Application of circ-UBE2I RNA sequence in preparation of medicine for inhibiting or treating glioma and medicine

Abstract

The invention provides application of a circ-UBE2I RNA sequence in preparation of a medicine for inhibiting or treating glioma and the medicine, and application of the circ-UBE2I RNA sequence in preparation of the medicine for inhibiting or treating glioma, the nucleotide sequence of the circ-UBE2I RNA sequence is as shown in SEQ ID No.1, the circ-UBE2I RNA sequence can effectively inhibit glioma cells from forming blood vessels to generate vascular mimicry, and the circ-UBE2I RNA sequence can effectively inhibit glioma cells from forming blood vessels to generate vascular mimicry. Therefore, the vascular mimicry is inhibited from participating in transportation of blood, oxygen and nutrient substances of tumor tissues, and the effect of inhibiting glioma or improving the survival rate of patients is achieved.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to the use of a circ-UBE2I RNA sequence in the preparation of a drug for inhibiting or treating glioma and a drug. Background Art

[0002] Glioma has abundant angiogenesis. Not only do vascular endothelial cells proliferate rapidly, but glioma cells can also form vascular mimicry (VM). VM is involved in the transportation of blood, oxygen and nutrients in tumor tissues. It is widely present in various malignant tumors such as breast cancer and melanoma, and is closely related to the low survival rate and poor prognosis of patients.

[0003] Because VM is different from ordinary blood vessels in structure, some current anti-cancer drugs targeting angiogenesis cannot achieve the expected effect. At present, the clinical treatment of glioma is mainly surgery, supplemented by postoperative radiotherapy and chemotherapy with temozolomide. Although these treatments can delay the progression of the disease, the median survival of GBM patients is still less than 14 months. From a histological point of view, glioma is a typical vascular-dependent solid tumor with microvascular proliferation as a pathological feature. In the past decade, anti-angiogenic therapy has been an important auxiliary treatment for high-grade gliomas at home and abroad, including the angiogenesis inhibitor bevacizumab, which has been used in clinical practice. However, a series of retrospective studies have found that the effects of the above treatments are not ideal.

[0004] Therefore, there is an urgent need for a drug to inhibit or treat it. Summary of the invention

[0005] The present application provides a use of a circ-UBE2I RNA sequence in the preparation of a drug for inhibiting or treating gliomas and a drug, which can effectively inhibit glioma cells from forming angiogenic vascular mimicry, thereby inhibiting vascular mimicry from participating in the transportation of blood, oxygen and nutrients in tumor tissues, thereby achieving the effect of inhibiting gliomas or improving patient survival rates.

[0006] In a first aspect, an embodiment of the present application proposes a use of a circ-UBE2I RNA sequence in the preparation of a drug for inhibiting or treating glioma, and the nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.1.

[0007] In this example, the circ-UBE2I RNA sequence has the ability to encode protein and can bind to the ribosome.

[0008] In this example, circ-UBE2I was found to be downregulated in glioma. circ-UBE2I may regulate the expression of VEGFR2 through an indirect pathway and regulate the VM formation of glioma cells.

[0009] Among them, the vascular endothelial growth factor (VEGF) signaling pathway is a crucial regulatory pathway in the formation of glioma VM. VEGFR2 and VEGFR1 are the most important receptors in this pathway. VEGFR2 is highly expressed in vascular endothelial cells. It is not only involved in regulating the differentiation, proliferation and migration of endothelial cells, but also closely related to the VM formation process. Studies have confirmed that VEGFR2 can be activated by VEGF, thereby promoting the activity of the PI3K-AKT pathway and promoting the VM formation of glioma cells. In addition, VEGFR2 also plays an important role in regulating the formation of VM in tumors such as gastric cancer and breast cancer.

[0010] Glioma VM can be understood as glioma vascular mimicry, or vascular mimicry for short, which can provide glioma cells with oxygen, nutrients, etc. This vascular mimicry can affect the development of gliomas and glioma cells.

[0011] In a second aspect, an embodiment of the present application proposes a recombinant vector, which includes a circ-UBE2I RNA sequence, and the nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.1.

