Application of NUP35 gene as glioblastoma diagnosis, prognosis and treatment target

Abstract

The invention discloses application of an NUP35 gene as a target spot for diagnosis, prognosis and treatment of glioblastoma, and discovers that the NUP35 gene plays an important role in proliferation and malignant progression of glioma by regulating fatty acid metabolism of glioma cells. The invention finds that the specific expression of NUP35 in glioma is high, and the expression is increased along with the increase of the pathological level of a patient. The specific knock-down NUP35 can effectively inhibit the growth of glioma cells and delay the lifetime of tumor-bearing mice at the same time. The NUP35 accelerates fatty acid metabolism in glioma cells by promoting expression of fatty acid metabolism related genes and promotes glioma growth. The invention finds that the NUP35 expression level can be used as a glioma prognostic marker, and the NUP35 can be used as an effective target spot for glioma treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of the NUP35 gene as a diagnostic, prognostic and therapeutic target for glioblastoma. Background Art

[0002] Glioblastoma multiforme (GBM) is the most common and highly malignant primary tumor in the central nervous system. It belongs to a type of astrocytoma and is graded as WHO grade IV. Its characteristics are highly invasive, unclear boundaries and multifocal growth. It occurs frequently in the deep white matter areas of the frontal and temporal lobes, and can also involve the basal ganglia and posterior cranial fossa (such as the cerebellar hemisphere and brainstem). The clinical manifestations include increased intracranial pressure symptoms (headache, projectile vomiting, visual decline), neurological dysfunction (hemiplegia, aphasia, sensory impairment) and epileptic seizures. The median survival of patients is only 12 - 16 months, and the five-year survival rate is less than 7%, with extremely poor prognosis.

[0003] Therefore, in-depth study of the molecular mechanism of the occurrence and development of GBM, excavation of new therapeutic targets, and development of molecular targeted therapy are major topics in this field at present and have important clinical significance. Summary of the Invention

[0004] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides the application of the NUP35 gene as a diagnostic, prognostic and therapeutic target for glioblastoma, which includes that the NUP35 gene is used as a glioblastoma-specific molecular marker.

[0006] Specifically, glioma is diagnosed by detecting the mRNA expression level of the NUP35 gene.

[0007] Specifically, the detection primers for the NUP35 gene are: forward primer: 5′-GGCCCGCGTTAGGATCTG-3′; reverse primer: 5′-ATCGAGGTTGTGGAGTCACC-3′.

[0008] Specifically, the NUP35 gene as a therapeutic target for glioblastoma includes inhibiting the growth of glioma cells by inhibiting the mRNA and protein expression levels of the NUP35 gene.

[0009] Specifically, the inhibition of the mRNA and protein expression levels of the NUP35 gene includes using nucleic acid molecules, small molecule chemical drugs, polypeptides, proteins or interfering lentiviruses to inhibit the mRNA and protein expression levels of the NUP35 gene.

[0010] Specifically, the nucleic acid molecule includes an antisense oligonucleotide, dsRNA, siRNA or shRNA.

[0011] Specifically, the sequence of shRNA is one or more of SEQ ID NO: 1 to 3.

[0012] As a preferred embodiment of the application of the NUP35 gene described in the present invention: The NUP35 gene regulates the expression of key genes in the process of fatty acid synthesis and decomposition, and regulates the fatty acid metabolism of glioma cells.

[0013] Beneficial effects of the present invention: The NUP gene family contains more than 100 genes. The present invention discovers that the NUP35 gene plays an important role in the proliferation and malignant progression of glioma by regulating the fatty acid metabolism of glioma cells. The present invention discovers that NUP35 is highly specifically expressed in glioma and increases with the increase of the patient's pathological grade. Functional experiments further confirm that overexpression of NUP35 promotes the proliferation of glioma cells. Specific knockdown of NUP35 can effectively inhibit the growth of glioma cells and at the same time delay the survival period of tumor-bearing mice. Further through transcriptome sequencing analysis and functional experiments, it is found that NUP35 accelerates the fatty acid metabolism in glioma cells by promoting the expression of genes related to fatty acid metabolism, and promotes the growth of glioma. The present invention discovers that the expression level of NUP35 can be used as a prognostic marker for glioma, and NUP35 can be used as an effective target for the treatment of glioma. Brief Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Among them:

[0015] Figure 1 NUP35 is highly expressed in glioma and is associated with poor prognosis of patients.

