CircRNA molecule for inhibiting vascular endothelial cell function and application thereof

Abstract

The invention discloses a circRNA (Ribonucleic Acid) molecule for inhibiting a vascular endothelial cell function and application of the circRNA molecule, the molecule is circGPBP1L1, and the nucleotide sequence of the molecule is as shown in SEQ ID NO. 1. Preferably, the circRNA molecule can regulate and control the proliferation, migration and / or tube forming ability of vascular endothelial cells. The invention proves that circGPBP1L1 can be used as a molecular treatment target and is used for preparing and screening medicines for regulating and controlling functions of vascular endothelial cells and resisting angiogenesis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a circRNA molecule that inhibits vascular endothelial cell function and an application thereof. Background Art

[0002] Angiogenesis is an important pathway for tumor growth and metastasis, and is an important cause of neovascular eye diseases. It is a process in which vascular endothelial cells migrate and proliferate to form new blood vessels in the form of budding or non-budding on the basis of existing capillaries or venules. It can provide metabolic conditions for tumor tissues to promote tumor progression and also cause irreversible damage to the eyes. Therefore, the function of vascular endothelial cells plays a key role in tumor angiogenesis, vascular eye diseases, etc. A variety of active molecules can participate in angiogenesis by regulating vascular endothelial cells, such as vascular endothelial growth factor A (VEGF-A) can bind to vascular endothelial growth factor receptor 2 (VEGF-R2) on the surface of vascular endothelial cells to promote cell migration, proliferation and neovascularization; fibroblast growth factor (FGF) promotes neovascularization through direct mitogen action; epidermal growth factor (EGF) promotes vascular endothelial cell proliferation and differentiation through epidermal growth factor receptor (EGFR), thereby participating in the occurrence of angiogenesis-dependent diseases such as tumors and retinopathy. At present, targeted treatment strategies for anti-angiogenesis such as VEGF and FGFR have been applied in clinical practice, such as sorafenib for the treatment of advanced colon cancer, and VEGF inhibitors such as ranibizumab and conbercept for the treatment of fundus diseases as ophthalmic biological drugs. However, due to the single protein target, the efficacy of single drugs is poor, and it is urgent to develop new molecules that regulate the function of vascular endothelial cells as new targets for anti-angiogenesis.

[0003] Circular RNA (circRNAs) is a new type of RNA molecule with a covalently closed ring structure. It can remove the inhibitory effect of miRNA on downstream target genes by adsorbing miRNA, can bind to functional proteins to regulate their biological activity, and can also serve as a translation template to encode functional peptides, participating in various pathological and physiological processes in the human body. For example, circDIDO1 can encode short peptides to bind to PARP1 protein to inhibit its activity, and can also directly bind to PRDX2 to promote RBX1-mediated ubiquitination degradation to inhibit gastric cancer growth and metastasis (Zhang Y, et al., 2021, Mol Cancer); circRNA ZNF609 upregulates STMN1 expression by adsorbing miR-145 to promote angiogenesis in nasopharyngeal carcinoma (Wang J, et al., 2021, Kaohsiung J Med Sci). Therefore, circRNA is expected to become a new target for disease treatment, but the role of circRNA in vascular endothelial cells is not yet fully understood. Summary of the invention

[0004] In order to clarify the regulatory effect of circRNA on vascular endothelial cell function, the present invention provides a circRNA molecule that inhibits vascular endothelial cell function. The specific scheme is as follows:

[0005] A circRNA molecule that inhibits vascular endothelial cell function, the molecule is circGPBP1L1, and its nucleotide sequence is shown in SEQ ID NO.1.

[0006] Preferably, the present invention also provides the use of the circRNA as a molecular target in the preparation or screening of anti-cancer angiogenesis, and the circRNA molecule can regulate the proliferation, migration and / or tube formation ability of vascular endothelial cells.

[0007] The circRNA molecule inhibits / increases the proliferation, migration and / or tube formation ability of vascular endothelial cells by upregulating / downregulating the expression level of circGPBP1L1.

[0008] Preferably, the circRNA molecule can inhibit angiogenesis and tumor size in cancer tissue.

[0009] On the other hand, the present invention provides an anti-angiogenic molecular targeted drug, which includes: circGPBP1L1, or a preparation that promotes the expression of circGPBP1L1.

[0010] Preferably, the agent for promoting the expression of circGPBP1L1 is a circGPBP1L1 overexpression plasmid.

