Polypeptide 220aa and application thereof in medicine for inhibiting or treating glioma cell neoplasia

Abstract

The invention provides polypeptide 220aa and application thereof in inhibiting or treating glioma cell neoplasia, the polypeptide 220aa can be used for inhibiting or treating glioma cell neoplasia, the amino acid sequence of the polypeptide 220aa is as shown in SEQ ID No.1, the polypeptide 220aa can effectively inhibit glioma cells from forming blood vessels to generate vascular mimicry, and the polypeptide 220aa can be used for inhibiting or treating glioma cell neoplasia. 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] This application relates to the field of biomedicine, and particularly to a polypeptide 220aa and its use in the preparation of a drug for inhibiting or treating glioma. Background Art

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

[0003] Due to the structure of VM being different from that of ordinary blood vessels, some current anti-cancer drugs targeting angiogenesis cannot achieve the expected effects. Currently, the clinical treatment of glioma mainly relies on surgery, supplemented by radiotherapy and chemotherapy with temozolomide after surgery. Although these treatment methods can delay the progression of the disease in patients, the median survival time of GBM patients is still less than 14 months. From a histological perspective, glioma is a typical angiogenesis-dependent solid tumor with microvascular proliferation as its pathological feature. In the past decade, anti-angiogenic therapy has been an important adjuvant treatment for high-grade gliomas at home and abroad, and the angiogenesis inhibitor bevacizumab has been applied clinically. However, a series of retrospective studies have found that the effects of the above treatment methods are not satisfactory.

[0004] Therefore, there is an urgent need for an effective treatment method to inhibit or treat it. Summary of the Invention

[0005] This application provides a polypeptide 220aa and its use in the preparation of a drug for inhibiting or treating glioma, which can effectively inhibit glioma cells from forming vasculogenic mimicry, thereby inhibiting the transportation of blood, oxygen, and nutrients in tumor tissues by vasculogenic mimicry, and achieving the effect of inhibiting glioma or improving the survival rate of patients.

[0006] In the first aspect, an embodiment of this application provides a polypeptide 220aa for inhibiting or treating the tumorigenesis of glioma cells, characterized in that the amino acid sequence of the polypeptide 220aa is as shown in SEQ ID No.1.

[0007] Inhibiting the formation of vascular mimicry (VM) in gliomas is an issue that cannot be ignored in the anti-angiogenic comprehensive treatment of gliomas. It is very important to clarify the molecular mechanism regulating VM formation. This application combines omics sequencing and molecular biology techniques to multi-dimensionally explain the molecular mechanism by which the molecular network composed of circ-UBE2I, polypeptide 220aa, and the VM growth-related factor VEGFR2 regulates the formation of VM in glioma cells at the cellular, tissue, and in vivo levels. It is found that it can not only reveal a new mechanism for the formation of VM in glioma cells but also provide new strategies and potential new targets for the comprehensive treatment of anti-glioma angiogenesis.

[0008] In this embodiment, the nucleotide sequence of the circ-UBE2I RNA sequence is shown in SEQ ID No.2. The circ-UBE2I RNA sequence has the ability to encode proteins, can bind to ribosomes, and can encode polypeptide 220aa, and its amino acid sequence is shown in SEQ ID No.1.

[0009] In this embodiment, it is found that circ-UBE2I, which is down-regulated in gliomas, may regulate the expression of VEGFR2 through an indirect pathway and regulate the formation of VM in glioma cells.

[0010] Among them, the vascular endothelial growth factor (VEGF) signaling pathway is a crucial regulatory pathway in the formation of VM in gliomas. Among them, VEGFR2 and VEGFR1 are the most important receptors in this pathway. VEGFR2 is highly expressed in vascular endothelial cells and not only participates in regulating the differentiation, proliferation, and migration of endothelial cells but is also closely related to the process of VM formation. Research has confirmed that VEGFR2 can be activated by VEGF, thereby promoting the activity of the PI3K-AKT pathway, and further promoting the formation of VM in 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.

