Bromo-cAMP-treated CREB knockdown and PDHA1-Ser293 / 300 hyperphosphorylated cells. EE represents PDHA1-S293E / S300E, AA represents PDHA1-S293A / S300A, WT represents wildtype, E represents Glutamate, A represents alanine, S represents serine, *** represents P<0.001, ** represents P<0.01, and * represents P<0.05.

[0037] Fig.23 is the cytoplasmic Ca 2+ The decrease of cAMP is the main reason for the inhibition of cAMP / PKA / CREB signaling pathway. The verification result diagram, in which A is ELISA detection of cAMP content in the cytoplasm and mitochondria of cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, B is Western blotting detection of cAMP effector protein activation in the cytoplasm and mitochondria of cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, C is ELISA detection of the effect of bicarbonate treatment on cAMP content in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, D is Western blotting detection of the effect of bicarbonate treatment on the activation of cAMP effector protein in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, E and F are cytoplasmic Ca 2+ probe (Fluo4 / AM) and mitochondrial Ca2+ The probe (Rhod2 / AM) monitors PDHA1-Ser293 / 300 hyperphosphorylation in the cytosol and mitochondria. 2+ Ion changes, G and H are cytoplasmic Ca 2+ probe (Fluo4 / AM) and mitochondrial Ca 2+ The probe (Rhod2 / AM) monitors MCU-i4 treatment of PDHA1-Ser293 / 300 and the simultaneous hyperphosphorylation of Ca in the cytosol and mitochondria. 2+ Ion changes, I is ELISA detection of cAMP changes in the cytoplasm and mitochondria of cells with hyperphosphorylated PDHA1-Ser293 / 300 treated with MCU-i4, J is Western blotting detection of cAMP effector protein activation in the cytoplasm and mitochondria of cells with hyperphosphorylated PDHA1-Ser293 / 300 treated with MCU-i4, K and L are tubule formation experiments to detect the functional recovery effect of MCU-i4 treatment on angiogenesis of cells with hyperphosphorylated PDHA1-Ser293 / 300, M~O are tubule formation experiments to detect the functional recovery effect of MCU-i4 and 8-Bromo-cAMP treatment on angiogenesis of cells overexpressing PDK2, EE represents PDHA1-S293E / S300E, WT represents wildtype, 8-Br-cAMP represents 8-Bromo-cAMP, Bicarb represents bicarbonate, *** represents P<0.001; ** represents P<0.01; * represents P<0.05;

[0038] Fig.24 The volcano plot shows the differentially expressed genes in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, where EE represents PDHA1-S293E / S300E and CON represents control;

[0039] Fig.25 GO enrichment analysis shows the biological function map of differentially expressed genes enrichment;

[0040] Fig.26 The signal pathway diagram showing the enrichment of differentially expressed genes is KEGG enrichment;

[0041] Fig. 27 It is the core hub differentially expressed gene-KEGG signaling pathway interaction network diagram;

[0042] Fig.28The results of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting FOS gene expression and inhibiting cell angiogenesis phenotype, where A is a volcano diagram of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting FOS gene expression, B is a qRT-PCR diagram of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting FOS gene expression, C is Western blotting of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting FOS gene expression, and D is Western Blotting was used to identify the overexpression of FOS gene in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300. E and F are Edu results showing the functional recovery effect of FOS overexpression on the proliferation of cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300. G and H are Transwell results showing the functional recovery effect of FOS overexpression on the migration of cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300. I-K are tubule formation results showing the functional recovery effect of FOS overexpression on the formation of vascular mimicry of cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300. EE indicates PDHA1-S293E / S300E, WT indicates wild type, *** indicates P<0.001; ** indicates P<0.01; * indicates P<0.05.

[0043] Fig.29 The schematic diagram of the cAMP signaling pathway shows that FOS is a downstream target gene of CREB, in which cAMP and FOS are marked in red, and cAMP effector protein PKA and its substrate protein CREB are marked in green;

[0044] Fig.30 The FOS transcription factors predicted by the JASPAR database, of which CREB is marked in red;

[0045] Fig.31 This is the FOS promoter sequence, in which the red mark is the CREB binding motif;

[0046] Fig.32The results of cell experiments prove that FOS, as a CREB target gene, participates in the regulation of cell angiogenesis by the cAMP / PKA / CREB signaling pathway, where A is qRT-PCR detection of FOS gene expression changes in CREB gene knockdown cells, B is Western blotting detection of FOS protein expression changes in CREB gene knockdown cells, C is qRT-PCR detection of FOS gene expression changes in CREB (or mutant CREB) overexpression cells, D is Western blotting detection of FOS protein expression changes in CREB (or mutant CREB) overexpression cells, E is a dual luciferase reporter gene activity assay to detect the effect of CREB (or mutant CREB) overexpression on FOS gene transcription, F is Western blotting detection of FOS expression changes in IBMX-treated PDHA1-Ser293 / 300 hyperphosphorylated cells, G is Western blotting detection of FOS expression changes in 8-Bromo-cAMP-treated PDHA1-Ser293 / 300 hyperphosphorylated cells, H is Western Blotting was used to detect the changes in FOS expression in PDHA1-Ser293 / 300 hyperphosphorylated cells treated with MCU-i4. I was Western blotting to identify PDHA1-Ser293 / 300 hyperphosphorylated cells with FOS gene knockdown. J-L were tubule formation experiments to detect the effect of FOS knockdown on the functional recovery of 8-Bromo-cAMP in PDHA1-Ser293 / 300 hyperphosphorylated cells. EE means PDHA1-S293E / S300E, WT means wild type, 8-Br-cAMP means 8-Bromo-cAMP, SHC means shRNA for control gene, *** means P<0.001; ** means P<0.01; * means P<0.05.

[0047] Fig.33The results of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation inhibiting CCN2 gene expression and inhibiting cell angiogenesis are shown in Figure 1, where A is the protein interaction network analysis of the interaction relationship between differentially low-expressed genes in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, B is qRT-PCR detection of CCN2 expression changes in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, C is Western blotting detection of CCN2 expression changes in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, D is qRT-PCR detection of CCN2 expression changes in FOS gene knockdown cells, E is Western blotting detection of CCN2 expression changes in FOS gene knockdown cells, and F is Western Blotting was used to detect the changes in CCN2 expression in cells treated with 8-Bromo-cAMP and hyperphosphorylated PDHA1-Ser293 / 300. G is Western blotting to detect the changes in CCN2 expression in cells treated with MCU-i4 and hyperphosphorylated PDHA1-Ser293 / 300. H is Western Blotting was used to identify cells with overexpression of CCN2 and hyperphosphorylation of PDHA1-Ser293 / 300. I and J are Transwell assays to detect the effect of overexpression of CCN2 on the migration of cells with overexpression of PDHA1-Ser293 / 300. K-M are tubule formation assays to detect the effect of overexpression of CCN2 on the formation of vascular mimicry in cells with overexpression of PDHA1-Ser293 / 300. N is Western blotting to identify cells with overexpression of PDHA1-Ser293 / 300 and hyperphosphorylation of FOS in cells with knockdown of CCN2. O and P are Transwell assays to detect the functional recovery of cell migration caused by overexpression of FOS in cells with knockdown of CCN2. Q-S are tubule formation assays to detect the functional recovery of cell vascular mimicry caused by overexpression of FOS in cells with knockdown of CCN2. EE indicates PDHA1-S293E / S300E. WT indicates wild type. 8-Br-cAMP indicates 8-Bromo-cAMP. SHC indicates shRNA for control. gene, *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05;

[0048] Fig.34 This is the result of Western blotting to detect the expression of cAMP / PKA / CREB and FOS / CCN2 proteins in PDK2-overexpressing cells;

[0049] Fig.35The results of the effects of different treatments on the angiogenic phenotype of PDK2 overexpressing cells are shown in Figure 1, where 8-Br-cAMP represents 8-Bromo-cAMP, *** represents P<0.001; ** represents P<0.01; * represents P<0.05;

[0050] Fig.36 This is a diagram of the overexpression of FOS and CCN2 in PDK2-overexpressing cells detected by Western blotting, where 8-Br-cAMP represents 8-Bromo-cAMP;

[0051] Fig.37 The results of the effect of FOS or CCN2 overexpression on the migration of PDK2 overexpressing cells are shown in Figure 1, where *** indicates P<0.001; ** indicates P<0.01; * indicates P<0.05;

[0052] Fig.38 The results of the tubule formation test of the effect of FOS or CCN2 overexpression on the vascular mimicry formation of PDK2 overexpressing cells, where *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05;

[0053] Fig.39 The results of glioma cells reducing the phosphorylation modification of PDHA1-Ser293 / 300 in hCMEC / D3 cells, where A is the two-dimensional electrophoresis analysis of T98G conditioned medium treatment to change the isoelectric point of PDHA1 in hCMEC / D3 cells, the red mark is the existence form of PDHA1 protein, a is the two-dimensional electrophoresis SDS-PAGE result of hCMEC / D3 cells treated with DMEM medium, b is the two-dimensional electrophoresis SDS-PAGE result of hCMEC / D3 cells treated with T98G conditioned medium, and c is the anti-PDHA 1 is the PVDF membrane of two-dimensional electrophoresis of hCMEC / D3 cells treated with DMEM medium labeled with anti-PDHA1 antibody, d is the PVDF membrane of two-dimensional electrophoresis of hCMEC / D3 cells treated with T98G conditioned medium labeled with anti-PDHA1 antibody, B is Western blotting detection of glioma cell conditioned medium treatment to reduce PDHA1-Ser293 and -Ser300 phosphorylation modifications in hCMEC / D3 cells, C is a structural model diagram of co-culture of glioma cells and hCMEC / D3, and D is Western blotting detection of glioma cells to reduce PDHA1-Ser293 and -Ser300 phosphorylation modifications in hCMEC / D3 cells;

[0054] Fig.40The results of the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 and overexpression of PDK2 inhibiting the pro-angiogenic function of glioma cells on hCMEC / D3 cells for tubulogenesis detection, where CM represents conditioned medium, EE represents PDHA1-S293E / S300E, WT represents wildtype, *** represents P<0.001; ** represents P<0.01; * represents P<0.05;

[0055] Fig.41 The results of the lactic acid content test in the conditioned medium of different cell lines are shown in FIG.

[0056] Fig.42 Results of the tubule formation test showing the effect of lactate on promoting angiogenesis in hCMEC / D3 cells;

[0057] Fig.43 This is the result of the tubule formation test of the promoting effect of lactate on HUVEC cell angiogenesis;

[0058] Fig.44 Figure 1 is the result of lactate regulating the existence form of PDHA1 protein in hCMEC / D3 cells, where A is the effect of lactate treatment on the existence form of PDHA1 protein in hCMEC / D3 cells detected by two-dimensional electrophoresis, the red arrow indicates the existence form of PDHA1 protein, a is the result of two-dimensional electrophoresis SDS-PAGE of hCMEC / D3 cells treated with PBS, b is the result of two-dimensional electrophoresis SDS-PAGE of hCMEC / D3 cells treated with lactate, c is the PVDF membrane of two-dimensional electrophoresis of hCMEC / D3 cells treated with PBS labeled with anti-PDHA1 antibody, d is the PVDF membrane of two-dimensional electrophoresis of hCMEC / D3 cells treated with lactate labeled with anti-PDHA1 antibody, and B is the effect of lactate treatment on PDHA1-Ser293 / 300 phosphorylation in hCMEC / D3 cells detected by Western blotting;

[0059] Fig.45The figures show that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits the angiogenic effect of lactate on hCMEC / D3 cells, A to C show that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits the angiogenic effect of lactate on hCMEC / D3 cells, D to F show that overexpression of PDK2 inhibits the angiogenic effect of lactate on hCMEC / D3 cells, G to I show that knockdown of PDK2 and dephosphorylation of PDHA1-Ser293 / 300 promote the angiogenic effect of lactate on hCMEC / D3 cells, CM indicates conditioned medium, EE indicates PDHA1-S293E / S300E, LA indicates lactic acid, WT indicates wild type, *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05. DETAILED DESCRIPTION

[0060] The present invention provides a PDHA1-Ser293 / 300 common phosphorylation protein marker for inhibiting tumor angiogenesis. The sequence of the PDHA1-Ser293 / 300 common phosphorylation protein marker is shown in SEQ ID NO: 1, and is specifically as follows:

[0061] MRKMLAAVSRVLSGASQKPASRVLVASRNFANDATFEIKKCDLHRLEEGPPVTTVLTREDGLKYYRMMQTVRRMELKADQLYKQKIIRGFCHLCDGQEACCVGLEAGINPTDHLITAYRAHGFTFTRGLSVREILAELTGRKGGCAKGKGGSMHMYAKNFYGGNGIVGAQVPLGAGIALACKYNGKDEVCLTLYGD GAANQGQIFEAYNMAALWKLPCIFICENNRYGMGTSVERAAASTDYYKRGDFIPGLRVDGMDILCVREATRFAAAYCRSGKGPILMELQTYRYHGHpSMSDPGVpSYRTREEIQEVRSKSDPIMLLKDRMVNSNLASVEELKEIDVEVRKEIEDAAQFATADPEPPLEELGYHIYSSDPPFEVRGANQWIKFKSVS.