[0011] Preferably, the preparation further comprises pharmaceutically addable excipients, which are one or more of diluents, excipients, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers or lubricants.

[0012] Beneficial effects:

[0013] The present invention provides an application of a circGPBP1L1 molecule as a therapeutic target in the preparation of drugs for regulating vascular endothelial cell function and anti-cancer angiogenesis. The present invention proves that circGPBP1L1 can be used as a molecular therapeutic target for the preparation and screening of drugs for regulating vascular endothelial cell function and anti-cancer angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1It is the molecular identification and detection of circGPBP1L1 in Example 1 of the present invention; wherein, A is the location of the circGPBP1L1 molecule in the genome; B is the Sanger sequencing to identify the specific junction sequence of circGPBP1L1; C is the RT-qPCR detection of the expression level of circGPBP1L1 in human retinal microvascular endothelial cells HRMEC compared with retinal epithelial cells RPE; D is the RNAFISH detection of the subcellular localization of circGPBP1L1 in human umbilical vein endothelial cells HUVEC; E is the HUVEC cell nucleocytoplasmic separation quantitative detection of circGPBP1L1 expression.

[0015] Figure 2 The effect of up-regulating circGPBP1L1 level on vascular endothelial cell function in Example 2 of the present invention; wherein, A is the efficiency of up-regulating circGPBP1L1 level by RT-qPCR detection of circGPBP1L1 expression plasmid; B is the effect of up-regulating circGPBP1L1 level on the activity of two vascular endothelial cells analyzed by CCK8 growth curve; C is the effect of up-regulating circGPBP1L1 level on the proliferation ability of two vascular endothelial cells detected by plate cloning experiment; D is the effect of up-regulating circGPBP1L1 level on the migration ability of two vascular endothelial cells evaluated by Transwell migration experiment, with a scale of 200 μm; E is the effect of up-regulating circGPBP1L1 level on the tube formation ability of two vascular endothelial cells analyzed by tubule formation experiment, with a scale of 20 μm. F is the gross view of subcutaneous tumors of gastric cancer cell lines in nude mice.

[0016] Figure 3 It is the effect of down-regulating circGPBP1L1 level on vascular endothelial cell function in Example 3 of the present invention; wherein, A is RT-qPCR detection of the efficiency of small interfering RNA down-regulating circGPBP1L1 level; B is CCK8 growth curve analysis of the effect of down-regulating circGPBP1L1 level on the activity of two vascular endothelial cells; C is plate cloning experiment detection of the effect of down-regulating circGPBP1L1 level on the proliferation ability of two vascular endothelial cells; D is Transwell migration experiment evaluation of the effect of down-regulating circGPBP1L1 level on the migration ability of two vascular endothelial cells, and the scale bar is 200μm; E is a tubule formation experiment analysis of the effect of down-regulating circGPBP1L1 level on the tube formation ability of two vascular endothelial cells, and the scale bar is 20μm. DETAILED DESCRIPTION

[0017] The present invention will be further explained below in conjunction with the accompanying drawings and embodiments.

[0018] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention.

[0019] Example 1: Molecular identification and detection of circGPBP1L1

[0020] 1.1 Experimental methods

[0021] 1.1.1 Genomic localization of circGPBP1L1:

[0022] The genomic location and sequence of hsa_circ_0000068 were obtained by searching the UCSC Genome Browser on Human (GRCh37 / hg19) database (https: / / genome.mdc-berlin.de / ). The nucleotide sequence is shown in SEQ ID NO.1.

[0023] SEQ ID NO.1:

[0024]

[0025] 1.1.2 CircGPBP1L1 quantitative analysis and product sequencing:

[0026] Human retinal microvascular endothelial cells HRMEC and retinal epithelial cells RPE in the logarithmic growth phase were collected, and RNA was extracted by adding RNA isolater (Vazyme, R401-01) according to the reagent instructions. 1 μg RNA was taken and reverse transcribed with Random Primer to synthesize cDNA using HisScript 1st StrandcDNA Synthesis Kit (Vazyme, R111-01). AceQ SYBR Green Master Mix Kit (Vazyme, Q111-02) was used to prepare the fluorescence quantitative PCR reaction system with 2 μL cDNA, and the real-time fluorescence quantitative PCR detection was performed using the American ABI 7500 fluorescence quantitative PCR instrument. The optimal annealing temperature was determined to be 60°C by gradient PCR, and the specificity of primer amplification was evaluated by amplification curves and melting curves. The PCR product was analyzed for amplification length by 1.5% agarose gel electrophoresis, and the product was sequenced by Sanger to evaluate the accuracy of amplification. The experimental data were analyzed by relative quantitative analysis. The primer sequences of circGPBP1L1 were shown in SEQ ID NO.2-3, β-actin was used as the internal reference gene (primer sequences were shown in SEQ ID NO.4-5), and △Ct=Ct circGPBP1L1 -Ct β-actin Indicates the test value of each sample, with 2 -(△CtHRMEC-△CtRPE) The expression fold of circGPBP1L1 in human retinal microvascular endothelial cells (HRMEC) compared with retinal epithelial cells (RPE) was calculated by Western blotting, and statistical analysis was performed using GraphPad Prism 5 and SPSS16.0.