[0011] Through research, it is found that circ-UBE2I encodes the same low-expressed polypeptide 220aa amino acid sequence. The low-expressed polypeptide 220aa amino acid sequence conjugates SUMO proteins through the UBC domain, regulates the SUMOylation level of VEGFR2, and finally regulates the formation of VM in glioma cells.

[0012] The UBC (Ubiquitin-conjugating enzyme) domain (28aa - 183aa) is the core catalytic domain of the E2 conjugating enzyme in the process of SUMOylation (also known as SUMOylation). SUMOylation is a post-translational modification process in which SUMO proteins (SUMO1 and SUMO2 / 3) are covalently and reversibly bound to substrate proteins under the mediation of E1 activating enzyme, E2 conjugating enzyme and E3 ligase, regulating protein stability and expression, and participating in the occurrence and development of various tumors.

[0013] In a second aspect, an embodiment of the present application provides a recombinant vector, which includes a polypeptide 220aa, and the amino acid sequence of the polypeptide 220aa is as shown in SEQ ID No.1.

[0014] In a third aspect, an embodiment of the present application provides a cell line, which includes a polypeptide 220aa, and the amino acid sequence of the polypeptide 220aa is as shown in SEQ ID No.1.

[0015] In some optional embodiments, the concentration of cells in the cell line is 1×105 / 100ul to 3×105 / 100ul.

[0016] In a fourth aspect, an embodiment of the present application provides a drug for inhibiting or treating glioma, which is characterized in that the drug includes the cell line of the third aspect, and the cell line includes a polypeptide 220aa, and the amino acid sequence of the polypeptide 220aa is as shown in SEQ ID No.1.

[0017] In some optional embodiments, the drug includes 1×105 / 100ul to 3×105 / 100ul of cells, and the cells are derived from the cell line.

[0018] Use of the polypeptide 220aa of the first aspect or the cell line of the third aspect in the preparation of a drug for inhibiting or treating the tumorigenesis of glioma cells.

[0019] This application has at least the following beneficial effects:

[0020] The polypeptide 220aa provided by the present application promotes the SUMOylation of VEGFR2, inhibits its protein stability and expression level, regulates the formation of VM in glioma cells, and thus inhibits or treats glioma. Description of the Drawings

[0021] Figure 1 Shows the identification and expression of CircUBE2I in glioma tissues and cells in the examples.

[0022] Figure 2Shows the effect of CircUBE2I of the embodiment on the VM formation ability of U343 cells.

[0023] Figure 3 Shows the effect of Circ-UBE2I of the embodiment on the expression of VM-related proteins.

[0024] Figure 4 Shows the expression and function of the novel polypeptide protein polypeptide 220aa encoded by Circ-UBE2I of the embodiment in glioma tissues. Detailed implementation manners

[0025] The following further describes in detail the implementation manners of the present application in conjunction with the accompanying drawings and embodiments. The detailed descriptions and drawings of the following embodiments are used to exemplarily 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.

[0026] Embodiment

[0027] The following embodiments more specifically describe the content disclosed in the present application. These embodiments are only for illustrative purposes, because various modifications and changes within the scope of the content disclosed in the present application are obvious to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized according to conventional methods and can be used directly without further treatment, and all instruments used in the embodiments are commercially available.