[0016] Figure 2 NUP35 promotes the growth of glioma cells.

[0017] Figure 3 NUP35 does not regulate the proliferation of glioma cells by affecting the formation of NPC and the integrity of the nuclear membrane.

[0018] Figure 4 NUP35 promotes fatty acid metabolism in glioma cells. Detailed Embodiments

[0019] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the following will describe the specific embodiments of the present invention in detail with reference to specific embodiments.

[0020] 1. Glioblastoma tissue acquisition and primary GBM cell culture: The glioma tissue was minced and digested with trypsin. After that, DMEM high-glucose medium (Bio-channel, catalog number BC-M-005) supplemented with 10% FBS (Gibco) was used for culture, and primary GBM cells were obtained.

[0021] 2. Plasmid construction and lentiviral transduction:

[0022] shRNA cloning: Three non-overlapping shRNA sequences were designed and synthesized based on the human NUP35 gene (Gene ID: 129401). After annealing into double-stranded oligo sequences, they were respectively ligated into the pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPRE (Shanghai Heyuan Biotechnology) (hereinafter referred to as pSLenti) vector linearized by double digestion (digested with Agel / EcoRI). They were respectively transformed into Stbl3 competent cells. After picking single colonies and verifying by sequencing, high-purity recombinant plasmids were extracted using the QIAGEN PlasmidMidiprep Kit, with a concentration ≥ 500 ng / μL. The recombinant pSLenti-shNUP35 plasmids were obtained.

[0023] shNUP35#1: 5′-TAGTGATTATCAGGTTATTTC-3′ (SEQ ID NO: 1)

[0024] shNUP35#2: 5′-GCACAGTATGGGAATATCT-3′ (SEQ ID NO: 2)

[0025] shNUP35#3: 5′-GCAGTGACAGATGTGCTTT-3′ (SEQ ID NO: 3)

[0026] OE-NUP35 cloning: The full CDS sequence of NUP35 was synthesized based on human NUP35 (GenBank ID: NM_138285.5) and an EcoRl restriction site was added. After annealing into double-stranded DNA, it was ligated into the GL130pSLenti-CMV-EGFP-3xFLAG-PGK-Puro-WPRE (hereinafter referred to as GL130) vector linearized by single digestion with EcoRl. It was transformed into Stbl3 competent cells. After picking single colonies and verifying by sequencing, high-purity recombinant plasmids were extracted using the QIAGEN PlasmidMidiprep Kit, with a concentration ≥ 500 ng / μL. The recombinant GL130-NUP35-WT (hereinafter referred to as NUP35) plasmids were obtained.

[0027] Lentivirus packaging and concentration: Mix recombinant pSLenti-shNUP35 / NUP35, psPAX2 (Addgene #12260), and pMD2.G (Addgene #12259) at a ratio of 3:2:1, and transfect them into HEK293T cells (70% confluence) using polyjet. Collect the viral supernatant in two steps at 48 h / 72 h.

[0028] Virus concentration: Filter the supernatant through a 0.45 μm filter membrane, then ultracentrifuge at 70,000×g for 2 h at 4°C, and store at -80°C.

[0029] Titer determination: Determine the number of virus particles (TU / mL) by qPCR, with the target titer ≥ 1×10^8 TU / mL.

[0030] Infect glioma cells: Infect U251 / GBM cells with lentivirus at an MOi of 10, add 4 μg / mL Polybrene, and screen with 5 μg / mL Puromycin for 3 days after 48 h. Detect the infection efficiency by fluorescence microscopy imaging.