[0027] SEQ ID NO.2: TTGGATGCCTCACCTTACT;

[0028] SEQ ID NO.3: GCCACACGAATCTATCACT;

[0029] SEQ ID NO.4: CACGAAAACTA CCTTCAACTCC;

[0030] SEQ ID NO. 5: CATACTCCTG CTTGCTGATC.

[0031] 1.1.3 RNA FISH detection of circGPBP1L1:

[0032] Human umbilical vein endothelial cells (HUVEC) in logarithmic growth phase were cultured at 1×10 3 Density seeding was carried out in 24-well plates containing crawling slices and cultured overnight. The culture medium was aspirated and washed 3 times with PBS. 4% (V / V) paraformaldehyde was added to fix at room temperature for 30 minutes and then aspirated. Washed 3 times with PBS, 100 μL 0.1% (V / V) Triton X-100 was added to each well for 15 minutes at room temperature, and then washed 3 times with PBS. Using the RNA FISH kit of Suzhou Jima Company, 100 μL 2×SSC was added to each well, incubated at 37°C for 30 minutes and then aspirated. 100 μL 70%, 85%, and 100% ethanol (V / V) were added to each well in turn, and the cells were allowed to stand at room temperature for 3 minutes and then aspirated. They were placed at 73°C for denaturation for 5 minutes. The 5'-end labeled Cy3 fluorescent circGPBP1L1 probe (sequence as shown in SEQ ID NO.6) synthesized by Suzhou Jima Company was used. 100 μL of 40 μg / mL probe mixture was added to each well, incubated at 37°C overnight and then discarded. 100 μL of 0.4×SSC / 0.35% (V / V) Tween 20 preheated at 65°C was added to each well, washed at room temperature for 2 min and then discarded. 100 μL of Hoechst 33342 staining solution was added to each well and stained in the dark for 10 min and then discarded. The wells were washed three times with PBS, and the slides were fixed on slides and photographed under a laser confocal microscope.

[0033] SEQ ID NO.6:

[0034] AGGCCACACGAAUCUAUCACUCUUAGCUGACUGUGGUGUUGA.

[0035] (4) Analysis of circGPBP1L1 by nuclear-cytoplasmic separation: Human umbilical vein endothelial cells (HUVEC) in the logarithmic growth phase were collected and resuspended in 300 μL pre-cooled Cell Fractionation Buffer using the PARIS Kit (Life Technologies, AM1921). The cells were placed on ice for 10 min and centrifuged at 500 × g for 5 min at 4°C. The supernatant was carefully aspirated as the cytoplasmic fraction. The precipitate as the nuclear fraction required the addition of 300 μL pre-cooled Cell Disruption Buffer. An equal volume of 2× Lysis / Binding Solution was added to the cytoplasmic and nuclear fractions at room temperature and mixed. An equal volume of anhydrous ethanol was added and mixed. The mixture was placed in a filter column and centrifuged at 12,000 g for 1 min. The lower filtrate was discarded and 700 μL Wash Solution 1 was added for washing and centrifugation. The lower filtrate was discarded and 500 μL Wash Solution 2 / 3 was added for washing and centrifugation twice. The empty tube was centrifuged again once and 50 μL 100°C preheated Elution was added. Solution, centrifuge for 30s to elute RNA twice, and store RNA at -80°C. RNA reverse transcription and quantitative PCR were performed as above, and the experimental data were analyzed by relative quantitative analysis method, with β-actin as the internal reference gene for the cytoplasmic component and U3 as the internal reference gene for the nuclear component (primer sequences are shown in SEQ ID NO.7-8), and the expression multiple of GPBP1L1 in the cytoplasm of human retinal microvascular endothelial cells HUVEC compared with the nucleus was calculated, and statistical analysis was performed using GraphPad Prism 5 and SPSS16.0.