[0028] Embodiment 1

[0029] Through circular RNA (circRNA) de-linearization sequencing, 24,205 co-expressed circRNAs and 3,117 abnormally expressed circRNAs were screened out from three pairs of glioma tissues and corresponding adjacent non-cancerous tissues derived from glioma patients. Further, by comparing with the circRNA database circBase, 2,739 differentially expressed circRNAs were screened out. Among them, the expression of circ-UBE2I (hsa_circ_0000665) was significantly down-regulated in gliomas. By using Sanger sequencing combined with agarose gel electrophoresis, the existence and specificity of circ-UBE2I in glioma cells were studied. After removing linear RNAs with RNaseR enzyme, qRT-PCR experiments were performed on normal brain tissues (NBTs, n = 3), WHO grade I (n = 4), WHO grade II (n = 3), WHO grade III (n = 5), and WHO grade IV (n = 5) glioma tissues. NBTs, WHO grade I (n = 4), WHO grade II (n = 3), WHO grade III (n = 5), and WHO grade IV represent normal adjacent tissues and glioma tissues of WHO grades I to IV, respectively. It was confirmed that the expression of circ-UBE2I was significantly down-regulated in human glioma tissues and was significantly down-regulated in HA, U251, U343, and A172 cells of glioma tissues. HA, U251, U343, and A172 represent human astrocytes and glioma cell lines, respectively. These down-regulations indicate that the expression of circ-UBE2I was significantly down-regulated in human glioma cells, and the results are as Figure 1 .

[0030] Figure 1 shown in Figure A of

[0031] Example 2: circ-UBE2I regulates the formation of vascular mimicry (VM) in glioma cells

[0032] The circ-UBE2I RNA sequence (hereinafter referred to as circ-UBE2I) was transfected into a vector and introduced into U343 cells, a type of glioma cell, through the vector. The cells were cultured to construct U343 cells with overexpressed circ-UBE2I.

[0033] The culture steps of U343 cells included seeding U343 cells into a 96-well plate at a density of 20,000 cells / well and continuing the culture. At the detection time point for each group of U343 cells, 1 / 10 volume of CCK-8 reagent was added to each well and cultured in a 37°C, 5% CO 2 incubator for 3 hours; the wavelength of 450 nm was selected, and the light absorption value of each well was measured on an enzyme-linked immunosorbent detector, and the results were recorded. A line graph was plotted with time as the abscissa and OD value as the ordinate to determine the subsequent density of U343 cells, such as the cell density during the CCK-8 experiment.

[0034] The angiogenesis experiment was carried out using a commercially available CCK-8 kit and a commercially available Transwell kit. The cell density during the CCK-8 experiment was 20,000 cells / well.

[0035] The cell density for the Transwell experiment was 0.5 - 2.0 x 10^6 / ml. Its experimental steps included:

[0036] 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.

[0037] 2. The cells were resuspended with serum-free medium and the cell density was adjusted to 0.5 - 2.0 x 10^6 / ml.

[0038] 3. 200 μL of the above cell suspension was added to the upper chamber, and 900 μL of medium containing 10% FBS was added to the lower chamber (culture plate).

[0039] 4. Incubate at 37°C for 24 hours.

[0040] 5. After incubation, carefully remove the cell chamber, aspirate the culture medium on the upper layer of the chamber, wash it once with PBS, fix it with 70% alcohol or 4% paraformaldehyde for 5 min, and stain it with 0.5% crystal violet (dissolved in 2% ethanol or PBS) for 20 min.

[0041] 6. Wash twice with PBS to remove the 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 for counting and calculate the average value.

[0042] Vascular mimicry formation experiment: Initial U343 cell density: 6×10 4 / ml The steps include: Cover a 96-well plate with 60 μl of Matrigel basement membrane matrix (BD Bioscience, MA, USA) before the experiment.

[0043] Resuspend U343 cells in 100 μl of FBS-free medium, take 100 μl of the cell suspension at a concentration of 6×10 4 cells / ml, and seed it on a 96-well culture plate. Incubate at 37 °C for 8 h, and capture the number of blood vessels formed under an inverted microscope (Olympus, Tokyo, Japan).

[0044] The study found that the expression of circ-UBE2I significantly inhibited the proliferation, migration and VM formation ability of U343 cells, and the results were as Figure 2 .