[0031] Silencing / overexpression efficiency detection: Western blot: Lyse cells with RIPA, separate proteins by SDS-PAGE, incubate with anti-NUP35 primary antibody overnight at 4°C, and develop with HRP secondary antibody to detect the NUP35 protein level.

[0032] 3. In vitro functional verification

[0033] The EdU, CCK-8, Transwell, and colony formation assays refer to (Xiuping Zhou, et al. Neuro-Onc0l. 2017).

[0034] 4. In vivo experiments

[0035] Nude mouse xenograft tumor model: Orthotopically implant GBM cells infected with shNUP35 (3×10^ 6 cells / mouse) into the right striatum of the nude mouse brain, and inject control group cells in the control group. Observe the tumor growth on the 7th, 14th, and 21st days after tumor implantation by small animal in vivo imaging, and record the survival period of the mice to draw the survival curve.

[0036] 5. Transcriptome sequencing and analysis:

[0037] Collect total RNA from U251 cells with downregulated NUP35 and control group for RNA-seq detection. Screen differentially expressed genes with a cut-off value of |log2FC| ≥ 1.2 and QValue < 0.05, and combine KEGG and GSEA enrichment analysis to screen the main pathways and key genes regulated by NUP35, and further detect them by qPCR.

[0038] 6. Transmission electron microscopy:

[0039] Centrifuge to collect the cells with down-regulated NUP35 and the control group cells. After fixing overnight at 4°C with 2.5% glutaraldehyde solution, rinse, fix with 1% osmium tetroxide solution, rinse, and dehydrate with gradient concentration ethanol; after treatment with pure acetone, embed, make sections, and observe under a transmission electron microscope.

[0040] 7. Preparation of tumor tissue microarray (TMA) and patient follow-up:

[0041] Collect the surgical specimens and clinical information of glioma patients, including imaging data, pathological features, etc. Refer to the published paper in this laboratory (Yan Wang, et al. Neuro-Oncology. 2022) to make TMA. For each sample, select two cores with typical pathological changes and a diameter of 1 mm according to H.E staining, and arrange them regularly in the prepared blank recipient wax block in a set order. Heat in a 52°C constant temperature oven to make the tissue cores tightly combine with the recipient wax block. Trim the tissue array wax block with a fully automatic tissue slicer to make each glioma tissue core fully exposed, section at a thickness of 4 μm, and attach the sections to adhesive glass slides after sectioning. Conduct telephone follow-up every three months after surgery to understand the symptoms and reexamination situation, and master the survival status and postoperative treatment situation of the patients.

[0042] 8. Immunofluorescence staining and imaging:

[0043] Prepare cell slides. After 24 h, fix with 4% paraformaldehyde at room temperature for 30 min, wash twice with PBS, then perforate and permeabilize with 0.5% Triton-X-100 for 10 min, and block with a blocking solution of 3% BSA + 0.05% Triton-X-100 at room temperature for 1 h. Incubate the primary antibody at 4°C overnight, wash 5 times with PBS, bind the secondary antibody at room temperature for 1 h, wash 5 times with PBS, and then mount the slides with an anti-fluorescence quencher containing DAPI. Finally, observe the cells through a laser confocal microscope (Leica Stellaris 5) and take pictures.

[0044] 9. Detection of NUP35 gene expression level: The primers for NUP35 gene detection are as follows: forward primer: 5′-GGCCCGCGTTAGGATCTG-3′; reverse primer: 5′-ATCGAGGTTGTGGAGTCACC-3′.