[0036] SEQ ID NO.7: GAGCACCGAAAACCACG;

[0037] SEQ ID NO. 8: CTCTCACTCCCCAATACG.

[0038] 1.2 Results Analysis

[0039] Gene localization analysis showed that circGPBP1L1 originated from the reverse splicing of exons 3 and 4 of the GPBP1L1 gene. Figure 1 A. The original sequence of circGPBP1L1 is shown in SEQ NO.1, and its unique junction sequence was verified by Sanger sequencing. Figure 1 B. Compared with human retinal epithelial cells RPE, the expression of circGPBP1L1 in human retinal microvascular endothelial cells HRMEC was significantly downregulated (p < 0.01). Figure 1 C. RNA FISH showed that circGPBP1L1 was expressed in the cytoplasm and nucleus of human umbilical vein endothelial cells (HUVEC). Figure 1 D. Nuclear-cytoplasmic separation and quantification showed that the expression of circGPBP1L1 in the cytoplasm of HUVEC was significantly higher than that in the nucleus. Figure 1 Middle E. (**p<0.01).

[0040] Example 2: Effect of up-regulation of circGPBP1L1 levels on endothelial cell function

[0041] 2.1 Experimental methods

[0042] 2.1.1 Construction of circGPBP1L1 expression plasmid:

[0043] Studies have shown that Alu sequences have the function of promoting circRNA circularization (Liang, et al, 2014, GenesDev). According to the characteristics of the circGPBP1L1 genome sequence, the Alu Sx3 intron sequence on the left side of GPBP1L1 exon 3 to the Alu Jb sequence on the right side of exon 4 was determined as the target fragment, and the sequence is shown in SEQ ID NO.9.

[0044] The synthetic target sequence (Nanjing GenScript) was cloned into the pcDNA3.1(+) vector to obtain the circGPBP1L1 expression plasmid, and the pcDNA3.1(+) empty vector was used as the control plasmid.

[0045] SEQ ID NO.9:

[0046]

[0047] 2.2.2 Plasmid transfection of endothelial cells:

[0048] Human umbilical vein endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMEC) in the logarithmic growth phase were digested and counted at 8×10 4 A number of cells were uniformly seeded in a 6-well plate and placed in a 37°C, 5% CO2 (V / V) incubator for overnight culture. After washing twice with sterile PBS, 2 mL of H-DMEM complete medium containing 10% (M / M) fetal bovine serum was added to each well and temporarily placed in an incubator. The system was configured using JetPRIME transfection reagent (Polyplus, 101000046), with 200 μL JetPRIMEbuffer containing 4 μL JetPRIME and 2 μg circGPBP1L1 expression plasmid (circGPBP1L1-OE) or pcDNA3.1 (+) control plasmid (EV) per well. After gentle mixing, the cells were placed at room temperature for 10 minutes and added to the corresponding wells for mixing. Subsequent cytological experiments were performed 48 hours later.

[0049] 2.2.3 Vascular endothelial cell function experiment:

[0050] CCK8 assay: Human umbilical vein endothelial cells (HUVEC) or human retinal microvascular endothelial cells (HRMEC) were digested and counted 48 hours after plasmid transfection. 2×10 3 A number of cells were evenly seeded in a 96-well plate. The circGPBP1L1-OE group and the EV group were set up for 5 days × 3 times, a total of 15 replicate wells. The outer circle of the well plate was filled with sterile PBS to prevent edge effect and placed in an incubator overnight. The next day, the medium was replaced with 100 μL H-DMEM complete medium containing 10 μL CCK8 reagent (Vazyme, A311-02) mixed solution. After incubation in the incubator for 2 hours, the absorbance OD at 450 nm was detected on a Cytation5 microplate reader. The detection was continued for 5 days, and the absorbance values ​​were recorded and the CCK8 growth curve was drawn.

[0051] Plate cloning experiment: Human umbilical vein endothelial cells (HUVEC) or human retinal microvascular endothelial cells (HRMEC) were digested and counted 48 hours after plasmid transfection. 3 The cells were uniformly seeded in a 3.5 cm cell culture dish containing 2 mL of H-DMEM complete medium. The medium was changed every 2 days. The culture was terminated after the appearance of single cell colonies visible to the naked eye after 10-14 days. The medium was discarded, and the cells were washed with PBS three times. The cells were fixed with 4% (V / V) paraformaldehyde at room temperature for 30 min, stained with 5% (V / V) crystal violet for 15 min, washed with PBS until the background was clear, dried at room temperature, and photographed, and the number of cell colonies in each dish was counted.