[0045] Figure 2 shown in Figure A of. CCK-8 experiments were performed on the cells of the CircUBE2I(+) NC group and the CircUBE2I(+) group respectively. The experimental results found that overexpression of the circUBE2I RNA sequence had a significant inhibitory effect on the proliferation ability of U343 cells; Figure B used the Transwell experiment to detect the effect of overexpression of circUBE2I on the migration ability of U343 cells, and found that overexpression of circUBE2I significantly inhibited the migration ability of U343 cells. Figure C used the angiogenesis experiment to detect the effect of overexpression of circUBE2I on the VM formation ability of U343 cells, indicating that overexpression of circUBE2I significantly inhibited the number of vascular mimics formed by U343 cells. The values are expressed as mean ± standard deviation (n = 3), **P < 0.01, compared with the CircUBE2I(+) NC group. Scale bar = 50 μm. Through research, it was found that the circUBE2I RNA sequence also had a comparable effect on U251 and A172 cells.

[0046] The above results indicate that the polypeptide 220aa expressed by the circ-UBE2I RNA sequence can significantly inhibit the biological behavior of glioma malignant cells, such as the poor ability to transport oxygen and water, etc., and has a positive effect on inhibiting glioma in organisms.

[0047] Example 3 circ-UBE2I alters the expression of VEGFR2 protein

[0048] To further understand the mechanism by which circ-UBE2I regulates VM formation in U343 cells, the effect of overexpressing circ-UBE2I on the protein expression of VEGFR2 in U343 cells was detected. The experiment confirmed that the protein expression of VEGFR2 in U343 cells was significantly downregulated by expressing circ-UBE2I.

[0049] 2. Add 1 ml of trypsin for digestion. After complete digestion, add 2 ml of complete medium to terminate the digestion.

[0050] 3. Centrifuge at 1200 rpm for 1 min and discard the supernatant.

[0051] 4. After cell counting, add RIPA according to the ratio of 1x10 7 cells / 500 μl of RIPA lysis buffer.

[0052] 5. Add 10 μl of PMSF, phosphatase inhibitor, and protease inhibitor to each ml of RIPA lysis buffer, invert and lyse for 2 h, centrifuge at 12000 rpm for 15 min, take the supernatant, and store at -20 °C.

[0053] (2) 1. Label the 1.5 ml EP tubes required for the experiment. The samples include the target sample, negative control, and positive control.

[0054] 2. Pipette 50 μl of the resuspended magnetic bead suspension into each 1.5 ml EP.

[0055] 3. Add 500 μl of RIP WashBuffer to each tube and vortex.

[0056] 4. Place the 1.5 ml EP tubes on the magnetic stand. Wait for the solution to clarify and discard the supernatant. Repeat once.

[0057] 5. Resuspend the magnetic beads with 500 μl of RIP Washbuffer, add 5 μg of the corresponding antibody (IgG antibody, VEGFR2 antibody) to each sample, and incubate at 4 °C for 4 h.

[0058] 6. Place the 1.5 ml EP tubes on the magnetic stand and discard the supernatant.

[0059] 7. Add 500 μl of RIP Wash Buffer, vortex and discard the supernatant. Repeat once.

[0060] 8. Add RIP Wash Buffer, vortex and place on ice.

[0061] (3) Binding of magnetic bead-antibody complex to protein

[0062] 1. Place the EP tubes from the previous step on the magnetic stand, remove the supernatant, and add 900 μl of RIP Immunoprecipitation Buffer to each tube.

[0063] 2. Quickly thaw the cell lysate prepared in the first step, centrifuge at 12,000 rpm for 10 min at 4°C. Pipette 100 μl of the supernatant into the magnetic bead - antibody complex from the previous step to make the total volume 1 ml. Incubate overnight at 4°C.

[0064] 3. After incubation, briefly centrifuge, place the EP tubes on the magnetic stand, wait for the solution to clarify, remove the supernatant, add 500 μl of RIP Wash Buffer, vortex, then place the EP tubes on the magnetic stand again, remove the supernatant, and repeat 5 times for a total of 6 washes.

[0065] (IV) RNA Elution

[0066] 1. Resuspend the above magnetic bead - antibody complex with 150 μl of Proteinase K Buffer and incubate at 55°C for 30 min.