[0012] In a third aspect, an embodiment of the present application proposes a cell line, which includes a circ-UBE2I RNA sequence, and the nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.1.

[0013] In some embodiments, the cell line expresses a polypeptide encoded by a circ-UBE2I RNA sequence, for example, polypeptide 220aa, whose amino acid sequence is shown in SEQ ID No.2.

[0014] Studies have found that circ-UBE2I encodes a 220aa amino acid sequence that is also low in expression. The low-expressed 220aa amino acid sequence conjugates with SUMO protein through the UBC domain, regulates the SUMOylation level of VEGFR2, and ultimately regulates the VM formation of glioma cells.

[0015] UBC (Ubiquitin-conjugating enzyme) domain (28aa-183aa) is the core catalytic domain of E2 conjugating enzyme in the ubiquitination (also known as SUMOylation) process. SUMOylation is a post-translational modification process, which refers to the reversible binding of SUMO proteins (SUMO1 and SUMO2 / 3) to substrate proteins with covalent bonds under the mediation of E1 activating enzyme, E2 conjugating enzyme and E3 ligase, and regulates protein stability and expression, and participates in the occurrence and development of various tumors.

[0016] In some embodiments, the concentration of cells in the cell line is 1×10 5 / 100 ul to 3×10 5 / 100 ul.

[0017] In a fourth aspect, an embodiment of the present application proposes a drug for inhibiting or treating glioma, wherein the drug includes a cell line, wherein the cell line contains a circ-UBE2I RNA sequence, and the nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.1.

[0018] In some embodiments, the drug comprises 1×10 5 / 100 ul to 3×10 5 / 100 ul of cells, and the cells are derived from the cell line.

[0019] In some embodiments, the cell line expresses a polypeptide encoded by a circ-UBE2I RNA sequence.

[0020] This application has at least the following beneficial effects:

[0021] The circular RNA circ-UBE2I provided in the present application can promote SUMOylation of VEGFR2 through its own ORF-encoded polypeptide protein 220aa, inhibit its protein stability and expression level, regulate the formation of glioma cell VM, and thus inhibit or treat glioma.

[0022] Inhibiting VM formation in gliomas is an issue that cannot be ignored in the comprehensive anti-angiogenic treatment of gliomas, and it is very important to clarify the molecular mechanism that regulates VM formation. This application combines omics sequencing and molecular biology techniques to explain the molecular mechanism of VM formation in glioma cells regulated by the molecular network composed of circ-UBE2I, polypeptide protein 220aa, and VM growth-related factor VEGFR2 at the cell, tissue, and in vivo levels. It was found that it can not only reveal a new mechanism of VM formation in glioma cells, but also provide new strategies and potential new targets for the comprehensive treatment of anti-angiogenesis in brain gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The identification and expression of circ-UBE2I in glioma tissues and cells of the embodiment are shown.

[0024] Figure 2 The effect of circ-UBE2I of the embodiment on the VM formation ability of U343 cells is shown.

[0025] Figure 3 The effect of Circ-UBE2I on the expression of VM-related proteins in the Example is shown.

[0026] Figure 4 The figure shows the expression of the novel polypeptide protein 220aa encoded by Circ-UBE2I in the example in glioma tissues.

[0027] Figure 5 The effect of Circ-UBE2I of the Example on the VM formation ability of U343 cells is shown.

[0028] Figure 6 The binding effects of 220aa and VEGFR2 and SUMO2 / 3 protein respectively in the example are shown. DETAILED DESCRIPTION

[0029] The following detailed description and drawings of the embodiments of the present application are used to illustrate the principles of the present application, but cannot be used to limit the scope of the present application, that is, the present application is not limited to the described embodiments.

[0030] Example

[0031] The following examples describe the disclosure of the present application in more detail, and these examples are only for illustrative purposes, as it is obvious to those skilled in the art that various modifications and variations are made within the scope of the disclosure of the present application. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on mass, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available.