[0062] In the present invention, the PDHA1-Ser293 / 300 co-phosphorylated protein marker is PDHA1-Ser293 / 300 co-hyperphosphorylation, and the PDHA1-Ser293 / 300 co-hyperphosphorylation is obtained by mutating the serine at the 293rd and 300th positions on the PDHA1 protein to glutamic acid.

[0063] The present invention provides the use of the PDHA1-Ser293 / 300 common phosphorylation protein marker as a molecular target in the preparation or screening of drugs for inhibiting tumor angiogenesis.

[0064] In the present invention, the PDHA1-Ser293 / 300 co-phosphorylated protein marker is PDHA1-Ser293 / 300 co-hyperphosphorylation, and the PDHA1-Ser293 / 300 co-hyperphosphorylation is obtained by mutating the serine at the 293rd and 300th positions on the PDHA1 protein to glutamic acid.

[0065] The present invention provides an application of a reagent causing the co-phosphorylation of PDHA1-Ser293 / 300 in the preparation of a drug for inhibiting tumor angiogenesis.

[0066] In the present invention, the reagent causing co-phosphorylation of PDHA1-Ser293 / 300 includes one or more of a nucleotide sequence capable of causing PDHA1 protein mutation, a plasmid containing the nucleotide sequence, and a recombinant cell containing the nucleotide sequence; the drug for inhibiting tumor angiogenesis is a drug for inhibiting glioma angiogenesis.

[0067] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0068] In the present invention, data were analyzed using Graphpadprism5 statistical software and R software. Measurement data were expressed as mean ± SD. For the comparison of two groups of continuous variables, the variables were in accordance with normal distribution and the significance statistics were analyzed by student t-test, and the differences of non-normal distribution variables were analyzed by Mann-Whitney U test (i.e. Wilcoxon rank sum test). Pearson correlation analysis was used to calculate the correlation coefficients of different genes. The prognosis survival curve was calculated using the Kaplan-Meier method, and the Log-rank method was used to compare the differences between the survival rates of the two. Multi-factor multi-component, variables were in accordance with normal distribution and two-way ANOVA was used for statistical analysis, univariate multi-component, variables were in accordance with normal distribution and one-way ANOVA was used, and statistical analysis between the two factors was performed by Tukey post-test according to the statistical results.

[0069] Example 1 Relationship between PDHA1-Ser293 / 300 phosphorylation and tumor vascular density in glioma VECs

[0070] In order to detect the potential function of PDHA1-Ser293 / 300 in regulating glioma angiogenesis, the present invention collected 10 clinical tissues of high-grade glioma (Department of Pathology, Shandong Cancer Hospital Affiliated to Shandong First Medical University) and used mIF technology to detect the phosphorylation levels of PDHA1-Ser293 and -Ser300 in intratumoral VECs and the effect of PDK2 protein expression on the expression abundance of endothelial marker protein CD31.

[0071] 1.1 Extraction of total cell RNA: refer to the instructions of RNA extraction kit (Super Fast Pure Cell RNA Isolation Kit, #RC102, Vazyme).

[0072] 1.2 cDNA library construction

[0073] 1) Use 1-2 μg total RNA as template, add Oligo(dT)18 and DEPC water, vortex to mix, and centrifuge (system see Table 1);

[0074] Table 1 cDNA chain generation system 1

[0075]

[0076] 2) React in a PCR instrument at 65°C for 5 min to open the RNA secondary structure; 3) Immediately place on ice to cool; 4) Centrifuge and collect the liquid at the bottom of the EP tube; 5) Add reagents to each tube according to the system in Table 2 below (final volume is 25 μL);

[0077] Table 2 cDNA chain generation system 2

[0078]

[0079] 6) Place in a PCR instrument and react at 42°C for 1 hour; 7) Continue to react at 75°C for 15 minutes; 8) Store the generated cDNA in a -40°C refrigerator.

[0080] 1.3 Quantitative real-time PCR (qRT-PCR)

[0081] 1) Mix the reaction reagents in a 0.1 mL eight-tube tube according to the reaction system in Table 3 and react with a PCR instrument;

[0082] Table 3 Fluorescence quantitative PCR reaction system

[0083]

[0084] 2) After the reaction is completed, the ct value representing the gene expression level is output and the -ΔΔctThe relative expression values ​​of related genes were calculated by the calculation method.

[0085] 1.4 Multiplex immunofluorescence

[0086] 1) After paraffin sections were dewaxed and hydrated with xylene (xylene 1 for 30 min, xylene II for 30 min, anhydrous ethanol + xylene for 5 min, anhydrous ethanol for 5 min, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, water), they were rinsed in PBS for 3 times, 3 min each time; 2) According to the requirements of each antibody, tissue antigens were repaired accordingly (3% H2O2 for 3 min); 3) PBS was rinsed for 3 times, 3 min each time; 4) The first blocking step: 10% bovine serum albumin (Bovine serum albumin) was used at room temperature. 5) Incubate the cells with the first primary antibody (dissolved in 1% BSA in PBST) in a humidified chamber for 1 hour at room temperature; 6) Pour out the first primary antibody solution and wash the tissue sections 3 times with PBS, 5 minutes each time; 7) Incubate the cells with the first secondary antibody (dissolved in 1% BSA in PBST) in the dark for 1 hour at room temperature; 8) Pour out the first secondary antibody solution and wash the cells 3 times with PBS, 5 minutes each time; 9) Second blocking step: Incubate the cells with 10% BSA in the dark for 30 minutes at room temperature; 10) Incubate the cells with the second primary antibody (dissolved in 1% BSA) in a humidified chamber for 1 hour at room temperature; 11) Pour out 1) Pour out the second primary antibody solution, wash the tissue sections with PBS in the dark for 3 times, 5 min each time; 12) Incubate the cells with the second secondary antibody (dissolved in 1% BSA) in the dark for 1 hour at room temperature; 13) Pour out the second secondary antibody solution, wash the cells with PBS in the dark for 3 times, 5 min each time; if more than two antigens need to be detected, continue the operation of the remaining antibodies according to steps 4) to 8); 14) Incubate the cells with 0.1-1 μg / mL Hoechst or DAPI (DNA stain) for 1 min; 15) Rinse the cells with PBS; 16) Seal the coverslip with a drop of mounting medium; 17) Seal the coverslip with nail polish to prevent the sample from drying out and moving under the microscope; 18) Store at 4°C in the dark.

[0087] According to the fluorescence quantitative results, the intratumoral blood vessels were divided into high PDK2 group and low PDK2 group according to the PDK2 expression level. The PDHA1-pSer300 and CD31 fluorescence quantitative statistical results showed that the PDHA1-pSer300 level in the PDK2 high expression group was significantly higher than that in the PDK2 low expression group ( Figure 1 The CD31 fluorescence intensity in the PDK2 high expression group was significantly lower than that in the PDK2 low expression group ( Figure 1A and F in Figure 3). The intratumoral blood vessels were divided into high phosphorylation group and low phosphorylation group according to the level of PDHA1-pSer300. The quantitative statistical results of PDK2 and CD31 fluorescence showed that the PDK2 level in the high phosphorylation group of PDHA1 was significantly higher than that in the low phosphorylation group ( Figure 1 A and C in Figure ), while the CD31 fluorescence intensity in the PDHA1 high phosphorylation group was significantly lower than that in the low phosphorylation group ( Figure 1 A and D in the figure). The intratumoral blood vessels were divided into CD31 high expression group and CD31 low expression group according to the CD31 level. The PDK2 and PDHA1-pSer300 fluorescence quantitative statistical results showed that the PDK2 and PDHA1-pSer300 in the CD31 high expression group were significantly lower than those in the low expression group ( Figure 1 Figure 5 (E and G) showed that PDK2 regulates the phosphorylation of PDHA1-pSer300 in glioma VECs.

[0088] Example 2 Effect of PDK2 overexpression on VECs angiogenesis and glioma

[0089] 2.1 Glioma intracranial tumor inoculation and detection

[0090] 1) 5-6 week old C57BL / J6 (purchased from Nanjing Animal Model Biology Institute) were placed under SFP standard conditions for 1 week to make the mice familiar with the environment; 2) Firefly luciferase-labeled glioma261 cells (#CBP60669; purchased from China Cell Bank) were cultured in large quantities; 3) Cells in the logarithmic growth phase were taken, washed with PBS, and digested with trypsin; 4) The digestion was terminated with complete medium and centrifuged at 1000 rpm for 10 min; 5) The cells were resuspended in serum-free medium containing 10% matrigel, counted, and the cell density was adjusted to 1×10 6 / μL; 6) Inject the cells at a density of 2μL / mouse into the mouse brain 3mm below the left cross line, i.e. 2×10 6 7) Observe the tumor formation in mice and detect the tumor growth using a small animal in vivo imaging system; 8) After 3 weeks of continuous detection, the mice were killed, the mouse brain tissues were dissected and separated, and the tissues were preserved in 4% paraformaldehyde for later use; 9) Detect the death of mice and draw the mouse survival curve.

[0091] 2.2 Immunohistochemical staining

[0092] 1) After the paraffin sections were dewaxed and hydrated with xylene (xylene Ι for 30 min, xylene Ⅱ for 30 min, anhydrous ethanol + xylene for 5 min, anhydrous ethanol for 5 min, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, water), they were rinsed with PBS for 3 times, each time for 3 min; 2) According to the requirements of each antibody, the tissue antigens were repaired accordingly (3% H2O2 for 3 min, EDTA repair); 3) PBS was rinsed for 3 times, each time for 3 min; 4) PBS was removed, and 50 μL of non-immune animal serum was added to each slide for 20 min; 5) Serum was removed, and 50 μL of specific primary antibody was added to each slide, and it was kept at 4°C overnight; 6) Primary antibody was removed, and PBS was rinsed for 3 times, each 7) Remove PBS, add 50 μL of biotin-labeled secondary antibody to each slide and incubate at room temperature for 10 min; 8) Remove secondary antibody, rinse with PBS 3 times, 3 min each time; 9) Remove PBS, add 50 μL of streptavidin-peroxidase solution to each slide and incubate at room temperature for 10 min; 10) Remove streptavidin-peroxidase solution, rinse with PBS 3 times, 3 min each time; 11) Remove PBS, add 100 μL of freshly prepared DAB solution to each slide and observe under a microscope for 3 to 10 min; 12) Rinse with tap water, counterstain with hematoxylin, and rinse with PBS or tap water to turn blue; 13) Make xylene transparent, fix with neutral resin and take photos with an optical microscope.

[0093] Adeno-associated virus (AAV) was used to infect C57BL / 6J mice to construct a model mouse model with specific overexpression of PDK2 in brain VECs. Two weeks later, the purchased firefly luciferase-labeled mouse glioma cells GL261 were transplanted into the right prefrontal cortex of the model mice. The growth of brain tumors in mice was regularly observed by small animal imaging technology ( Figure 2 and Figure 3 When the mouse intracranial tumor grew to a certain dimension, the whole brain tissue of the model mouse was obtained by dissection. After preparing paraffin-embedded sections, the mIF technology was used to detect the expression of PDK2 in VECs to determine whether the model mouse was successfully constructed. The results showed that specific PDK2 overexpression occurred in the VECs of the brain tumor of the PDK2-overexpressing mouse ( Figure 4 and Figure 5 The growth of intracranial tumors in mice was detected by in vivo imaging of small animals. The results showed that the growth of brain tumors in the PDK2 overexpression group was significantly slow. Figure 6 ). The statistical results of mouse survival showed that the survival time of mice in the PDK2 overexpression group was significantly prolonged ( Figure 7 The nuclei of the mouse brain tissue obtained by dissection were marked, and the area with dense nuclei was the tumor tissue area. The results showed that the infiltration area of ​​brain tumors in the PDK2 overexpression group of mice was significantly reduced ( Figure 8 ). mIF was used to further analyze the tumor vascular density marked by CD31, and the results showed that the vascular density in the brain tumors of mice in the PDK2 overexpression group was significantly reduced ( Fig. 9 ).