[0045] Figure 1Analysis revealed that NUP35 was highly expressed in gliomas and was associated with poor prognosis of patients. A. mRNA expression levels of the NUP35 gene in non-tumor brain tissue and glioma tissue samples in the TCGA database. B. mRNA expression levels of the NUP35 gene in glioma tissue samples of different grades in the CGGA database. C. Correlation analysis of the mRNA level of the NUP35 gene and patient survival in the TCGA database. D. Correlation analysis of the mRNA level of the NUP35 gene and patient survival in the CGGA database. E. Western blot was used to detect the NUP35 protein expression levels in non-tumor brain tissue and glioma patient samples. F. TMA-immunohistochemical staining was used to detect the NUP35 protein expression levels in non-tumor brain tissue and glioma tissue samples of different grades. G. Statistical results of the protein expression levels of NUP35 in non-tumor brain tissue and glioma tissue samples (E). Data are expressed as mean ± SD ＊＊ p < 0.01, t-test. H. Quantitative statistical results of NUP35-positive cells in Figure F, comparing the percentages of NUP35-positive cells in non-tumor brain tissue and glioma tissue of different grades. Data are expressed as mean ± SD ＊＊＊ p < 0.001. I. Relationship between NUP35 expression level and overall patient survival was shown. J. ROC curve analysis of NUP35 expression level as a glioma marker. The diagnostic accuracy of NUP35 as a glioma marker was 95%, the diagnostic specificity was 94%, and the sensitivity was 91%.

[0046] Figure 2 Overexpression of NUP35 promoted the malignant progression of gliomas. A. WB was used to detect the protein expression levels of NUP35 in different glioma cell lines. B. WB was used to verify the overexpression results of NUP35 in U87 cells. C. Observation of the overexpression of NUP35 in U87 glioma cells and the subcellular localization of NUP35-GFP under a fluorescence microscope. D. CCK-8 was used to detect the effect of overexpressing NUP35 on the proliferation of U87 cells. Data are expressed as the mean ± SD of three independent experiments, ＊＊＊ p < 0.001, t-test. E. EdU assay was used to detect the effect of overexpressing NUP35 on the proliferation of U87 cells. F. Quantitative statistical results of the number of EdU-positive cells in Figure E are shown, and data are expressed as the mean ± SD of three independent experiments, ＊＊＊ p < 0.001, t-test. G. Transwell assay was used to detect the effect of overexpressing NUP35 on the invasion and migration of U87 cells. H. Scratch assay was used to detect the effect of overexpressing NUP35 on the migration of U87 cells. I. Statistical results of the effect of NUP35 on cell invasion and migration ability in Figure G are shown, and data are expressed as the mean ± SD of three independent experiments, ＊ p < 0.05, ＊＊p < 0.01, t-test. J. shows the quantitative statistical results of NUP35 on cell migration in H. The data are presented as the mean ± SD of three independent experiments. ＊＊ p < 0.01, t-test.

[0047] Figure 3 NUP35 does not regulate glioma cell proliferation by affecting the formation of NPCs and the integrity of the nuclear membrane. A. WB verifies the results of NUP35 downregulation in U251 and GBM cells. B. Colony formation assay detects the effects of NUP35 downregulation on the proliferation of U251 and GBM cells respectively. C. Shows the statistical results of the cell clone sizes in Figure B. The data are presented as the mean ± SD of three independent experiments. ＊＊ p < 0.01, ＊＊＊ p < 0.001, t-test. D. EdU assay detects the effects of NUP35 downregulation on the proliferation of U251 and GBM cells respectively. E. Shows the statistical results of the number of EdU-positive cells in Figure D. The data are presented as the mean ± SD of three independent experiments. ＊＊ p < 0.01, t-test. F. In vivo imaging of small animals shows the sizes of orthotopic xenografts in mice at different times after NUP35 downregulation. G. Statistical quantification of tumor growth by bioluminescence value shows the results. The data are presented as the mean ± SD, *p < 0.05. ＊＊＊ p < 0.001, t-test (n = 9 per group). H. Animal survival curves show the overall survival periods of animals with NUP35 downregulation and the control group. I. Immunofluorescence results of mAb414 / NUP35 show the nuclear membrane morphology after NUP35 downregulation. J. Transmission electron microscopy results show the NPC status after NUP35 downregulation. K. Shows the statistical results of the number of NPCs in Figure J. The data are presented as the mean ± SD of three independent replicates. ns indicates no statistical difference, t-test. L. Shows the statistical results of the diameters of NPCs in Figure J. The data are presented as the mean ± SD of three independent replicates. ns indicates no statistical difference, t-test.