[0052] Cell migration assay: Human umbilical vein endothelial cells (HUVEC) or human retinal microvascular endothelial cells (HRMEC) were digested and counted 48 hours after plasmid transfection, and then resuspended in serum-free H-DMEM blank medium. Each well was cultured with 8×10 cells per well in 200 μL medium. 4 A number of cells were evenly seeded in the chamber of the Transwell migration plate, 600 μL of H-DMEM complete medium was added to the lower chamber, and the plate was cultured in an incubator for 12 h. After washing with PBS three times, 600 μL of 4% (V / V) paraformaldehyde was added to fix the chamber at room temperature for 30 min, and the chamber was stained with 5% (V / V) crystal violet for 15 min. After washing with PBS until no purple liquid flowed out, the non-migrated cells on the inside of the chamber were carefully wiped off with a cotton swab. After drying at room temperature, three random fields of view were photographed under a microscope, and the number of cells in each field of view was counted.

[0053] Tubule formation assay: serum-free H-DMEM blank medium was taken up with a pre-cooled pipette tip and diluted with pre-cooled Matrigel (BD Biosciences, 356234) at a ratio of 1:1. 50 μL per well was spread on the bottom of the corresponding well of a 96-well plate and placed at 37°C for solidification for more than 30 min. Human umbilical vein endothelial cells (HUVEC) or human retinal microvascular endothelial cells (HRMEC) were digested and counted 48 hours after plasmid transfection, and 100 μL of H-DMEM complete medium containing 4×10 4 A number of cells were evenly seeded in the corresponding wells of the 96-well plate treated as above and photographed under a microscope after 4-6 hours.

[0054] 2.2.4 Anti-cancer angiogenesis experiment:

[0055] Nude mouse subcutaneous tumor experiment: Six 4-6 week old male BALB / c nude mice (Changzhou Cavens) were randomly divided into two groups, 3 mice in each group, and kept in a sterile barrier environment of an SPF animal experiment center. The experimental process complied with animal ethics and the experimental plan was approved by the Animal Experiment Ethics Committee. The gastric cancer cell line MGC-803 in the logarithmic growth phase was digested and centrifuged, and 6×10 5The cells were uniformly seeded in 10 cm cell culture dishes and placed in an incubator for overnight culture. After washing twice with sterile PBS, 8 mL of H-DMEM complete medium containing 10% (V / V) fetal bovine serum was added to each dish and temporarily placed in an incubator. The JetPRIME transfection reagent (Polyplus, 101000046) was used to configure the system. Each dish contained 500 μL of JetPRIME buffer containing 20 μL of JetPRIME and 8 μg of circGPBP1L1 expression plasmid (circGPBP1L1-OE) or pcDNA3.1 (+) control plasmid (EV). After gentle mixing, the cells were placed at room temperature for 10 min and added to the corresponding dish for mixing. The cells were placed in an incubator for 48 h. The above transfected gastric cancer cells were digested and centrifuged, washed twice with sterile PBS, the supernatant was discarded, and the cells were resuspended in an appropriate amount of sterile PBS to adjust the density to 5×10 4 / μL, 200μL of cell suspension was inoculated into the right axilla of each nude mouse in each group, and the tumor formation was observed. After 2 weeks, the nude mice were dislocated by cervical dislocation, and the tumor tissues were removed and photographed.

[0056] 2.2 Results Analysis

[0057] Transfection of circGPBP1L1 expression plasmid can significantly increase the level of circGPBP1L1 in human umbilical vein endothelial cells HUVEC by more than 60 times (p < 0.001), and can increase the level of circGPBP1L1 in human retinal microvascular endothelial cells HRMEC by more than 200 times (p < 0.01). Figure 2 A. Upregulation of circGPBP1L1 levels can significantly inhibit the growth activity of human vascular endothelial cells HUVEC and HRMEC. The results are shown in Figure 2 B. Upregulation of circGPBP1L1 levels can significantly inhibit the clonal proliferation of human vascular endothelial cells HUVEC and HRMEC. Figure 2 C. Upregulation of circGPBP1L1 levels can significantly inhibit the migration ability of human vascular endothelial cells HUVEC and HRMEC. The results are shown in Figure 2 D. Upregulation of circGPBP1L1 levels can significantly inhibit the tubule formation ability of human vascular endothelial cells HUVEC and HRMEC. The results are shown in Figure 2 In the nude mouse subcutaneous tumor model, upregulation of circGPBP1L1 levels can significantly inhibit angiogenesis and tumor size in gastric cancer tissues. Figure 2 Middle F. (*p<0.05, **p<0.01, ***p<0.001).