[0094] Example 3 Effect of simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 on angiogenesis in hCMEC / D3 cells

[0095] 3.1 Experimental methods

[0096] 3.1.1 Cell culture

[0097] 1) Cell recovery: (1) Take out the frozen hCMEC / D3 cells (#CL0843, purchased from Wuhan Pronocell Life Science Co., Ltd.) from liquid nitrogen and immediately thaw them in a 37°C water bath; (2) Transfer the thawed cells into a 15 mL sterile centrifuge tube and add 2 mL of complete culture medium (89% DMEM + 10% FBS + 1% PS); (3) Centrifuge at 1000 rpm for 10 min; (4) Discard the supernatant, add complete culture medium to resuspend, place in a 10 cm cell culture dish, and culture in a 37°C incubator (5% CO2) for 24 h.

[0098] 2) Cell culture: (1) Observe the cell growth status under a microscope; (2) Subculture the cells when the degree of polymerization reaches more than 95%; (3) Remove the culture supernatant and wash with PBS 1 to 2 times; (4) Digest with trypsin for 1 min; (5) Add complete culture medium to terminate digestion and culture the cells in a new cell culture dish.

[0099] 3) Cell freezing: (1) When the cells grow to the logarithmic phase, remove the culture medium; (2) Remove the culture supernatant and wash with PBS 1 to 2 times; (3) Trypsin digest for 1 minute; (4) Add complete culture medium to terminate digestion; (5) Centrifuge at 1000 rpm for 10 minutes; (6) Resuspend the cells in freshly prepared cell freezing solution (90% FBS + 10% DMSO) and aspirate the cells into cell freezing tubes; (7) Place the frozen cells in a programmed cooling box at -80°C and transfer them to a liquid nitrogen tank for long-term storage after 24 hours.

[0100] 3.1.2 E. coli transformation experiment

[0101] 1) Open the 42℃ water bath in advance and sterilize with UV light in the bacterial clean bench; 2) Take out the competent cells DH5α (purchased from Tiangen Biochemical Technology Co., Ltd.) from the -80℃ refrigerator and thaw on ice. Be careful not to shake violently; 3) Carefully add DNA to the competent cells and let it stand on ice for 35 minutes; 4) Heat shock at 42℃ for 90 seconds and immediately put it on ice; 5) After standing for 2-3 minutes, add 600μLL basal medium and shake at 37℃ 150rpm for 50 minutes; 6) Spread the above bacterial solution on Amp+ resistant solid medium (1L medium formula: 10g peptone, 5g yeast powder, 10g sodium chloride, 100mg / mL ampicillin sodium) taken out of the 4℃ refrigerator in advance, and place it in a 37℃ constant temperature incubator for 14±2h; 7) Observe the growth of plaques in the culture dish, pick independent and round monoclonal plaques for shaking at 37℃ 150rpm, and then detect the connection effect.

[0102] 3.1.3 Construction of shRNA stable expression cell lines

[0103] 1) Map of eukaryotic lentiviral shRNA expression vectors Fig.10 As shown, the shRNA structure formed in the cell is as follows Fig.11 .

[0104] 2) Knockdown plasmid construction

[0105] (1) The purchased lentiviral shRNA vector backbone (#10878, Addgene) was double-digested (Age Ι and Eco Ι), the system is shown in Table 4;

[0106] Table 4 AgeΙ / EcoRΙ double restriction enzyme digestion plasmid system and reaction conditions

[0107]

[0108] (2) Design the target gene shRNA sequence (21 bp) through the NEB official website, and customize the shRNA primers according to the following format;

[0109] Forwardprimer: 5'CCGG-21bp sense-CTCGAG-21bp antisense-TTTTTG3'

[0110] Reverse primer: 5'AATTCAAAAA-21bp sense-CTCGAG-21bp antisense3'

[0111] (3) After primer synthesis, annealing is performed (the system and reaction procedure are shown in Table 5); the annealing reaction procedure is: 95°C for 5 min and then slowly cooled to room temperature;

[0112] Table 5 Primer annealing reaction system

[0113]

[0114] (4) Connecting the annealed primers to the vector fragments recovered after enzyme digestion according to the ligation reaction system (see Table 6 for the ligation reaction system and reaction procedure);

[0115] Table 6 AgeΙ / EcoRΙ double restriction enzyme plasmid ligation reaction system and reaction conditions

[0116]

[0117] (5) E. coli transformation (refer to 3.1.2 E. coli transformation experiment); (6) Sequencing analysis.

[0118] 2) Lentivirus packaging: (1) HEK293T cells (#CBP60439, purchased from China Cell Bank) in the logarithmic growth phase were diluted at a 1:3 volume ratio and inoculated in a 25 cm 2 The cells were placed in a cell culture flask and cultured in a 37°C cell culture incubator; (2) when the cell growth degree of polymerization reached 70%, the virus packaging plasmid (pLK0.1-shRNA: PsPAX2: PMD2.G = 4:3:1, the plasmid was purchased from Hunan Fenghui Biotechnology Co., Ltd.); (3) the cells were cultured in a 37°C incubator for 12 hours, the supernatant was discarded, and fresh complete culture medium (89% DMEM + 10% FBS + 1% PS) was replaced; (4) after continuing to culture in a 37°C incubator for 24 hours, the supernatant suspension containing the virus was collected, temporarily stored at 4°C, and fresh complete culture medium was added again; (5) after continuing to culture for 24 hours, the supernatant suspension containing the virus was continued to be collected, and it was combined with the supernatant suspension containing the virus collected the day before, and centrifuged at 1000 rpm for 10 minutes; (6) the supernatant obtained after centrifugation was filtered through a 0.45 μm filter and temporarily stored at 4°C (long-term storage at -80°C, no repeated freezing and thawing).

[0119] 3) Infect target cells with lentivirus: (1) hCMEC / D3 cells in logarithmic growth phase were inoculated in 25 cm 2 Cell culture flask; (2) When the cells grow to a polymerization degree of 70% to 80%, they are infected with viruses and polybrene is added at a final concentration of 2 μg / mL; (3) 48 hours after viral infection, the cell culture medium is replaced with 2 μg / mL puromycin for cell screening; (4) After screening for at least 48 hours, the cells are collected for detection of target gene mRNA or protein levels, and the screening is continued for 1 week.

[0120] 4) Monoclonal cell screening: (1) The cell lines obtained by puromycin screening are inoculated into a 96-well cell culture plate at a cell density of 1 cell / well, and puromycin drug screening is performed simultaneously; (2) After the cells adhere to the plate, the monoclonal cells are marked and cultured continuously; (3) After the cultured cells grow to a cell growth density of 90%, the gene mRNA or protein level is detected, and finally monoclonal cell lines with different expression levels of the target gene are screened.

[0121] 3.1.4 Construction of gene knockout cell lines

[0122] 1) Eukaryotic gene knockout is performed using the CRISPR / Cas9 / sgRNA system. See the vector map for details. Fig.12 .

[0123] 2) CRISPR / Cas9 / sgRNA plasmid construction

[0124] (1) The purchased vector CRISPR-v2 (#52961, Addgene) backbone was treated with enzymes (BsmbΙ), the system is shown in Table 7;

[0125] Table 7 BsmbI restriction enzyme plasmid system and reaction conditions

[0126]

[0127] (2) sgRNA sequence synthesis primer, annealing, the system is the same as Table 5; according to the ligation reaction system, the annealed primer is connected to the vector fragment recovered after enzyme digestion (the system is the same as Table 6); (3) Escherichia coli transformation (refer to 3.1.2 Escherichia coli transformation experiment); (4) Sequencing analysis.

[0128] 3) Lentiviral packaging (refer to 2) Lentiviral packaging in 3.1.3 Construction of shRNA stable expression cell line).

[0129] 4) Infect target cells with lentivirus (refer to 3) Infect target cells with lentivirus in 3.1.3 Construction of shRNA stable expression cell line).

[0130] 5) Gene knockout efficiency detection: (1) Cell genome extraction; (2) PCR amplification of sgRNA region DNA fragments, the system is shown in Table 8, and the primer design method used is the same as above; (3) PCR product annealing (the system is the same as Table 5); (4) T7 nuclease detection of CRISPR / Cas9 / sgRNA efficiency, the system is shown in Table 9; (5) Agarose gel electrophoresis to detect T7 nuclease digestion results, and successful digestion is considered to have potential CRISPR / Cas9 / sgRNA knockout efficiency.

[0131] Table 8 PCR amplification reaction system

[0132]

[0133] Table 9T7 endonuclease reaction system

[0134]

[0135] 6) Monoclonal cell screening: (1) The cell lines obtained by puromycin screening are inoculated into a 96-well cell culture plate at a cell density of 1 / well, and puromycin drug screening is performed simultaneously; (2) After the cells adhere to the plate, the monoclonal cells are marked and cultured continuously; (3) After the cultured cells grow to a cell growth density of 90%, the protein level is detected, and finally monoclonal cell lines with different expression levels of the target gene are screened.

[0136] 3.1.5 Construction of gene overexpression cell lines

[0137] 1) Construction of gene overexpression plasmid

[0138] (1) Design specific primers according to the gene sequence insertion site in the pCDH-CMV-MCS-EF1-turboRFP-T2A-Puro vector backbone, and amplify the target gene fragment using cDNA as a template. The synthesis system is shown in Table 10;

[0139] Table 10 Target fragment amplification reaction system

[0140]

[0141] (2) Vector linearization (BamHI was used), and the enzyme digestion system is shown in Table 11;

[0142] Table 11 Vector linearization restriction enzyme digestion reaction system

[0143]

[0144] (3) tapping the linearized plasmid fragments to recover the fragments;

[0145] (4) Connecting the linearized vector to the gene fragment synthesized by PCR using a recombinase system. The connection system is shown in Table 12;

[0146] Table 12T4 ligase ligation reaction system

[0147]

[0148] (5) Transform the concatenated product into E. coli (refer to 3.1.2 E. coli transformation experiment);

[0149] (6) Pick the positive monoclonal clones and analyze the connection results by sequencing;

[0150] (7) The plasmid returned by sequencing is amplified to obtain a vector plasmid of transfection purity.

[0151] 2) Lentiviral packaging (refer to 2) Lentiviral packaging in 3.1.3 Construction of shRNA stable expression cell line).

[0152] 3) Lentiviral infection (refer to 3) Infection of target cells with lentivirus in 3.1.3 Construction of shRNA stable expression cell line).

[0153] 3.1.6 Construction of gene site-directed mutagenesis cell lines

[0154] 1) Construction of target gene knockout cell line (refer to 3.1.4 Construction of gene knockout cell line).

[0155] 2) Construction of target gene site-directed mutagenesis overexpression plasmid (taking PDHA1 as an example):

[0156] (1) Design and synthesis of a mimetic of hyperphosphorylated PDHA1 S293E / S300E and simulated dephosphorylation of PDHA1 S293A / S300A The site-directed mutagenesis primers were used to amplify the PCR fragment using the constructed pCDH-CMV-PDHA1-EF1-turboRFP-T2A-Puro plasmid as a template (the system is shown in Table 13);

[0157] Table 13 Target fragment amplification reaction system

[0158]

[0159] (2) Add 2 μL of DpnI endonuclease to the PCR amplification product and react at 37°C for 15 min; (3) Purify and recover the cleavage product and measure its concentration; (4) Circularize the cleavage product (see Table 14 below for the system); (5) Transform into Escherichia coli (refer to 3.1.2 Escherichia coli transformation experiment); (6) Pick positive clones; (7) Sequencing and identification.

[0160] Table 14 PCR product cyclization system

[0161]

[0162] 3) Lentiviral packaging (refer to 2) Lentiviral packaging in 3.1.3 Construction of shRNA stable expression cell line).

[0163] 4) Lentiviral infection (refer to 3) Infection of target cells with lentivirus in 3.1.3 Construction of shRNA stable expression cell line).

[0164] 3.1.7 Western blotting

[0165] 1) Extraction of total cell protein (hCMEC / D3 cells as an example): (1) hCMEC / D3 cells were digested with trypsin and placed in a 1.5 mL EP tube and centrifuged at 1000 rpm for 10 min; (2) After washing with PBS 1 to 2 times, RIPA lysis buffer was added to the cells and lysed on ice for 30 min, with vortexing for 1 min every 10 min; (3) After cell lysis was completed, ultrasonic disruption was performed; (4) Centrifuged at 12000 rpm for 30 min at 4°C; (5) The supernatant was taken, 25 μL of protein loading buffer was added, and the cells were boiled in boiling water for 5 min and stored at -80°C for later use.

[0166] 2) BCA protein quantification: (1) Dilute the undenatured protein 20-fold; (2) Add the diluted protein and standard to a 96-well cell culture plate according to the instructions of the BCA assay kit (#P0009, purchased from Shanghai Biotech Biotechnology Co., Ltd.); (3) After adding the reaction solution, incubate at 25°C for 10 min; (4) Measure with an ELISA reader; (5) Calculate the protein concentration.