[0032] Example 1

[0033] Through circRNAs delinear sequencing, 24,205 co-expressed circRNAs and 3,117 abnormally expressed circRNAs were screened from 3 pairs of glioma tissues and corresponding adjacent non-cancerous tissues from patients with brain glioma. Further, by comparing the circRNAs database circBase, 2,739 differentially expressed circRNAs were screened, among which circ-UBE2I (hsa_circ_0000665) was significantly downregulated in glioma. Using Sanger sequencing combined with agarose gel electrophoresis, the presence and specificity of circ-UBE2I in glioma cells were found. After using RNaseR enzyme to remove linear RNA, qRT-PCRs experiments were performed in four groups of glioma tissues, including normal human brain tissues (NBTs, n=3), WHO grade I (n=4), WHO II (n=3), WHO III (n=5) and WHO IV (n=5). NBTs, WHO grade I (n=4), WHO II (n=3), WHO III (n=5) and WHO IV represented normal adjacent cancer tissues and glioma tissues of WHO grade I to IV, respectively. It was confirmed that circ-UBE2I expression was significantly downregulated in human glioma tissues; it was significantly downregulated in HA, U251, U343, and A172 cells of brain glioma tissues. HA, U251, U343, and A172 represent human astrocytes and glioma cell lines, respectively. These downregulations indicate that circ-UBE2I expression is significantly downregulated in human glioma cells. The results are shown in Figure 2 Figure 1 .

[0034] Figure 1 Figure A in Figure 1 uses Sanger sequencing combined with agarose gel electrophoresis to identify the presence of circ-UBE2I RNA sequence (hsa_circ_0000665) in glioma cells. Figure B in Figure 1 uses QRT-PCR to detect circ-UBE2I in normal human brain tissue.

[0035] (normal brain issues, NBTs, n = 3), WHO level I (n = 4), WHO II (n = 3), WHO III

[0036] (n=5) and WHO IV (n=5) grade glioma tissues. Values ​​are expressed as mean ± standard deviation, **P<0.01. In Figure B, the lower figure uses QRT-PCR to detect the expression level of circ-UBE2I in human astrocytes (HA) and brain glioma cells (U251, U343, A172). Values ​​are expressed as mean ± standard deviation, **P<0.01.

[0037] Example 2circ-UBE2I regulates VM formation in glioma cells

[0038] The circ-UBE2I RNA sequence (hereinafter referred to as circ-UBE2I) was transferred into a vector, which was then introduced into U343 cells, a glioma cell, and the cells were cultured to construct U343 cells overexpressing circ-UBE2I.

[0039] The culturing steps of U343 cells include planting U343 cells in a 96-well plate at a density of 20,000 cells / well and continuing to culture. At the detection time point, 1 / 10 volume of CCK-8 reagent is added to each well of each group of U343 cells, and cultured in a 37°C, 5% CO2 incubator for 3 hours; a wavelength of 450nm is selected, and the light absorption value of each well is measured on an enzyme-linked immunosorbent monitor, and the results are recorded, and a line graph is drawn with time as the horizontal axis and OD value as the vertical axis. , so as to determine the density of subsequent U343 cells, such as the cell density in the CCK-8 experiment.

[0040] Angiogenesis assay was performed using a commercially available CCK-8 kit and a commercially available Transwell kit. Cell density for CCK-8 assay: 20,000 cells / well.

[0041] The cell density of the Transwell experiment is 0.5-2.0x 10^6 / ml. The experimental steps include:

[0042] 1. The cells were washed once with PBS, digested with EDTA or 0.05% trypsin, and then neutralized with serum-free medium containing 5% BSA and centrifuged at 1500 rpm for 5-10 min.

[0043] 2. Resuspend the cells in serum-free medium and adjust the cell density to 0.5-2.0x 10^6 / ml.

[0044] 3. Take the above 200uL of cell solution and add it to the small chamber, and add 900uL of culture medium containing 10% FBS to the lower chamber (culture plate).

[0045] Incubate at 437°C for 24 hours.

[0046] 5. After incubation, carefully remove the cell chamber, remove the culture medium on the upper layer of the chamber, wash once with PBS, fix with 70% alcohol or 4% paraformaldehyde for 5 minutes, and stain with 0.5% crystal violet (dissolved in 2% ethanol or PBS) for 20 minutes.