[0067] 2. After incubation, place the EP tubes on the magnetic stand, pipette the supernatant into a new 1.5 - ml EP tube, and add 250 μl of RIP Wash Buffer to each tube.

[0068] 3. Then add 400 μl of phenol:chloroform:isoamyl alcohol (125:24:1), vortex for 15 s, and centrifuge at 12,000 rpm for 10 min at room temperature.

[0069] 4. Carefully pipette the upper aqueous phase into another new 1.5 - ml EP tube, add 400 μl of chloroform to each tube, vortex for 15 s, and centrifuge at 12,000 rpm for 10 min at room temperature.

[0070] 5. Carefully pipette the upper aqueous phase into another new 1.5 - ml EP tube, add 850 μl of anhydrous ethanol (RNase - free) to each tube, mix well, and precipitate overnight at - 20°C.

[0071] 6. After incubation, centrifuge at 12,000 rpm for 15 min at 4°C, and carefully remove the supernatant.

[0072] 7. Add 900 μl of 80% ethanol, centrifuge at 12,000 rpm for 15 min at 4°C, carefully remove the supernatant, open the lid, let it stand for 3 min, and air - dry.

[0073] 8. Add 10 μl of DEPC water for dissolution and store at -80 °C. RNA immunoprecipitation confirmed that, as observed from the figure: The circUBE2I expression was the highest in the Input group (positive control, total RNA group); meanwhile, compared with the IgG group (negative control), there was no significant difference in the relative expression level of circUBE2I in the immunoprecipitation group using the VEGFR2 antibody (P>0.05), indicating that there was no binding interaction between circ-UBE2I and VEGFR2. The results are as Figure 3 shown.

[0074] Figure 3 Figure A in

[0075] detected the effect of overexpressed circ-UBE2I on the expression of VEGFR2 protein in U343 cells by Western blot assay. The values are expressed as mean ± standard deviation (n = 3), **P<0.01, compared with the circ-UBE2I(+)NC group. (B) RNA immunoprecipitation confirmed the detection of the binding interaction between circ-UBE2I and VEGFR2.

[0076] To deeply study the mechanism of circ-UBE2I regulating the formation of VM in U343 cells, in the early stage of the project, by searching the circRNADb database and TransCirc software, it was found that circ-UBE2I had a potential ORF and two IRES sequences and might translate a polypeptide protein with a length of 220 amino acids, polypeptide 220aa. By analyzing the amino acid sequence encoded by the circRNA circularization site, a specific antibody for detecting polypeptide 220aa was prepared in the early stage. Western blot assay found that polypeptide 220aa was significantly lowly expressed in glioma tissues. The results are as Figure 4 .

[0077] The database CircRNADb and the software TransCirc found that circUBE2I contained an ORF and two IRESs and encoded a polypeptide 220aa with a length of 220 amino acids.