[0167] 3) SDS-PAGE vertical electrophoresis: (1) Prepare stacking gel and separation gel according to the instructions of the gel preparation kit (#P0012A, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) (the concentration of separation gel is related to the size of the protein); (2) Load the protein at a volume of 40 μg / well; (3) Perform electrophoresis at 80 V for 10 min. After the protein crosses the boundary between the stacking gel and the separation gel, switch to 150 V for 60 min; (4) Transfer to the membrane at 250 mA for 90 min; (5) Block with 5% BSA for 1 to 2 h; (6) Wash with 0.5% PBST 1 to 2 times, then add the primary antibody and incubate at 4°C overnight; (7) Recover the primary antibody and wash with 0.5% PBST 4 times, 10 min each time; (8) Add the secondary antibody and incubate at 25°C for 1 to 2 h; (9) Wash with 0.5% PBST 4 times, 10 min each time; (10) ECL color development.

[0168] 3.1.8 CCK-8 detection of cell viability

[0169] 1) Cells in the logarithmic growth phase were seeded into a 96-well cell culture plate at a cell density of 1500 cells / well (depending on the cells), and at least 4 replicates were performed in each group as parallel experiments; 2) After the cells grew stably, 10% CCK-8 reagent was added to each well and incubated at 37°C for 1 to 2 hours; 3) OD450 was measured by an ELISA reader; 4) Cell viability was calculated by subtracting the blank control value from the data reading of each group.

[0170] 3.1.9 Transwell assay for cell migration

[0171] 1) Take cells with a growth density of 80%, wash with PBS, and then digest with 1 mL of trypsin for 1 min; 2) Add 3 mL of complete culture medium to terminate digestion, and immediately centrifuge at 1000 rpm for 10 min; 3) Resuspend the cells with 1 mL of serum-free culture medium, and centrifuge at 1000 rpm for 10 min; 4) Repeat step 3 2 to 3 times until the serum in the cells is completely washed away; 5) Count the cells and adjust the cell density to 5×10 5 / mL; 6) Take 200μL of cells and add them to the upper chamber of the Transwell chamber covered with matrix gel, and add 600μL of complete culture medium (matrigel concentration is 1:20) to the lower chamber; 7) Place in a 37℃ cell culture incubator and culture for 48h; 8) Take out the Transwell chamber and carefully wipe the cells in the upper chamber with a cotton swab; 9) Fix the cells on the back of the chamber (invading cells) with 4% paraformaldehyde for 30min; 10) Stain with 0.1% crystal violet for 10min; 11) Wash with deionized water 2-3 times; 12) Dry at room temperature and take pictures under a microscope, count and analyze.

[0172] 3.1.10 Edu detection of cell proliferation

[0173] 1) The cells to be tested were seeded into a 24-well cell culture plate at a cell density of 50,000 / well; 2) When the cells grew to the logarithmic growth phase, the Edu detection reagent was added according to the operating instructions of the Edu detection kit (#C0071S, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) and incubated in a cell culture environment for 2 hours; 3) The cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes; 4) The cells were washed 3 times with cell washing solution (3% BSA prepared in PBS), each time for 5 minutes; 5) The cells were treated with 1% PBST for 10 minutes; 6) After washing 3 times with cell washing solution, the prepared Edu staining solution was added; 7) The cells were washed 3 to 5 times with cell washing solution, each time for 5 minutes; 8) 1×Hochest was added to treat the cells for nuclear staining; 9) The cells were washed 3 to 5 times with cell washing solution, each time for 5 minutes; 10) Edu fluorescence was photographed using an inverted fluorescence microscope.

[0174] 3.1.11 Tubulogenesis Detection of Vascular Endothelial Cell Vascular Mimicry Formation Ability

[0175] 1) Spread the thawed matrix gel into a 48-well cell culture plate in advance, add 60 μL to each well, and place it in a cell culture incubator for 30 minutes; 2) Digest the cells in the logarithmic growth phase and centrifuge them; 3) Carefully add the centrifuged cells to the cell culture plate containing matrix gel at a cell density of 70,000 / well and culture for 6 hours; 4) Use an inverted microscope to photograph the cell tubule formation; 5) Use Image J biological software to count the tubule formation indicators.

[0176] 3.2 Construction of PDHA1 knockout hCMEC / D3 cells

[0177] In order to study the regulatory effect of PDHA1-Ser293 / 300 phosphorylation modification on the angiogenic phenotype of VECs, the present invention uses gene editing and lentiviral packaging technology to construct VECs cell lines that simulate its hyperphosphorylation and dephosphorylation. First, CRISPR / Cas9 gene knockout technology was used to construct PDHA1 knockout hCMEC / D3 cells. In the present invention, PDHA1-sgRNA targets in three different regions were designed ( Fig.13 A), the synthesized PDHA1-sgRNA was cloned into the lentiCRISPR-v2 gene knockout vector, and the lentiviral packaging technology was used to construct the PDHA1 gene knockout hCMEC / D3 cell line with different sgRNA targeting. The CRISPR / Cas9 gene knockout efficiency was detected by T7 nuclease, and the results showed that the CRISPR / Cas9 targeted by PDHA1-sgRNA#1 had a higher cutting efficiency ( Fig.13 Western blotting results further showed that PDHA1-sgRNA#1 targeting CRISPR / Cas9 significantly reduced the expression level of PDHA1 protein in hCMEC / D3 cells ( Fig.13 C), in subsequent studies, the PDHA1 knockout cells constructed with PDHA1-sgRNA#1 were used as a basis to further construct an hCMEC / D3 cell line that simulates the simultaneous hyperphosphorylation and dephosphorylation of PDHA1-Ser293 / 300.

[0178] Subsequently, hCMEC / D3 cells infected with CRISPR / Cas9-PDHA1-sgRNA#1 were cultured for monoclonal cell lines to screen for cell lines with homozygous knockout of PDHA1. Five PDHA1 homozygous knockout hCMEC / D3 cell lines were obtained by Western blotting identification ( Fig.13D in it). In order to analyze the genomic mutation mode of PDHA1 knockout, the present invention uses Sanger sequencing technology to sequence and analyze the PDHA1 genome in these 5 PDHA1 homozygous knockout hCMEC / D3 cells. The upstream and downstream DNA fragments of the PDHA1-sgRNA#1 region in the cells were amplified using fragment-specific primers, and the TA cloning molecular technology was used to insert it into the multiple cloning site in the pMD19-T vector, and the single clone was picked for Sanger sequencing analysis. The results showed that 3 PDHA1 homozygous knockout cell lines showed the insertion of 1 base at a specific site in the PDHA1 genome, which caused the premature termination of its mRNA translation and prevented protein synthesis; 1 cell line showed the deletion of 14 bases at a specific site in the PDHA1 genome, which caused the premature termination of its mRNA translation and prevented protein synthesis; 1 cell line showed the deletion of 9 bases at a specific site in the PDHA1 genome, which did not cause the premature termination of its mRNA translation. However, Western blotting detection found that the PDHA1 protein expression in the cell line was missing ( Fig.13 E).

[0179] PDHA1 knockout inhibits angiogenesis in hCMEC / D3 cells

[0180] In order to study the role of PDHA1 in VECs angiogenesis, the present invention first analyzed the angiogenesis-related phenotypes of PDHA1 homozygous knockout hCMEC / D3 cells, including cell proliferation, cell migration, and cell tubule formation ability.

[0181] The Edu experiment was used to detect cell proliferation. The results showed that the cell proliferation rate of the control group (sgCON) hCMEC / D3 cells was about 55% 48 hours after implantation, while the proliferation rate of hCMEC / D3 cells with homozygous knockout of PDHA1 was about 40-45%. The statistical results showed that homozygous knockout of PDHA1 led to a statistically significant decrease in the proliferation ability of hCMEC / D3 cells ( Fig.14 A and B in ).

[0182] CCK8 assay was used to further detect cell proliferation. The results showed that compared with the control hCMEC / D3 cells, the cell viability of PDHA1 homozygous knockout cells was significantly reduced at DAY2 and lasted until DAY4. The statistical results showed that PDHA1 homozygous knockout led to a statistically significant decrease in the growth rate of hCMEC / D3 cells ( Fig.14 C).

[0183] Transwell assay was used to detect cell migration. The results showed that the 48h cell migration rate of hCMEC / D3 cells in the control group (sgCON) was about 40%, while the migration rate of hCMEC / D3 cells with homozygous knockout of PDHA1 was about 10%. The statistical results showed that homozygous knockout of PDHA1 led to a statistically significant decrease in the migration ability of hCMEC / D3 cells ( Fig.14 D and E in the figure). Tubulogenesis can simulate the ability of angiogenesis in vivo. Therefore, the present invention uses the experimental technique of tubulogenesis to detect the effect of homozygous knockout of PDHA1 on the angiogenesis ability of hCMEC / D3 cells. The results showed that the control group hCMEC / D3 formed a complete tubular morphology 5 hours after implantation, and the cell confluence was 700 and the branch length was nearly 80,000, while the confluence of PDHA1 homozygous knockout cells was 300-600 and the branch length was nearly 40,000-50,000. The statistical analysis results showed that homozygous knockout of PDHA1 led to a statistically significant decrease in the ability of hCMEC / D3 cells to form mimicry ( Fig.14 F~H in the figure). The results showed that the proliferation, cell migration, and cell tubulogenesis of hCMEC / D3 cells with homozygous knockout of PDHA1 were inhibited, suggesting that PDHA1 protein plays an important role in the regulation of angiogenic behavior of hCMEC / D3 cells.

[0184] 3.4 Simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits hCMEC / D cell angiogenesis

[0185] To further analyze the effect of PDHA1 phosphorylation modification on hCMEC / D3 cell angiogenesis, the present invention designed and synthesized a protein that simulates the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 (mutating Ser293 and Ser300 sites to Glu293 and Glu300, respectively, named PDHA1-S293E / S300E, referred to as EE) and the dephosphorylation of PDHA1 (mutating Ser293 and Ser300 to Glu293 and Glu300, respectively, named PDHA1-S293E / S300E, referred to as EE) based on hCMEC / D3 cells with homozygous knockout of PDHA1. The sites were mutated to Ala293 and Ala300, respectively, named PDHA1-S293A / S300A, referred to as AA) with site-directed mutagenesis primers (the single site mutations were named S293A, S293E, S300A, S300E), and the lentiviral packaging plasmids for overexpression of S293A, S293E, S300A, S300E, PDHA1-S293E / S300E, and PDHA1-S293A / S300A were constructed according to the instructions of the point mutagenesis reagent. Fig.15A in Figure 2 was constructed, and a site-directed mutant cell line was constructed using lentiviral packaging technology. The hCMEC / D3 cell line that mimics the simultaneous hyperphosphorylation and dephosphorylation of PDHA1-Ser293 / 300 was obtained by Western blotting. Fig.15 B).

[0186] Depend on Fig.15 As shown in C~H, by detecting the phenotypes of cells with different genotypes of PDHA1, it was found that homozygous knockout of PDHA1 significantly inhibited the proliferation, migration, and tubule formation ability of hCMEC / D3 cells. Ectopic overexpression of PDHA1 (WT) in PDHA1 homozygous knockout cells restored the angiogenesis phenotype of cells inhibited by homozygous knockout of PDHA1. In PDHA1 homozygous knockout cells, ectopic overexpression of PDHA1 protein (EE) that simulated simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 was performed, and the inhibited angiogenesis phenotype of cells could not be restored. Ectopic overexpression of PDHA1 protein (AA) that simulated simultaneous dephosphorylation of PDHA1-Ser293 / 300 in PDHA1 homozygous knockout cells restored the angiogenesis phenotype of cells inhibited by homozygous knockout of PDHA1. It shows that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 sites inhibits the angiogenesis phenotype of hCMEC / D3 cells.

[0187] 3.5 Effects of single site mutations of PDHA1-Ser293 and -Ser300 on angiogenesis of hCMEC / D cells

[0188] To further analyze the effect of PDHA1-Ser293 / 300 single site mutation on hCMEC / D3 cell angiogenesis phenotype, the present invention analyzed the hCMEC / D3 cell angiogenesis phenotype of constructed S293A, S293E, S300A, S300E genotypes. CCK8 analysis results showed that the growth of cells with hyperphosphorylation or dephosphorylation of PDHA1-Ser293 / 300 single site was consistent with WT, and did not affect the growth of hCMEC / D3 cells ( Fig.16 A in Figure 2). Edu detection results showed that the proliferation of cells with hyperphosphorylation or dephosphorylation of a single site of PDHA1-Ser293 / 300 was consistent with that of WT, and did not affect the proliferation of hCMEC / D3 cells ( Fig.16 B and C in Figure 2). Transwell analysis results showed that the migration ability of cells with hyperphosphorylation or dephosphorylation of PDHA1-Ser293 / 300 was consistent with that of WT cells, and did not affect the migration of hCMEC / D3 cells ( Fig.16D and E in Figure 2). The results of the study on tubule formation showed that the ability of cells to form vascular mimicry when the single site of PDHA1-Ser293 / 300 was hyperphosphorylated or dephosphorylated was consistent with that of WT cells, and did not affect the angiogenesis of hCMEC / D3 cells ( Fig.16 F~H in it).