[0047] 6. Wash twice with PBS to remove excess dye, immediately wipe off the cells on the upper layer of the filter membrane with a cotton swab, and let it air dry naturally. Observe the cells on the lower layer of the filter membrane under a microscope, randomly select different fields of view to count and calculate the average value.

[0048] Vascular mimicry formation experiment: Initial U343 cell density: 6×10 4 The / ml step included: coating a 96-well plate with 60 μl of Matrigel basement membrane matrix (BD Bioscience, MA, USA) before the experiment.

[0049] Resuspend U343 cells in 100 μl of FBS-free medium at a concentration of 6 × 10 4 100ul of cell suspension with a concentration of 10 cells / ml was inoculated into 96-well culture plates, incubated at 37°C for 8h, and the number of angiogenesis was captured under an inverted microscope (Olympus, Tokyo, Japan).

[0050] The study found that the expression of circ-UBE2I significantly inhibited the proliferation, migration and VM formation ability of U343 cells. Figure 2 .

[0051] Figure 2 Figure A, the CCK-8 assay was used to detect the effect of overexpression of circ-UBE2I on the proliferation of U343 cells in the circ-UBE2I(+)NC group and the circ-UBE2I(+) group, respectively, indicating that the proliferation of cells in the circ-UBE2I overexpression group was significantly reduced. Figure B used the Transwell assay to detect the effect of overexpression of circ-UBE2I on the migration ability of U343 cells. Figure C used the angiogenesis assay to detect the effect of overexpression of circ-UBE2I on the VM (vascular mimicry) formation ability of U343 cells, indicating that the vascular mimicry formation in the circ-UBE2I(+) group was reduced. Values ​​are expressed as mean ± standard deviation (n = 3), **P < 0.01, compared with the circ-UBE2I(+)NC group.

[0052] In summary, it is shown that the circ-UBE2I RNA sequence can significantly inhibit the biological behavior of malignant glioma cells and has a positive effect on inhibiting glioma in organisms.

[0053] Studies have shown that the circ-UBE2I RNA sequence also has a significant effect on U251 and A172 cells.

[0054] Example 3: circ-UBE2I changes VEGFR2 protein expression

[0055] To further understand the mechanism by which circ-UBE2I regulates VM formation in U343 cells, the effect of overexpression of circ-UBE2I on VEGFR2 protein expression in U343 cells was detected. The experiment confirmed that circ-UBE2I expression significantly downregulated VEGFR2 protein expression in U343 cells.

[0056] RIP experimental steps:

[0057] (I) 1. Cultivate the cells until they are in good condition, remove the culture medium, add 5 ml of PBS, shake left and right, and wash the cells once

[0058] 2. Add 1 ml of trypsin for digestion. After digestion is complete, add 2 ml of complete culture medium to terminate digestion.

[0059] 3. Centrifuge at 1200 rpm for 1 min and remove the supernatant.

[0060] 4. After cell counting, calculate the number of cells per 1x10 7 RIPA was added at a ratio of 10 cells / 500 μl RIPA lysis buffer.

[0061] 5. Add 10 μl of PMSF, phosphatase inhibitors and protease inhibitors to each ml of RIPA lysis buffer, flip and lyse for 2 hours, centrifuge at 12,000 rpm for 15 minutes, take the supernatant and store at -20°C.

[0062] (II) 1. Label the 1.5 ml EP tubes required for the experiment. Samples include the target sample, negative control and positive control.

[0063] 2. Pipette 50 μl of the resuspended magnetic bead suspension into each 1.5 ml EP.

[0064] 3. Add 500 μl RIPWash Buffer to each tube and vortex.

[0065] 4. Place the 1.5ml EP tube on the magnetic rack, wait for the solution to clarify and remove the supernatant, repeat once.

[0066] 5. Resuspend the magnetic beads with 500 μl of RIP Wash buffer, add 5 μg of corresponding antibodies (IgG antibody, VEGFR2 antibody) to each sample, and incubate at 4°C for 4 h.