[0078] Figure 4Among them, in Figure A, it was found that the expression of 220aa was significantly lower in gliomas than in normal tissues. T is the expression level of the polypeptide 220aa in tumor tissues, N is the expression level of the polypeptide 220aa in normal tissues, T1, T2, T3, and T4 are multiple groups of parallel experimental groups, and N1, N2, N3, and N4 are multiple groups of parallel experimental groups. In Figure B, the Blank group represents the blank group, that is, no vector was introduced into this cell line. The cirUBE2I-ΔATG group was the cell line into which an empty vector without the cirUBE2I RNA sequence was introduced, and other experimental conditions were the same as those of the cirUBE2I-220aa group. Experiments were conducted on the U343 and U252 glioma cell lines in Figure B, and it was found that overexpression of 220aa significantly inhibited the cell proliferation ability of glioma cells, indicating that the cirUBE2I-220aa group, that is, the expression of the polypeptide 220aa in the cell line was beneficial to inhibiting the cell proliferation of glioma cells. In Figure C, the cell migration conditions and the number of migrating cells of the Blank group, cirUBE2I-ΔATG group, and cirUBE2I-220aa group in the U343 and U252 glioma cell lines were shown. It was found that the cirUBE2I-220aa group, that is, overexpression of 220aa significantly inhibited the migration and invasion abilities of glioma cells. In Figure D, the conditions of vasculogenic mimicry of the Blank group, cirUBE2I-ΔATG group, and cirUBE2I-220aa group in the U343 and U252 glioma cell lines were shown, showing that overexpression of 220aa in the cirUBE2I-220aa group reduced the formation of vasculogenic mimicry. In addition, Figure E showed that overexpression of 220aa significantly inhibited the protein expression levels of VEGFR2 and VE-cadherin. Among them, GAPDH was the content of the internal reference protein. VEGFR2 is the main receptor of vascular endothelial growth factor. In the tumor microenvironment, VEGF promotes the proliferation, migration, and neovascularization of vascular endothelial cells by binding to VEGFR2. High expression of VEGFR2 is usually associated with high-grade gliomas (such as glioblastoma, GBM), indicating stronger tumor invasiveness and poorer prognosis. VE-cadherin is a key protein in the intercellular junctions of vascular endothelial cells and participates in regulating the endothelial barrier function. In gliomas, its abnormal expression may lead to the disruption of the blood-brain barrier and an increase in tumor vascular permeability. VE-cadherin may participate in the increase in tumor vascular permeability in the tumor microenvironment, promoting the migration of tumor cells through blood vessels to distant organs to form metastases. Therefore, overexpression of 220aa in the cirUBE2I-220aa group significantly inhibited the protein expression levels of VEGFR2 and VE-cadherin, which was beneficial to inhibiting or treating gliomas.

[0079] In summary, it was found in the study that the circular RNA circ-UBE2I was down-regulated in glioma tissues and cells. Overexpression of circUBE2I significantly inhibited the proliferation, migration and VM formation ability of glioma cells, and down-regulated the protein expression level of VEGFR2. Further, it was found that the novel polypeptide protein polypeptide 220aa, which may be encoded by circ-UBE2I ORF, was down-regulated in glioma tissues. Expression of polypeptide 220aa significantly inhibited the VM formation ability of glioma cells and down-regulated the expression of VEGFR2. In addition, the binding ability of polypeptide 220aa to SUMO proteins (SUMO2 / 3) was preliminarily demonstrated, and bioinformatics software was used to predict that polypeptide 220aa may conjugate SUMO proteins through its own UBC domain, promoting the molecular mechanism of SUMOylation modification of VEGFR2.

[0080] Although the present application has been described with reference to the preferred embodiments, various modifications can be made to it and components thereof can be replaced with equivalents without departing from the scope of the present application. In particular, as long as there is no structural conflict, the technical features mentioned in each embodiment can be combined in any way. The present application is not limited to the specific embodiments disclosed in the text, but includes all technical solutions falling within the scope of the claims.

Claims

1. A polypeptide 220aa for inhibiting or treating glioma cell tumorigenesis, characterized in that: The amino acid sequence of the polypeptide 220aa is shown in SEQ ID No.

1.

2. A recombinant vector, characterized in that: The recombinant vector comprises polypeptide 220aa, and the amino acid sequence of the polypeptide 220aa is shown as SEQ ID No.

1.

3. A cell line, characterized in that The cell line comprises polypeptide 220aa, and the amino acid sequence of the polypeptide 220aa is shown as SEQ ID No.

1.

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

5. A drug for inhibiting or treating glioma, characterized in that: The drug includes the cell line according to claim 3 or 4, and the cell line includes polypeptide 220aa, and the amino acid sequence of the polypeptide 220aa is shown in SEQ ID No.

1.

6. The drug according to claim 5, 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.

7. Use of the polypeptide 220aa according to claim 1 or the cell line according to claim 3 or 4 in the preparation of a drug for inhibiting or treating glioma cell tumorigenesis.

Images