[0189] Effect of PDK2 overexpression on angiogenesis in hCMEC / D3 cells

[0190] According to the above-mentioned phosphorylation proteome results and the results of in vivo studies in mice, PDK2 regulates the phosphorylation modification of PDHA1-Ser293 / 300 and participates in the regulation of VECs angiogenesis. To further verify this conclusion, the present invention uses lentiviral packaging technology to construct PDK2 overexpression and shRNA knockdown cell lines respectively. The lentiviral packaging plasmid for ectopic overexpression of PDK2 was constructed by molecular cloning technology, and the lentiviral packaging technology was used to obtain PDK2 overexpression lentiviral particles by lentiviral packaging. Subsequently, hCMEC / D3 cells with ectopic overexpression of PDK2 were constructed by viral infection, and Western blotting identification showed that the cell line was successfully constructed ( Fig.17 A in the figure). The construction of the PDK2 knockdown cell line is similar to that of the present invention. The present invention constructs a PDK2-specific shRNA expression plasmid by molecular cloning technology, obtains PDK2 knockdown lentiviral particles after lentiviral packaging by lentiviral packaging technology, and then constructs PDK2 knockdown hCEMCD / 3 cells by viral infection, and performs Western blotting identification to prove that the cell line is successfully constructed ( Fig.17 B).

[0191] Western blotting analysis showed that the phosphorylation levels of PDHA1-Ser293 and -Ser300 in PDK2 overexpressing cells were significantly increased, while those in PDK2 knockdown cells were significantly decreased ( Fig.17 A and B in Figure 2 indicate that PDHA1-Ser293 and -Ser300 are PDK2 substrates, and their phosphorylation modification is regulated by changes in PDK2 expression. Edu was used to detect cell proliferation, and the results showed that PDK2 overexpression significantly inhibited the proliferation of hCMEC / D3 cells ( Fig.17 C and D). Transwell results showed that PDK2 overexpression significantly inhibited the migration of hCMEC / D3 cells ( Fig.17 E and F in Figure 4). The results of the tubule formation experiment further showed that PDK2 overexpression significantly inhibited the vascular mimicry formation ability of hCMEC / D3 cells ( Fig.17In order to verify that the regulatory effect of PDK2 on cell angiogenesis depends on its phosphorylation modification of PDHA1-Ser293 / 300, the present invention further overexpressed PDK2 in cells where PDHA1-Ser293 / 300 was simultaneously overphosphorylated ( Fig.17 A in Figure 2). Analysis of angiogenic phenotypes showed that overexpression of PDK2 in hCMEC / D3 cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 did not further inhibit the angiogenic phenotype ( Fig.17 I~O in the figure), indicating that the inhibitory effect of PDK2 overexpression on the angiogenic phenotype depends on its modification of PDHA1-Ser293 / 300 phosphorylation.

[0192] Further analysis of the angiogenesis phenotype of PDK2 gene knockdown cells revealed that low expression of PDK2 had no effect on cell angiogenesis ( Fig.17 The results showed that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibited the angiogenic behavior of hCMEC / D3 cells, while the simultaneous dephosphorylation or single site changes had no effect on the angiogenic behavior of cells.

[0193] Example 4 Effects of simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 on mitochondrial OXPHOS and glycolysis in hCMEC / D3 cells

[0194] 4.1 Protein co-immunoprecipitation

[0195] 1) Collect hCMEC / D3 cells and centrifuge at 1000rpm for 10min; 2) After washing with PBS 1-2 times, add 1mL of Co-IP protein lysis buffer to lyse cells on ice for 30min, and vortex for 1min every 10min; 3) Ultrasonic disruption for 10min; 4) Centrifuge at 12000rpm for 30min at 4℃; 5) Take the supernatant and put it in a new EP tube that has been placed on ice in advance; 6) Take 1% protein lysis buffer in a new EP tube as input; The remaining protein lysis buffer is evenly divided into two parts, one for the target protein group, which is used for target protein antibody incubation, and the other for IgG group, which is used for subsequent IgG incubation; 7) Add the target protein antibody and IgG that have been bound to Protein A in advance to the corresponding protein lysis buffer, 2μL per tube, and incubate at 4℃ overnight; 8) Centrifuge at 12000rpm at 4℃ for 5min; 9) Wash Protein A with protein lysis buffer at 4℃ for 5min, 12000rpm Centrifuge at 4°C for 5 min (repeat the operation 5 times); 10) add an equal volume of protein loading buffer to the precipitate containing Protein A, boil in boiling water for 10 min, centrifuge at 4°C, 12000 rpm, and take the supernatant for Western blotting analysis.

[0196] 4.2 Isolation of cell mitochondria

[0197] 1) According to the instructions of the mitochondrial extraction kit (#SM0020, purchased from Beijing Solebow Technology Co., Ltd.), 5×10 7 1) Add 1 mL of cell lysis buffer to each cell; 2) Grind and break the cells; 3) Centrifuge at 4°C, 1000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL EP tube; 4) Centrifuge at 4°C, 1000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL EP tube; 5) Centrifuge at 4°C, 13000 rpm for 10 min to obtain the crude mitochondrial precipitate, and transfer the supernatant to a new 1.5 mL EP tube (cytoplasmic component); 6) Wash the crude mitochondria with washing buffer and centrifuge at 4°C, 1000 rpm for 10 min; 7) Transfer the supernatant to a new 1.5 mL EP tube, and centrifuge at 4°C, 13000 rpm for 10 min to obtain the mitochondrial precipitate.

[0198] 4.3 Mitochondrial membrane potential detection

[0199] 1) The cells to be tested were seeded in a 24-well cell culture dish at a cell density of 50,000 / well; 2) When the cells grew to the logarithmic phase, the probe was added according to the operating instructions of the mitochondrial membrane potential detection kit (#C2880S, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.), and incubated in a cell culture incubator at 37°C for 1 hour; 3) The cells were washed three times with cell culture medium; 4) The mitochondrial membrane potential was photographed with an inverted fluorescence microscope; 5) The fluorescence intensity was analyzed using Image J biological software.

[0200] 4.4 ROS content detection

[0201] 1) The cells to be tested were seeded in a 24-well cell culture dish at a cell density of 50,000 / well; 2) When the cells grew to the logarithmic phase, the probe was added according to the operating instructions of the ROS detection kit (#S0035S, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) and incubated in a cell culture incubator at 37°C for 30 minutes; 3) The cells were washed three times with PBS; 4) ROS in the cells were photographed with an inverted fluorescence microscope; 5) The fluorescence intensity was analyzed using Image J biological software.

[0202] 4.5 Pyruvate dehydrogenase complex activity detection

[0203] 1) Digest the cells in the logarithmic growth phase and centrifuge them, then wash with PBS to obtain the cell pellet; 2) 6 100 μL of cell lysis buffer was added to each cell, and the cells were disrupted by ultrasound (5s / 5s, 30 times, 200V); 3) the cell supernatant was obtained after centrifugation at 4°C, 10000 rpm for 10 min; 4) the activity of pyruvate dehydrogenase complex in the sample was detected according to the operating instructions of the kit.

[0204] 4.6 Lactic acid content detection

[0205] 1) Digest the cells in the logarithmic growth phase and centrifuge them, then wash with PBS to obtain the cell pellet; 2) 6 100 μL of cell lysis buffer was added to each cell and the cells were disrupted by ultrasound (5s / 5s, 30 times, 200V); 3) the cell supernatant was obtained after centrifugation at 4°C, 10000 rpm for 10 min; 4) the lactic acid content in the sample was detected according to the operating instructions of the lactic acid content (LA) detection kit (#BC2235, purchased from Beijing Solebow Technology Co., Ltd.).

[0206] In order to study whether the inhibitory effect of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation on angiogenesis is related to the conversion of mitochondrial energy metabolism, the present invention first analyzed the effect of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation on PDHc activity. The results showed that PDHA1-Ser293 / 300 simultaneous hyperphosphorylation significantly inhibited PDHc activity in hCMEC / D3 cells ( Fig.18 A in Figure 1). Co-IP was used to investigate the binding interaction between PDHA1 and another important protein component of E1, pyruvate dehydrogenase E1 subunitbeta (PDHB), in cells where PDHA1-Ser293 / 300 was simultaneously overphosphorylated. It was found that simultaneous overphosphorylation of PDHA1-Ser293 / 300 affected its binding to PDHB ( Fig.18 B), suggesting that this may be an important reason why PDHA1 hyperphosphorylation inhibits PDHc activity.

[0207] The present invention then analyzed the oxidation rate (oxygen consumption rate, OCR) in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 using a high-resolution tissue energy metabolism analysis system (O2K). The results showed that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 led to a decrease in cell OCR ( Fig.18 C in Figure 3). The analysis results further showed that PDHA1-Ser293 / 300 simultaneously over-phosphorylates the basic respiration of cells ( Fig.18 D in), maximum breathing ( Fig.18 E in), respiratory reserve capacity ( Fig.18 F), and oxidative phosphorylation of complex II ( Fig.18 The results of the present invention showed that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibited mitochondrial respiration and OXPHOS in cells, which is consistent with the relevant literature reports. However, the results of the present invention showed that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibited cellular glycolysis and lactate formation ( Fig.18 The reason for this is that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits the conversion of pyruvate to Acetyl-CoA, which may lead to excessive accumulation of pyruvate and inhibit cell glycolysis through feedback regulation.

[0208] The changes in OXPHOS and respiratory chain byproduct ROS in cells were further detected using ROS-specific probes. The results showed that ROS accumulation in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 was significantly reduced ( Fig.18N and O in the mitochondrial membrane potential). The results of mitochondrial membrane potential detection further showed that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 led to a decrease in the mitochondrial membrane potential of cells ( Fig.18 P and Q in ).

[0209] Example 5: Simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 by blocking Ca 2+ cAMP-dependent synthesis inhibits cAMP / PKA / CREB signaling pathway in the cytoplasm

[0210] 5.1 Detection Method

[0211] 5.1.1 Non-targeted metabolome analysis

[0212] 1) Digest the cells in the logarithmic growth phase and centrifuge at 1000 rpm for 10 min; 2) Wash with PBS and centrifuge to obtain the cell pellet, immediately freeze it with liquid nitrogen and store it in a -80°C refrigerator; 3) Use SHIMADZUNexera UHPLC and TripleTOF 6600 mass spectrometer (SCIEX) with electrospray ionization source interface to detect the metabolites in the cells; 4) Output the obtained mass spectrometry data to Progenesis QI software was used for further processing, and metabolic features were annotated using an internal library. In the metabolomics study based on mass spectrometry technology, in order to obtain reliable and high-quality metabolomics data, QC samples were used for quality control during the detection process; 5) Combined with the VIP value OPLS-DA of multivariate statistical analysis and the P value t test of univariate statistical analysis, significant differences in metabolites between groups were screened (screening threshold: VIP ≥ 1, P-value < 0.05); 6) The differences in metabolites between groups were normalized (z-score), cluster analysis was performed, and R-pheatmap was used to draw heat maps; 7) The correlation of differential metabolites was calculated using cor() in R language, and the cor.test() function in R language was used for statistical analysis; 8) According to the qualitative results of the metabolites, the corresponding compound ID (C_id) was found from the KEGG database. After multiple tests and corrections, the pathways with Q-value ≤ 0.05 were defined as their significantly enriched signal pathways.

[0213] 5.1.2 Calcium ion content detection

[0214] 1) The cells to be tested were seeded in a 24-well cell culture dish at a cell density of 50,000 / well; 2) When the cells grew to the logarithmic phase, the probes of calcium ion probe Fluo4-AM (S1-61S, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) and Rhod2-AM (#HY-D0989, purchased from MedChemExpress) were added according to the operating instructions, and the cells were incubated at 37°C in a cell culture incubator for 10 minutes; 3) The cells were washed three times with PBS; 4) Cell culture medium was added and cultured at 37°C in a cell culture incubator for 30 minutes; 4) Fluo4-AM and Rho2-AM in the cells were photographed with an inverted fluorescence microscope; 5) The fluorescence intensity was analyzed using Image J biological software.

[0215] 5.1.3 cAMP content detection

[0216] 1) Digest the cells in the logarithmic growth phase for 1 min, centrifuge at 1000 rpm for 10 min, and wash with PBS to obtain the cell pellet; 2) 6 100 μL of pre-cooled PBS was added to each cell and the cells were disrupted by ultrasound (5s / 5s, 30 times, 200V); 3) the cell supernatant was obtained after centrifugation at 4°C, 10000 rpm for 10 min; 4) the cAMP content in the sample was detected according to the operating instructions of the cAMP ELISA kit (E-EL-0056, purchased from Elabscience).