[0067] 6. Place the 1.5ml EP tube on the magnetic rack and discard the supernatant.

[0068] 7. Add 500 μl RIPWash Buffer, vortex and discard the supernatant, and repeat once.

[0069] 8. Add RIPWash Buffer, vortex and place on ice.

[0070] (III) Binding of magnetic bead antibody complex to protein

[0071] 1. Place the EP tube from the previous step on the magnetic rack, remove the supernatant, and add 900 μl of RIP Immunoprecipitation Buffer to each tube.

[0072] 2. Rapidly thaw the cell lysate prepared in the first step, centrifuge at 12000rpm, 4℃ for 10min. Pipette 100μl of the supernatant into the magnetic bead-antibody complex in the previous step to make the total volume 1ml. Incubate at 4℃ overnight.

[0073] 3. After incubation, centrifuge briefly, place the EP tube on a magnetic rack, wait for the solution to clarify and remove the supernatant, add 500μl RIPWash Buffer, vortex and place the EP tube on a magnetic rack, remove the supernatant, repeat 5 times, and wash 6 times in total.

[0074] (IV) RNA elution

[0075] 1. Resuspend the magnetic bead-antibody complex with 150 μl of Proteinase K Buffer and incubate at 55°C for 30 min.

[0076] 2. After incubation, place the EP tube on the magnetic rack, transfer the supernatant to a new 1.5 ml EP tube, and add 250 μl RIPWash Buffer to each tube.

[0077] 3. Add 400 μl of phenol:chloroform:isoamyl alcohol (125:24:1), vortex for 15 seconds, and centrifuge at 12,000 rpm for 10 minutes at room temperature.

[0078] 4. Carefully aspirate the upper aqueous phase and transfer it to another new 1.5 ml EP tube. Add 400 μl of chloroform to each tube, vortex for 15 seconds, and centrifuge at 12,000 rpm for 10 minutes at room temperature.

[0079] 5. Carefully aspirate the upper aqueous phase and transfer it to another new 1.5 ml EP tube. Add 850 μl of anhydrous ethanol (RNase-free) to each tube, mix well, and precipitate at -20°C overnight.

[0080] 6. After incubation, centrifuge at 12000rpm and 4℃ for 15min and carefully remove the supernatant.

[0081] 7. Add 900 μl of 80% ethanol, centrifuge at 12,000 rpm and 4°C for 15 min, carefully remove the supernatant, open the lid, let it stand for 3 min, and dry.

[0082] 8. Add 10 μl DEPC water to dissolve and store at -80℃.

[0083] RNA immunoprecipitation experiments confirmed that the expression of circUBE2I was the highest in the Input group (positive control, whole RNA group). At the same time, compared with the IgG group (negative control), there was no statistical difference in the relative expression of circUBE2I in the immunoprecipitation group using VEGFR2 antibody (P>0.05), indicating that there was no binding interaction between circ-UBE2I and VEGFR2. Figure 3 shown.

[0084] Figure 3 Figure A shows the effect of overexpression of circ-UBE2I on VEGFR2 protein expression in U343 cells using Western blot. Values ​​are expressed as mean ± standard deviation (n = 3), **P < 0.01, compared with the circ-UBE2I (+) NC group. (B) RNA immunoprecipitation confirmed the binding of circ-UBE2I to VEGFR2.

[0085] Example 4 Peptide protein 220aa encoded by circ-UBE2I

[0086] In order to further study the mechanism of circ-UBE2I regulating VM formation in U343 cells, the project initially searched the circRNADb database and TransCirc software and found that circ-UBE2I has a potential ORF and two IRES sequences, and may translate a 220-amino acid polypeptide protein 220aa. By analyzing the amino acid sequence encoded by the circRNA cyclization site, a specific antibody for detecting 220aa was prepared in the early stage. Western blot experiments found that 220aa was significantly lowly expressed in glioma tissues. The results are as follows Figure 4 .