[0048] Figure 4.NUP35 promotes fatty acid metabolism in glioma cells. A. Heatmap showing differentially expressed genes regulated by NUP35, with 709 genes down-regulated and 924 genes up-regulated (DEGs Cut-off: |log2FC|>1.2, FDR<0.05). B. KEGG pathway enrichment analysis showing the top 10 signaling pathways enriched by DEGs regulated by NUP35. C. Heatmap showing fatty acid metabolism-related genes regulated by NUP35 among DEGs. D. GSEA analysis shows that the oxidative phosphorylation pathway is significantly inhibited after NUP35 knockdown. E. RT-qPCR was used to detect the effect of down-regulating NUP35 on the mRNA levels of key enzymes in fatty acid metabolism, FASN and CPT1A. Data are presented as the mean ± SD of three independent replicates, ＊＊ p < 0.01, t-test. F. RT-qPCR was used to detect the effect of overexpressing NUP35 on the mRNA levels of key enzymes in fatty acid metabolism, FASN and CPT1A. Data are presented as the mean ± SD of three independent replicates, ＊＊ p < 0.01, t-test. G. WB was used to detect the effect of down-regulating or overexpressing NUP35 on the protein expression of key enzymes in fatty acid metabolism, FASN and CPT1A, with β-actin as the internal reference protein control. H. Seahorse energy metabolism analysis was used to detect the effect of down-regulating NUP35 on the oxygen consumption rate (OCR) of cells. I. Seahorse energy metabolism analysis was used to detect the effect of overexpressing NUP35 on the oxygen consumption rate (OCR) of cells. J. BODIPY 493 / 503 fluorescence staining showed the changes in lipid droplet content in cells after down-regulating or overexpressing NUP35, respectively (scale bar: 10 μm). K. CCK-8 assay was used to detect the effect of treating cells overexpressing NUP35 with Orlistat (20 μM) on cell proliferation. Data are presented as the mean ± SD of three independent replicates, ＊＊＊ p < 0.001, t-test.

[0049] The present invention designed shRNA according to the CDS region of the NUP35 gene, packaged it into a lentiviral vector, and made it enter eukaryotic cells to silence the expression of NUP35. The shRNA of the present invention can silence the protein expression of NUP35 and significantly inhibit the proliferation of immortalized and primary glioma cells. Therefore, the drug with this shRNA as the active ingredient can be used as an anti-tumor drug for the treatment of glioma diseases, which is of great significance for providing new drug action targets for the treatment of glioma diseases and can solve the problem of adverse reactions of anti-tumor drugs in the prior art.

[0050] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. Application of NUP35 gene as a target for diagnosis, prognosis and treatment of glioblastoma, characterized by: The NUP35 gene is used as a glioblastoma-specific molecular marker.

2. The use according to claim 1, characterized in that: Glioma is diagnosed by detecting the mRNA expression level of the NUP35 gene.

3. The use according to claim 1, characterized in that: The detection primers of NUP35 gene are: upstream primer: 5′-GGCCCGCGTTAGGATCTG-3′; downstream primer: 5′-ATCGAGGTTGTGGAGTCACC-3′.

4. The use according to any one of claims 1 to 3, characterized in that: The NUP35 gene is a therapeutic target for glioblastoma, including inhibiting the growth of glioma cells by inhibiting the mRNA and protein expression levels of the NUP35 gene.

5. The use according to claim 4, characterized in that: The method of inhibiting the mRNA and protein expression levels of the NUP35 gene includes using nucleic acid molecules, small molecule chemical drugs, polypeptides, proteins or interfering lentiviruses to inhibit the mRNA and protein expression levels of the NUP35 gene.

6. The use according to claim 5, characterized in that: The nucleic acid molecule includes an antisense oligonucleotide, dsRNA, siRNA or shRNA.

7. The use according to claim 6, characterized in that: The sequence of the shRNA is one or more of SEQ ID NOs: 1 to 3.

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