[0217] 5.2 Non-targeted metabolomic analysis of changes in metabolites in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300

[0218] In order to study the pathway by which PDHA1-Ser293 / 300 phosphorylation regulates hCMEC / D3 cell angiogenesis by affecting mitochondrial energy metabolism, the present invention uses non-targeted metabolome analysis to detect changes in cell metabolites in wild-type (WT), PDHA1-Ser293 / 300 hyperphosphorylation (EE) and dephosphorylation (AA) groups. Based on the detection threshold (Fold change>3 and P-value<0.05), 16 metabolites were identified to be reduced in EE cells, 5 metabolites were found to be reduced in both EE and AA groups, 5 metabolites were found to be increased in EE cells, and 1 metabolite was found to be reduced in AA cells ( Fig.19 and Fig. 20 ).

[0219] Subsequently, the present invention conducted KEGG signaling pathway enrichment analysis on these 27 metabolites. The results showed that these metabolites were mainly enriched in 35 signaling pathways, including cAMP signaling pathway, chemokine signaling pathway, Rap1 signaling pathway, etc. ( Fig.21 ). Analysis of the differential metabolites in these signaling pathways revealed that the intracellular second messenger cAMP plays a core regulatory role in these signaling pathways. Further analysis of the changes in its content revealed that the cAMP content in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 was significantly reduced ( Fig.19 The above research results indicate that cAMP may be the main cellular metabolite affected by simultaneous hyperphosphorylation of PDHA1-Ser293 / 300.

[0220] 5.3 Simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 participates in the regulation of hCMEC / D3 cell angiogenesis by inhibiting the cAMP / PKA / CREB signaling pathway

[0221] Based on the non-target metabolome analysis results in 5.2, the present invention further verified the changes in cAMP content in cells using ELISA, and the results were consistent with the metabolome analysis results, that is, cAMP in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 was significantly reduced ( Fig. 22 A in the figure). cAMP is a classic intracellular second messenger. cAMP in the cytoplasm is synthesized by adenylate cyclase (sAC) using ATP as a substrate. Its downstream classic effector protein is PKA. After being bound by cAMP, the protein releases its catalytic subunit (such as PKACA, PKACB) and then phosphorylates its substrate protein. CREB is a classic substrate protein of PKA. The activated protein acts as a transcription factor to initiate the expression of target genes in the cell nucleus and promote changes in cell phenotype ( Fig. 22 B in the figure). Western blotting was then used to detect the activation of the cAMP downstream effector protein PKA. The results showed that the content of PKA catalytic subunits PKACA and PKACB in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 was significantly reduced, and the phosphorylation level of its substrate protein CREB at serine 133 (CREB-Ser133) was also significantly reduced ( Fig. 22 C). This indicates that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits the downstream PKA / CREB signaling pathway by reducing the cellular cAMP content, which may be an important molecular pathway for inhibiting cellular angiogenesis.

[0222] In order to verify the biological effect of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation on inhibiting the angiogenic phenotype of cells by inhibiting the cAMP / PKA / CREB signaling pathway, the present invention uses the PKA activator 8-Bromo-cAMP (cell permeable cAMP analog) to analyze the functional recovery effect of activation of this pathway on the angiogenic phenotype of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation cells. The results showed that 8-Bromo-cAMP treatment restored the suppressed angiogenic phenotype in PDHA1-Ser293 / 300 simultaneous hyperphosphorylation cells ( Fig. 22 D to F in Figure 1), indicating that the cAMP / PKA signaling pathway is the main pathway for PDHA1-Ser293 / 300 phosphorylation to inhibit cell angiogenesis. Site-specific shRNA was further used to construct a CREB gene knockdown PDHA1-Ser293 / 300 hyperphosphorylated cell line ( Fig. 22 G), found that CREB protein deficiency hindered the function of 8-Bromo-cAMP to restore the angiogenic phenotype ( Fig. 22 H~J in the figure indicate that the cAMP / PKA signaling pathway regulates the angiogenic phenotype of cells by regulating the phosphorylation of CREB protein. These results indicate that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 reduces the intracellular cAMP content and inhibits the cAMP / PKA / CREB signaling pathway, which is its main way to inhibit cell angiogenesis.

[0223] 5.4 The main reason for the inhibition of cAMP / PKA / CREB signaling pathway is the cytoplasmic Ca 2+ reduce

[0224] cAMP exists in multiple subcellular structures in cells, including cell membranes, cytoplasm, mitochondria, etc. According to the above analysis results, the cAMP content in cells where PDHA1-Ser293 / 300 are simultaneously overphosphorylated is reduced. However, it is not clear in the prior art which subcellular structures of the cell cAMP is affected. Since the simultaneous overphosphorylation of PDHA1-Ser293 / 300 leads to mitochondrial energy metabolism disorders, it is speculated that cAMP in mitochondria may be affected by the simultaneous overphosphorylation of PDHA1-Ser293 / 300. To verify this speculation, the present invention first separated the mitochondria in the cells, and detected the changes in cAMP in the mitochondria and cytoplasm, respectively. The ELISA analysis results showed that the cAMP content in the cytoplasm of cells where PDHA1-Ser293 / 300 was simultaneously overphosphorylated was significantly reduced, while the cAMP content in the mitochondria was significantly increased ( Fig.23A in the figure). The expression levels of its effector proteins were further analyzed by Western blotting. The results showed that PDHA1-Ser293 / 300 simultaneously overphosphorylated the effector protein PKA in the cytoplasm of cAMP and inhibited it, while the PKA protein in the mitochondria was activated ( Fig.23 These results indicate that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 affects cAMP synthesis in hCMEC / D3 cytoplasm and inhibits its downstream PKA / CREB signaling pathway, while its hyperphosphorylation promotes cAMP synthesis in hCMEC / D3 cell mitochondria and activates its downstream PKA protein.

[0225] Since the cAMP synthesis pathways in both mitochondria and cytosol are regulated by bicarbonate and Ca 2+ The simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 blocks the conversion of pyruvate to Acetyl-CoA, thereby inhibiting the TCA cycle and oxidative phosphorylation. It is speculated that the generation of bicarbonate may be inhibited and cAMP synthesis may be blocked. To verify this speculation, bicarbonate treatment was used to detect the recovery of the cAMP / PKA / CREB signaling pathway in hCMEC / D3 cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300. The results showed that bicarbonate treatment did not increase the cAMP content and the content of the catalytic subunit of its effector protein in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 ( Fig.23 C and D), indicating that bicarbonate may not be the main factor inhibiting cAMP synthesis in this cell.

[0226] Ca 2+ is another important factor in activating sAC activity, and Ca 2+ The content of is regulated by the energy metabolism state. 2+ It may be the main reason for the change of cAMP content in cytoplasm and mitochondria. 2+ probe (Fluo4 / AM) and mitochondrial Ca 2+ The probe (Rhod2 / AM) monitors PDHA1-Ser293 / 300 hyperphosphorylation in the cytosol and mitochondria. 2+ The results showed that PDHA1-Ser293 / 300 simultaneously overphosphorylated Ca in the cytoplasm. 2+ significantly decreased, while mitochondrial Ca 2+ Increased content ( Fig.23 E and F in Figure 1). It can be seen that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 may lead to the increase of Ca in the cytoplasm through the disorder of mitochondrial energy metabolism.2+ To confirm this conclusion, the present invention uses mitochondrial Ca 2+ The hCMEC / D3 cells with hyperphosphorylated PDHA1-Ser293 / 300 were treated with the channel inhibitor MCU-i4, and the Ca 2+ The results showed that MCU-i4 treatment led to the increase of Ca2+ in mitochondria and the recovery of cAMP signaling pathway. 2+ The content of Ca 2+ Increased content ( Fig.23 G and H); further MCU-i4 treatment increased the cAMP content and the activity of its effector proteins in the cytoplasm, while decreasing the cAMP content and the activity of its effector proteins in the mitochondria ( Fig.23 I and J in Figure 1) indicate that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 leads to Ca2+ accumulation in the cytosol through mitochondrial energy metabolism disorder. 2+ The cytoplasmic cAMP / PKA / CREB signaling pathway is inhibited by the influx of MCU-i4 into mitochondria. To further demonstrate the inhibitory effect of this molecular mechanism on the angiogenesis phenotype of cells, the present invention used MCU-i4 to treat hCMEC / D3 cells with hyperphosphorylated PDHA1-Ser293 / 300, and analyzed its function of restoring cell angiogenesis through tubule formation experiments. The results showed that MCU-i4 treatment rescued the suppressed angiogenesis phenotype in cells with hyperphosphorylated PDHA1-Ser293 / 300 and PDK2 overexpression ( Fig.23 K~O in it).

[0227] Combined with the above research results, it is shown that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 blocks OXPHOS and promotes Ca 2+ Influx into mitochondria, leading to cytosolic Ca 2+ Depletion of cAMP leads to damage of cAMP / PKA / CREB pathway by inhibiting the synthesis of cAMP, thereby inhibiting cell angiogenesis.

[0228] Example 6: Simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits cAMP / PKA / CREB-dependent FOS gene expression and inhibits angiogenesis

[0229] 6.1 Dual luciferase reporter gene activity assay

[0230] 1) hCMEC / D3 cells overexpressing CREB and CREB(S133A) were seeded in 12-well cell culture plates respectively; 2) cells were grown to a polymerization degree of 80% for cell transfection, and pGL3 and TK plasmids were transfected into target cells at a ratio of pGL3:TK=10:1; 3) cells were lysed using the cell lysis buffer in the dual luciferase reporter gene detection kit (#RG088S, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) after 12 hours, and the cell suspension was transferred to a 1.5 mL EP tube; 4) Renilla luciferase substrate and firefly luciferase substrate were added in sequence according to the operating instructions of the kit to measure the fluorescence value; 5) the firefly luciferase reporter gene activity was calculated according to the operating instructions of the kit.

[0231] 6.2 Transcriptome high-throughput sequencing analysis of DEGs in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300

[0232] To identify DEGs in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, the present invention performed transcriptome high-throughput sequencing analysis on the cells (EE) and control cells (CON). According to the screening threshold (fold change>2; P-value<0.05), the present invention identified 156 differentially highly expressed genes and 9 differentially underexpressed genes in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 ( Fig.24 ). GO enrichment and KEGG pathway enrichment analysis were performed on these DEGs, and the results showed that these 165 DEGs were mainly involved in regulating cell division and cell cycle, microtubule cytoskeleton organization, inflammatory response, etc. ( Fig.25 and Fig.26 ). The core hub DEGs were further analyzed through the PPI network, and the interaction network was constructed together with the KEGG signaling pathway. The results showed that the core hub DEGs were involved in the signaling pathways of inflammatory response, including chemokine signaling pathway, IL-17 signaling pathway, NF-kB signaling pathway, TFN signaling pathway and cytokine-cytokine receptor interaction ( Fig. 27 ). Fig. 27 Analysis found that the hub molecule AP-1 transcription factor subunit FOS plays an important role in these pathways.

[0233] 6.3 Simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits FOS gene expression and suppresses the angiogenic phenotype of cells

[0234] Through transcriptome high-throughput sequencing analysis, the present invention screened out the core hub DEGs that may affect the phenotype of PDHA1-Ser293 / 300 simultaneously overphosphorylated cells. At the same time, the transcription factor family gene FOS, as a core hub gene, had a significantly reduced gene expression level in PDHA1-Ser293 / 300 simultaneously overphosphorylated cells ( Fig.28 To verify this result, the present invention used qRT-PCR and Western blotting experiments to analyze the FOS gene expression level and protein expression level in hCMEC / D3 cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300. The results showed that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibited the FOS gene expression level ( Fig.28 B) and protein expression levels ( Fig.28 C). Therefore, the reduction of FOS gene expression may be involved in the inhibitory effect of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation on the angiogenic phenotype of hCMEC / D3 cells by inhibiting the expression of its pro-angiogenic target genes.

[0235] In order to prove the regulatory role of FOS in inhibiting hCMEC / D3 cell angiogenesis by simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, the present invention uses molecular cloning and lentiviral packaging technology (see Example 3 for experimental methods) to construct cells with ectopic overexpression of FOS gene and simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 ( Fig.28 D in the figure). By analyzing the angiogenesis phenotype of cells, the functional restoration effect of FOS ectopic overexpression on angiogenesis of cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 was further detected. The Edu detection results showed that FOS ectopic overexpression restored the inhibitory effect of PDHA1-Ser293 / 300 simultaneous hyperphosphorylation on the proliferation of hCMEC / D3 cells ( Fig.28 Transwell analysis results showed that ectopic overexpression of FOS restored the inhibitory effect of PDHA1-Ser293 / 300 hyperphosphorylation on hCMEC / D3 cell migration ( Fig.28 G and H in Figure 3). The results of tubulogenesis analysis showed that ectopic overexpression of FOS restored the inhibitory effect of PDHA1-Ser293 / 300 hyperphosphorylation on the vascular mimicry formation ability of hCMEC / D3 cells ( Fig.28 These results indicate that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits cell angiogenesis by suppressing FOS gene expression.