[0087] Figure 4 Figure A in the middle is a Western blot experiment to detect the expression level of polypeptide protein 220aa in NBTs, low-grade glioma tissues (WHOI-II, low-grade gliomas, LGGs) and high-grade glioma tissues (WHOIII-IV, high-grade gliomas, HGGs), and Figure B shows the different numbers of vascular mimicry cells. The values ​​are expressed as mean ± standard deviation (n = 3), **P < 0.01, compared with the NBTs group. In summary, it shows that the polypeptide protein 220aa

[0088] Example 5 220aa amino acid sequence regulates VM formation in glioma cells

[0089] We constructed U343 cells overexpressing 220aa, and after culturing for 24 hours, we found through a series of experiments that overexpression of 220aa significantly inhibited the migration and VM formation ability of U343 cells, and downregulated the expression level of VEGFR2. Further immunoprecipitation experiments (IP) revealed that there was a significant binding between 220aa and VEGFR2. Figure 5 .

[0090] Figure 5 Figure A in the figure uses Transwell assay to detect the effect of overexpression of 220aa on the migration ability of U343 cells. Figure B uses 3D angiogenesis assay. Values ​​are expressed as mean ± standard deviation (n = 3), **P < 0.01, compared with the 220aa (+) NC group. Scalebar = 50μm. Figure C uses Western blot to detect the effect of overexpression of 220aa on the expression of VM-related protein VEGFR2 in U343 cells. Values ​​are expressed as mean ± standard deviation (n = 3), **P < 0.01 or *P < 0.05, compared with the 220aa (+) NC group. Figure D uses IP assay to detect the binding effect between 220aa and VEGFR2.

[0091] Example 6 220aa regulates VEGFR2 by binding to SUMO protein via the UBC domain

[0092] In order to further explore the mechanism of 220aa binding to VEGFR2 and regulating its expression, the applicant previously analyzed the full-length 220aa sequence through the NCBI database and found that it has only one UBC domain, suggesting that it may play a role by conjugating SUMO protein. The study found that VEGFR2 has two SUMOylation sites, K1110 and K1270, combined with GPS-SUMO prediction, and further verified the binding of VEGFR2 and SUMO1 protein in U343 cells through Co-IP experiments. The results are as follows Figure 6 .

[0093] Co-IP experimental process:

[0094] Note: Please operate the samples on ice or at 4°C.

[0095] 1. Sample Preparation

[0096] 1) Lyse the above cell lines.

[0097] Tip 1: High concentrations of detergents will interfere with immunoprecipitation. Therefore, you can first use a small volume of RIPA (CatNo.PR20001) to lyse cells to prepare protein lysates, and then add 1X PBS (CatNo.PR20023) or incubation solution to dilute to the required volume before starting IP capture.

[0098] Tip 2: Use sufficient protein lysate: For single-tube IP experiments, the recommended initial volume of lysate is 0.2–0.4 ml, containing 1–3 mg of total protein. Bradford or BCA assays can be used to determine total protein concentration.

[0099] Tip 3: Make sure that protease inhibitors (Cat No. PR20016) are added to the lysate. The amount of protease inhibitors can be 1.5-2 times that of normal WB experimental sample preparation.

[0100] Lysate pretreatment: Cut off the end of the sterilized pipette tip at a 45° angle, quickly draw up the resuspended Protein Asepharose beads (Cat No. PR40023) suspension, and add it to the EP tube containing the lysate 1). Usually 1-3 mg of total protein lysate needs to be added with 30 μl of resuspended Protein A sepharose beads suspension.

[0101] 2) Cut off the end of the sterilized pipette tip at a 45° angle to prevent beads from clogging the pipette tip and causing uneven aspiration.

[0102] 3) Incubate at 4°C with rotation for 60 min (vertical rotating mixer, low speed rotation is recommended).

[0103] 4) Centrifuge at 4°C, 500 g for 1 min and transfer the supernatant into a new EP tube.

[0104] Note: For IgG-rich tissues, pretreatment with Protein A beads or Protein G beads (Cat No. PR40024) is recommended.