[0058] Example 3 Effect of downregulation of circGPBP1L1 levels on vascular endothelial cell function

[0059] 3.1 Experimental methods

[0060] 3.1.1 Transfection of endothelial cells with small interfering RNA:

[0061] Human umbilical vein endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMEC) in the logarithmic growth phase were digested and counted at 8×10 4 The cells were uniformly seeded in a 6-well plate and placed in a 37°C, 5% (V / V) CO2 incubator for overnight culture. After washing twice with PBS, 2 mL of H-DMEM complete medium containing 10% (V / V) fetal bovine serum was added to each well and temporarily placed in an incubator. The system was configured using JetPRIME transfection reagent (Polyplus, 101000046), with 200 μL of JetPRIME buffer per well containing 4 μL of JetPRIME and 25 nM circGPBP1L1-si1 (sequence as shown in SEQ ID NO.10-11) or circGPBP1L1-si2 (sequence as shown in SEQ ID NO.12-13) or negative control siNC (sequence as shown in SEQ ID NO.14-15), gently mixed, and then placed at room temperature for 10 min before adding to the corresponding wells for mixing. Subsequent cytological experiments were performed 48 hours later.

[0062] SEQ ID NO.10: CAGUCAGCUAAGAGUGAUATT;

[0063] SEQ ID NO.11: UAUCACUCUUAGCUGACUGTT;

[0064] SEQ ID NO.12:UCAGCUAAGAGUGAUAGAUTT;

[0065] SEQ ID NO.13: AUCUAUCACUCUUAGCUGATT;

[0066] SEQ ID NO.14: UUCUCCGAACGUGUCACGUTT;

[0067] SEQ ID NO. 15: ACGUGACACGUUCGGAGAATT.

[0068] 3.1.2 Vascular endothelial cell function experiment:

[0069] After digestion and counting, human umbilical vein endothelial cells HUVEC or human retinal microvascular endothelial cells HRMEC transfected with small interfering RNA for 48 hours were subjected to CCK8 assay, plate cloning assay, cell migration assay and tubule formation assay. The specific operation was the same as that in Example 2.

[0070] 3.2 Results Analysis

[0071] Transfection of circGPBP1L1 small interference gene could significantly downregulate the level of circGPBP1L1 in human umbilical vein endothelial cells HUVEC and human retinal microvascular endothelial cells HRMEC by more than 90% (p < 0.001). Figure 3 A. Downregulation of circGPBP1L1 levels can increase the growth activity of human vascular endothelial cells HUVEC and HRMEC. The results are shown in Figure 3 B. Downregulation of circGPBP1L1 levels can significantly promote the clonal proliferation of human vascular endothelial cells HUVEC and HRMEC. The results are shown in Figure 3 C. Downregulation of circGPBP1L1 levels can significantly enhance the migration ability of human vascular endothelial cells HUVEC and HRMEC. The results are shown in Figure 3 D. Downregulation of circGPBP1L1 levels can significantly increase the tubule formation ability of human vascular endothelial cells HUVEC and HRMEC. The results are shown in Figure 3 Middle E. (*p<0.05, **p<0.01, ***p<0.001)

[0072] 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 circRNA molecule that inhibits vascular endothelial cell function, characterized in that: The molecule is circGPBP1L1, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The use of the circRNA molecule according to claim 1, characterized in that: The circRNA molecules can regulate the proliferation, migration and / or tube formation ability of vascular endothelial cells.

3. The use according to claim 2, characterized in that: The circRNA molecule inhibits / increases the proliferation, migration and / or tube formation ability of vascular endothelial cells by upregulating / downregulating the expression level of circGPBP1L1.

4. The use according to claim 2, characterized in that: The circRNA molecules can inhibit angiogenesis and tumor size in cancer tissues.

5. An anti-angiogenic molecular targeted drug, characterized in that: The molecular targeted drug includes: circGPBP1L1, or a preparation that promotes the expression of circGPBP1L1.

6. The molecular targeted drug according to claim 5, characterized in that: The preparation for promoting circGPBP1L1 expression is a circGPBP1L1 overexpression plasmid.

7. The molecular targeted drug according to claim 6, characterized in that: The preparation further comprises pharmaceutically addable auxiliary materials, which are one or more of diluents, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants.

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