[0236] 6.4 Joint analysis of non-target metabolism and high-throughput data proves that FOS is a target gene of the cAMP / PKA / CREB signaling pathway

[0237] The present invention further combined the non-target metabolome analysis results with the transcriptome high-throughput sequencing results and found that FOS is a downstream target gene of cAMP / PKA / CREB in the cAMP signaling pathway ( Fig.29 ). The results showed that the decrease in FOS gene expression in hCMEC / D3 cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 may be due to the inhibition of the cytoplasmic cAMP / PKA / CREB signaling pathway.

[0238] 6.5 Analysis of CREB as a putative FOS transcription factor using JASPAR online biological database

[0239] Since CREB is a transcription factor, it is speculated that FOS gene expression may be caused by the inhibition of CREB, which leads to the inhibition of its transcription. To verify this speculation, the present invention first studied the transcription factors that regulate FOS gene expression through the JASPAR biological online database, and found that CREB is indeed a transcription factor of FOS ( Fig.30 ). We further downloaded the FOS promoter sequence from the NCBI database and analyzed the characteristics of the FOS promoter region. We found that the FOS gene promoter region contained two putative CREB binding motifs ( Fig.31 ).

[0240] 6.6 Cell experiments prove that FOS, as a CREB target gene, participates in the regulation of cAMP / PKA / CREB signaling pathway on cell angiogenesis

[0241] qRT-PCR results showed that CREB gene knockdown significantly inhibited FOS gene expression in hCMEC / D3 cells ( Fig.32 Western blotting results further showed that the expression level of FOS protein in hCMEC / D3 cells with CREB gene knockdown was reduced ( Fig.32 B in the figure). We further used a gene overexpression strategy to ectopically overexpress CREB in hCMEC / D3 cells, and constructed hCMEC / D3 cells with a point mutation that simulated the dephosphorylation of CREB serine 133 (CREB-Ser133, PKA substrate) as a control to analyze the regulation of FOS gene expression in hCMEC / D3 cells by CREB overexpression and CREB-Ser133 mutation overexpression. qRT-PCR results showed that ectopic overexpression of CREB significantly promoted FOS gene expression in hCMEC / D3 cells ( Fig.32C), while overexpression of CREB-Ser133 mutant plasmid could not promote the expression level of FOS gene in cells ( Fig.32 C in Figure 3). Western blotting results further showed that the expression level of FOS protein in hCMEC / D3 cells overexpressing CREB gene was increased, while overexpression of CREB-Ser133 mutant plasmid could not increase the expression level of FOS protein in hCMEC / D3 cells ( Fig.32 D in Figure 4). Dual luciferase reporter gene activity assay further confirmed this result ( Fig.32 E) These results indicate that FOS is a target gene of CREB and its gene expression is controlled by the activity status of CREB protein.

[0242] In order to prove that the reduction of FOS gene expression level in PDHA1-Ser293 / 300 simultaneously hyperphosphorylated cells is caused by the inhibition of cAMP / PKA / CREB signaling pathway, the present invention further uses cAMP activating reagents IBMX and 8-Bromo-cAMP to treat PDHA1-Ser293 / 300 simultaneously hyperphosphorylated cells, and detects the changes in FOS protein expression level. The results show that IBMX and 8-Bromo-cAMP treatment restore the suppressed FOS gene expression level in PDHA1-Ser293 / 300 simultaneously hyperphosphorylated cells ( Fig.32 F and G in Figure 3). MCU-i4 was further used to treat hCMEC / D3 cells with hyperphosphorylated PDHA1-Ser293 / 300, and it was found that the suppressed FOS protein expression level in the cells was restored ( Fig.32 In order to verify that the regulatory effect of cAMP / PKA / CREB signaling pathway on cell angiogenesis depends on FOS gene expression, the present invention uses gene site-specific shRNA to construct a cell line in which FOS is knocked down and PDHA1-Ser293 / 300 is hyperphosphorylated (see Example 3 for the method), and the tubule formation experiment is used to detect the recovery effect of 8-Bromo-cAMP treatment on the angiogenesis phenotype of FOS-deficient PDHA1-Ser293 / 300 hyperphosphorylated cells. The results show that FOS gene knockdown hinders the recovery effect of 8-Bromo-cAMP on the angiogenesis of PDHA1 hyperphosphorylated cells ( Fig.32 I~L).

[0243] Example 7 CCN2 as a FOS target gene performs anti-angiogenic behavior of PDHA1-Ser293 / 300 simultaneous phosphorylation

[0244] To further explore the molecular mechanism of FOS regulating angiogenesis, the present invention conducted protein interaction analysis between the differentially underexpressed genes identified in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 and FOS to identify genes that may be regulated by FOS and underexpressed in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300. The analysis results showed that CCN2 and FOS have a close protein interaction relationship ( Fig.33 A in the figure indicates that CCN2 may be a target gene of FOS. Further analysis of CCN2 gene expression in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 and cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 by overexpression of FOS showed that the expression level of CCN2 gene was reduced in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, while FOS overexpression restored the reduced CCN2 gene expression in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 ( Fig.33 B and C in the figure). This result proves that the reduction in CCN2 gene expression level in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 is caused by the loss of FOS protein. To verify that CCN2 is the target gene of FOS in hCMEC / D3 cells, the present invention analyzed the gene expression level and protein expression level of CCN2 in hCMEC / D3 cells with FOS knockdown by qRT-PCR and Western blotting, respectively. The results showed that both the gene and protein expression levels of CCN2 in hCMEC / D3 cells with FOS knockdown were significantly reduced ( Fig.33 D and E in the figure), the results showed that CCN2, as a target gene of FOS, is regulated by FOS protein in its expression level. Therefore, it is speculated that the reduced expression level of CCN2 gene in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 may be due to the inhibition of cAMP / PKA / CREB signaling pathway, which leads to the reduced expression level of FOS gene. To verify this hypothesis, the present invention used 8-Bromo-cAMP and MCU-i4 to treat cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, respectively, and detected the changes in CCN2 protein expression in the cells. The results showed that 8-Bromo-cAMP and MCU-i4 treatment restored the suppressed CCN2 protein expression in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300, however, FOS gene knockdown inhibited the rescue effect of 8-Bromo-cAMP and MCU-i4 treatment on CCN2 expression level ( Fig.33 F and G). The results showed that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 reduced CCN2 gene expression in cells by inhibiting the expression of FOS dependent on the cAMP / PKA / CREB signaling pathway.

[0245] Since CCN2 has been reported to play an important regulatory role in the migration of VECs cells, and VECs cell migration is an important way to promote angiogenesis, it is speculated that the downregulation of CCN2 gene expression levels in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 may inhibit the angiogenesis phenotype of cells by affecting the migration phenotype of hCMEC / D3 cells. The present invention uses lentiviral packaging technology to ectopically overexpress CCN2 in cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 ( Fig.33 H in the figure), and further explored the role of CCN2 in the inhibition of hCMEC / D3 cell angiogenesis by simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 by detecting cell migration and tubule formation ability. Transwell results showed that ectopic overexpression of CCN2 restored the cell migration phenotype inhibited by simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 ( Fig.33 I and J in Figure 2). The results of tubulogenesis showed that ectopic overexpression of CCN2 restored the ability of cells to form vascular mimicry while hyperphosphorylation of PDHA1-Ser293 / 300 inhibited the formation of vascular mimicry ( Fig.33 The results showed that low expression of CCN2 gene was the main reason for the inhibition of angiogenic behavior of hCMEC / D3 cells by simultaneous hyperphosphorylation of PDHA1-Ser293 / 300.

[0246] In order to verify that the main molecular pathway for simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 to inhibit cell angiogenesis is the low expression of CCN2 regulated by FOS, the present invention further performed gene knockdown of CCN2 in cells with ectopic overexpression of FOS and simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 ( Fig.33 N in the figure), and the effect of CCN2 deficiency on the recovery of FOS function was studied by detecting cell migration and tubule formation ability. Transwell analysis results showed that FOS ectopic overexpression restored the cell migration ability inhibited by PDHA1-Ser293 / 300 hyperphosphorylation, while CCN2 deficiency further inhibited the recovery function of FOS on its cell migration ( Fig.33 In addition, the results of the present invention show that knockdown of CCN2 gene inhibits the migration ability of hCMEC / D3 cells, further verifying the important role of this gene in cell migration. The results of tubule formation analysis showed that ectopic overexpression of FOS restored the ability of cells to form vascular mimicry, which was inhibited by hyperphosphorylation of PDHA1-Ser293 / 300, while CCN2 deficiency further inhibited the function of FOS in restoring its vascular mimicry. Fig.33Similar to the cell migration phenotype, CCN2 gene knockdown inhibited the formation of vascular mimicry in hCMEC / D3 cells, further demonstrating the regulatory role of CCN2 in angiogenesis of hCMEC / D3 cells.

[0247] Example 8 The FOS / CCN2 gene expression axis regulated by cAMP / PKA / CREB is an important molecular mechanism for PDK2 overexpression to inhibit cell angiogenesis

[0248] Based on the above analysis results of the present invention, PDK2 overexpression inhibits cell angiogenesis by controlling PDHA1-Ser293 / 300 phosphorylation. Further studies have shown that PDHA1-Ser293 / 300 hyperphosphorylation simultaneously inhibits cytoplasmic Ca 2+ The cAMP / PKA / CREB signaling pathway is dependent on PDK2 to inhibit the FOS / CCN2 gene expression axis, thereby inhibiting the angiogenic behavior of hCMEC / D3 cells. Therefore, it is speculated that PDK2 overexpression may also inhibit the angiogenic behavior of hCMEC / D3 cells through this molecular pathway. To verify this speculation, the present invention first detected the expression changes of cAMP effector proteins and their downstream FOS / CCN2 in hCMEC / D3 cells overexpressing PDK2 by Western blotting experiments. The results are consistent with the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 in cells, and PDK2 overexpression leads to a decrease in the expression level of cAMP effector proteins, its substrate CREB phosphorylation modification level, and the downstream target gene axis FOS / CCN2 protein ( Fig.34 ), indicating that the FOS / CCN2 gene expression axis regulated by cAMP / PKA / CREB in PDK2-overexpressing cells is also in an inhibited state. Further treatment of PDK2-overexpressing hCMEC / D3 cells with 8-Bromo-cAMP and MCU-i4, respectively, revealed that the inhibited cAMP effector protein, the phosphorylation modification level of its substrate CREB, and the protein expression level of the downstream target gene axis FOS / CCN2 were restored ( Fig.34 ).

[0249] To verify that this pathway is the main way for PDK2 overexpression to inhibit cell angiogenesis, the present invention used 8-Bromo-cAMP and MCU-i4 to treat hCMEC / D3 cells overexpressing PDK2, and detected the ability of cell tubule formation. The results showed that both 8-Bromo-cAMP and MCU-i4 treatments restored the angiogenesis behavior of cells inhibited by PDK2 overexpression ( Fig.35), this result indicates that the inhibition of cAMP / PKA / CREB signaling pathway is an important pathway for PDK2 overexpression to inhibit hCMEC / D3 cell angiogenesis. To prove the regulatory role of FOS / CCN2 gene expression axis, the present invention further ectopically overexpressed FOS and CCN2 in PDK2 overexpressing cells (refer to Example 3, Fig.36 ), Transwell and tubule formation assays were used to detect the restorative effects of FOS and CCN2 overexpression on angiogenesis in PDK2-overexpressing cells. Transwell analysis results showed that ectopic overexpression of FOS and CCN2 restored the cell migration ability inhibited by PDK2 overexpression ( Fig.37 The results of tubule formation analysis showed that ectopic overexpression of FOS and CCN2 restored the ability of cells to form vascular mimicry that was inhibited by overexpression of PDK2 ( Fig.38 ). This indicates that PDK2 overexpression inhibits the angiogenic behavior of hCMEC / D3 cells by inhibiting the FOS / CCN2 gene expression axis. The above studies indicate that the FOS / CCN2 axis regulated by cAMP / PKA / CREB is the main molecular mechanism by which PDK2 overexpression inhibits angiogenesis.