[0105] Specific steps of co-immunoprecipitation:

[0106] 1) Take 200-400μl of lysate (or pretreatment) containing 1-3mg total protein, add it to an EP tube, add 1-4μg of specific antibody and 150-300μl of incubation solution. The optimal amount of antibody should be determined by the antibody titer. Take an equal amount of protein lysate, add an equal amount of incubation solution and an equal amount of isotype IgG to incubate as the Control IgG control group. Rotate at 4℃ for 2-4h or overnight.

[0107] 2) Then add 50 μl of resuspended Protein A agarose beads and incubate with rotation at 4°C for 1-4 h.

[0108] 3) Centrifuge at 4°C, 500 g for 30 seconds and discard the supernatant.

[0109] 4) Wash the precipitated complex with 1 mL 1×TBST (Cat No. PR20017, now with protease inhibitor (Cat No. PR20016)) each time, centrifuge at 4°C, 500g for 30 seconds, and discard the supernatant; repeat the wash 4-5 times. After the last wash / centrifugation, keep about 80ul of solution in the EP tube (discard the rest by aspiration).

[0110] 5) Add 20 μl 5x SDS Sample Buffer, resuspend the IP complex beads, boil in 95–100°C water bath for 5 min, then centrifuge at 8,000-10,000 g for 3 min, and carefully pipette the supernatant into a new EP tube.

[0111] 3. Western blot analysis, the results are as follows.

[0112] Figure 6 The immunoprecipitation experiment in the paper verifies the binding of VEGFR2 and SUMO1 proteins in U343 cells. GAPDH represents the internal reference protein. It can be observed from the figure that the expression of SUMO1 protein is the highest in the Input group (positive control, which can also be understood as the whole protein group). Compared with the IgG group (negative control), the relative expression of SUMO1 protein in the immunoprecipitation group using VEGFR2 antibody is significantly increased, which illustrates the binding of VEGFR2 and SUMO1 in U343 cells.

[0113] In summary, the study found that the expression of circular RNA circ-UBE2I was downregulated in glioma tissues and cells, and overexpression of circ-UBE2I significantly inhibited the proliferation, migration and VM formation ability of glioma cells, and downregulated the protein expression level of VEGFR2; it was further found that the novel polypeptide protein 220aa, which may be encoded by circ-UBE2IORF, was downregulated in glioma tissues, and the expression of 220aa significantly inhibited the VM formation ability of glioma cells and downregulated the expression of VEGFR2. In addition, the binding ability of 220aa to SUMO protein (SUMO2 / 3) was preliminarily proved, and the molecular mechanism by which 220aa may conjugate SUMO protein through its own UBC domain to promote SUMO modification of VEGFR2 was predicted with the help of bioinformatics software.

[0114] Although the present application has been described with reference to preferred embodiments, various modifications may be made thereto and parts thereof may be replaced with equivalents without departing from the scope of the present application. In particular, the various technical features mentioned in the various embodiments may be combined in any manner as long as there is no structural conflict. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. Use of a circ-UBE2I RNA sequence in the preparation of a drug for inhibiting or treating glioma, characterized in that: The nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.

1.

2. A recombinant vector, characterized in that: The recombinant vector includes a circ-UBE2I RNA sequence, and the nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.

1.

3. A cell line, characterized in that The cell line includes a circ-UBE2I RNA sequence, and the nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.

1.

4. The cell line according to claim 3, characterized in that The cell line expresses a polypeptide encoded by the circ-UBE2I RNA sequence.

5. The cell line according to claim 4, characterized in that The concentration of cells in the cell line is 1×10 5 / 100 ul to 3×10 5 / 100 ul.

6. A drug for inhibiting or treating glioma, characterized in that: The drug includes the cell line according to any one of claims 3 to 5, wherein the cell line contains a circ-UBE2I RNA sequence, and the nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.

1.

7. The drug according to claim 6, characterized in that The medicine comprises 1×10 5 / 100 ul to 3×10 5 / 100 ul of cells, and the cells are derived from the cell line.

8. The drug according to claim 6, characterized in that The cell line expresses a polypeptide encoded by a circ-UBE2I RNA sequence.

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