[0250] Example 9 Glioma cells reduce phosphorylation modification of PDHA1-Ser293 / 300 in hCMEC / D3 cells

[0251] 9.1 Two-dimensional electrophoresis

[0252] 1) Protein lysis buffer was used to lyse the cell proteins in a 4°C refrigerator for 1 h; 2) 2-DQuant Kit was used to quantify the protein; 3) the protein was added to the protein loading hole of the isoelectric focusing instrument at a content of 750 μg / group, and the dry gel strips (pH 3-11) prepared in advance were gently added thereto, and the gel was sealed with mineral oil and then hydrated and isoelectric focusing were performed respectively; 4) the gel strips were taken out, and the reducing agent and alkylating agent were used to reduce and alkylate them respectively; 5) the gel strips were placed on the upper layer of the SDS-PAGE gel prepared in advance for two-dimensional separation; 6) the protein in the SDS-PAGE gel was transferred to the PVDF membrane, and the gel without transferred protein was stained with Coomassie Brilliant Blue; 7) the PVDF membrane was blocked with 0.3% BSA blocking solution; 8) after washing with PBST, the specific primary antibody was used for incubation at 4°C overnight; 9) after washing with PBST, the secondary antibody labeled with alkaline phosphatase was used for incubation at room temperature for 1 hour; 10) the protein changes were detected with alkaline phosphatase substrate; 11) the scanner scanned the stained protein in the PVDF membrane and the protein in the SDS-PAGE gel respectively.

[0253] 9.2 Collection of glioma cell conditioned medium

[0254] 1) Transferring the culture supernatant of glioma cells in the logarithmic growth phase to a 15 mL centrifuge tube; 2) Centrifuging at 3000 rpm and 4° C. for 10 min; 3) Filtering through a 0.45 μm filter to obtain the cell-conditioned medium.

[0255] Tumor VECs angiogenesis is usually regulated by tumor malignant cells, and the above results of the present invention show that PDHA1-Ser293 / 300 phosphorylation is reduced in glioma tumor angiogenesis. Therefore, the present invention assumes that PDHA1 phosphorylation modification in VECs is controlled by glioma cells. To verify this hypothesis, the present invention first collects and obtains the supernatant of T98G conditioned culture of glioma cells, and detects its effect on the existence form of PDHA1 protein in hCMEC / D3 cells by two-dimensional electrophoresis. The results show that T98G conditioned medium treatment significantly changes the isoelectric point of PDHA1 protein in hCMEC / D3 cells ( Fig.39 A in (marked by a red box), indicating that glioma cells regulate the presence of PDHA1 protein in VECs. In order to prove the regulatory effect of glioma cells on the phosphorylation of PDHA1-Ser293 / 300 in hCMEC / D3 cells, the present invention further collected conditioned medium from different glioma cells (U251, U118, and T98G), and analyzed its effect on the phosphorylation of PDHA1-Ser293 / 300 in hCMEC / D3 cells by Western blotting experimental technology. The results showed that treatment with conditioned medium from glioma cells U251, U118, and T98G reduced the phosphorylation modification levels of PDHA1-Ser293 and -Ser300 sites in hCMEC / D3 cells to varying degrees ( Fig.39 B). Glioma cells were then co-cultured with hCMEC / D3 cells using a Transwell chamber to further analyze the effect of the phosphorylation modification of PDHA1-Ser293 / 300 in hCMEC / D3 cells. Fig.39 As shown in Figure C, glioma cells were added to the upper layer of the Transwell chamber, and hCMEC / D3 cells were added to the lower layer. After 48 hours of co-culture, hCMEC / D3 cells were collected and their total proteins were extracted for Western blotting analysis. The results showed that glioma cells reduced the phosphorylation modification of PDHA1-Ser293 and -Ser300 in hCMEC / D3 cells ( Fig.39 D). The results showed that glioma cells regulated the phosphorylation modification of PDHA1-Ser293 / 300 in hCMEC / D3 cells.

[0256] Example 10 Simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits the angiogenic effect of glioma cells on hCMEC / D3

[0257] The above results show that glioma cell treatment leads to a decrease in the phosphorylation level of PDHA1-Ser293 / 300 in hCMEC / D3 cells, indicating that PDHA1-Ser293 / 300 phosphorylation may be involved in the regulation of glioma cells on VECs-induced angiogenesis. Therefore, the present invention further analyzes the effect of glioma cells on hCMEC / D3 cell angiogenesis. The results of the tubule formation experiment showed that both U118 and T98G cell CM treatment promoted the angiogenic phenotype of hCMEC / D3 cells, while simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 or overexpression of PDK2 significantly inhibited its promoting effect on hCMEC / D3 angiogenesis ( Fig.40 This result indicates that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 regulated by PDK2 in VECs inhibits the pro-angiogenic effect of glioma cells.

[0258] Example 11 Lactic acid promotes VECs angiogenesis

[0259] Lactic acid is a type of metabolite released by malignant tumor cells and is a common angiogenic factor in TME. In order to prove that glioma TME contains high concentrations of lactic acid, the present invention first detects the lactic acid content released in glioma cells. CM from different types of glioma cells were collected, and three normal cell lines (293T, HUVEC, and hCMEC / D3) were selected for CM collection. The CM was set to the content released by the same number of cells by counting the cells. The difference in lactic acid content of the collected CM was detected by a lactic acid detection kit. The results showed that compared with the control cells, the lactic acid content released by glioma cells was about 10 times that released in normal cells ( Fig.41 ). This suggests that glioma TME contains high concentrations of lactate.

[0260] In order to prove that high concentrations of lactic acid in TME promote the angiogenesis of VECs, the present invention uses lactate to simulate high concentrations of lactic acid in TME, treats hCMEC / D3 and HUVEC cells respectively, and detects changes in cell angiogenesis behavior by tubule formation experiments. The results show that different concentrations of lactate (1, 2, 4mM) have inconsistent regulatory effects on the angiogenesis of hCMEC / D3 cells. 1mM lactate treatment affects the angiogenesis phenotype of the cells, while 2mM and 4mM treatments promote the angiogenesis phenotype of the cells. However, only 4mM lactate treatment has a statistically significant promoting effect on the angiogenesis phenotype of the cells ( Fig.42 ). Unlike hCMEC / D3 cells, treatment of HUVEC cells with 2-8 mM lactate significantly increased the length of tubule branches, while the effect on cell aggregation did not show a particularly obvious concentration dependence ( Fig.43 ). These results indicate that lactate, as a pro-angiogenic factor, promotes the angiogenic phenotype of hCMEC / D3 and HUVEC cells in a dose-dependent manner.

[0261] Example 12 Lactic acid regulates the existence form of PDHA1 protein in hCMEC / D3 cells

[0262] Based on the results of the above examples, it is speculated that lactic acid, as a pro-angiogenic factor in TME, will also affect the phosphorylation modification of PDHA1-Ser293 / 300 in glioma neovascularization. To verify this speculation, the present invention first used a two-dimensional electrophoresis experiment to detect the effect of lactic acid treatment on the presence of PDHA1 protein in hCMEC / D3 cells. The results showed that lactic acid treatment changed the presence of PDHA1 protein in hCMEC / D3 cells, and this change in PDHA1 was consistent with the trend of glioma cell treatment ( Fig.44 A in the figure shows that lactic acid and glioma cell treatment consistently change the form of PDHA1 protein in VECs. To further analyze the effect of lactic acid treatment on the phosphorylation modification of PDHA1-Ser293 / 300 in VECs cells, the present invention uses Western blotting to detect the changes in the phosphorylation level of PDHA1-Ser293 / 300 in hCMEC / D3 cells treated with lactic acid. The results are consistent with the treatment of glioma cells. Lactic acid treatment reduces the phosphorylation modification of PDHA1-Ser293 and -Ser300 in hCMEC / D3 cells ( Fig.44 B). The results showed that lactate treatment could regulate the phosphorylation modification of PDHA1-Ser293 / 300 in VECs cells.

[0263] Example 13: Simultaneous phosphorylation of PDHA1-Ser293 / 300 inhibits the angiogenic effect of lactate on hCMEC / D3 cells

[0264] In order to study the regulatory effect of PDHA-Ser293 / 300 phosphorylation on the angiogenic effect of lactate, the present invention detected the angiogenic effect of lactate in hCMEC / D3 cells with simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 by tubulogenesis assay. The results showed that lactate treatment promoted angiogenesis of hCMEC / D3 cells, while simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibited the angiogenic effect of lactate on cells ( Fig.45At the same time, the present invention detected the angiogenic behavior of lactate in hCMEC / D3 cells overexpressing PDK2, and the results were similar to those of the present invention. Lactic acid treatment promoted angiogenesis in hCMEC / D3 cells, while PDK2 overexpression significantly inhibited the angiogenic behavior of lactate on cells ( Fig.45 In contrast, PDHA1-Ser293 / 300 dephosphorylation and PDK2 knockdown still promoted the angiogenic effect of lactate on hCMEC / D3 cells ( Fig.45 G~I in it).

[0265] The present invention uses molecular biology technology to study the biological function and mechanism of action of DPP PDHA1-Ser293 / 300 phosphorylation identified by quantitative phosphorylation proteomics in VECs angiogenesis. First, at the clinical level, the present invention uses mIF technology to prove that the phosphorylation level of PDHA1-Ser293 / 300 is significantly correlated with the vascular density of glioma. Subsequently, it was proved through a small animal intracranial tumor model that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 in VECs inhibits tumor angiogenesis and tumor progression, and prolongs the survival time of mice. Finally, at the cellular level, gene editing strategies and cell phenotypic analysis techniques were used to further prove that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits the angiogenic phenotype of hCMEC / D3 cells. Through mitochondrial energy metabolism stress detection, non-target metabolome analysis and transcriptome high-throughput sequencing analysis, combined with cell function recovery experimental verification, it was finally proved that the simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 leads to Ca in the cytoplasm by inhibiting mitochondrial OXPHOS. 2+ The molecular mechanism by which cAMP is depleted by influx into mitochondria, thereby inhibiting the cytoplasmic cAMP synthesis pathway, inhibiting the FOS / CCN2 gene expression axis by inhibiting the cAMP / PKA / CREB signaling pathway, and ultimately inhibiting the cellular angiogenesis behavior.

[0266] In addition, the present invention further adopts a two-dimensional electrophoresis proteome separation method, and combines Western blotting experimental technology to prove the effect of glioma cells and lactate treatment on the existence form of PDHA1 protein in hCMEC / D3 cells and the regulatory effect of PDHA1-Ser293 / 300 phosphorylation modification, so as to reveal the potential molecular factors of glioma TME on the regulation of PDHA1-Ser293 / 300 phosphorylation modification in new blood vessels. Further cell phenotypic experiments have shown that simultaneous hyperphosphorylation of PDHA1-Ser293 / 300 inhibits the angiogenic effects of glioma cells and lactate on hCMEC / D3 cells, suggesting that the reduction of PDHA1-Ser293 / 300 phosphorylation levels in glioma new blood vessels may be a biological effect in response to the pro-angiogenic TME. These results provide important clues for further research on the response mechanism of PDHA1-Ser293 / 300 phosphorylation to the formation of glioma pro-angiogenic TME.

[0267] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A PDHA1-Ser293 / 300 common phosphorylated protein marker for inhibiting tumor angiogenesis, characterized in that: The sequence of the PDHA1-Ser293 / 300 co-phosphorylated protein marker is shown in SEQ ID NO:

1.

2. The PDHA1-Ser293 / 300 co-phosphorylated protein marker according to claim 1, characterized in that: The PDHA1-Ser293 / 300 co-phosphorylated protein marker is PDHA1-Ser293 / 300 co-hyperphosphorylation, and the PDHA1-Ser293 / 300 co-hyperphosphorylation is obtained by mutating the serine at the 293rd and 300th positions on the PDHA1 protein to glutamic acid.

3. Use of the PDHA1-Ser293 / 300 co-phosphorylated protein marker according to claim 1 as a molecular target in the preparation or screening of drugs for inhibiting tumor angiogenesis.

4. The use according to claim 3, characterized in that: The PDHA1-Ser293 / 300 co-phosphorylated protein marker is a PDHA1-Ser293 / 300 co-overphosphorylated protein, and the PDHA1-Ser293 / 300 co-overphosphorylated protein is obtained by mutating the serine at the 293rd and 300th positions on the PDHA1 protein to glutamic acid.

5. The use according to claim 3, characterized in that: The drug for inhibiting tumor angiogenesis is a drug for inhibiting glioma angiogenesis.

6. Use of the reagent causing the co-phosphorylation of PDHA1-Ser293 / 300 according to claim 2 in the preparation of a drug for inhibiting tumor angiogenesis.

7. The use according to claim 6, characterized in that: The reagent causing the co-phosphorylation of PDHA1-Ser293 / 300 includes one or more of a nucleotide sequence capable of causing PDHA1 protein mutation, a plasmid containing the nucleotide sequence, and a recombinant cell containing the nucleotide sequence.

8. The use according to claim 6, characterized in that: The drug for inhibiting tumor angiogenesis is a drug for inhibiting glioma angiogenesis.

Images