A computer-assisted drug screening method, system, computer equipment and storage medium for screening new anti-colorectal cancer drugs

Abstract

The present invention discloses a computer-assisted drug screening method, system, computer equipment and storage medium for screening new anti-colorectal cancer drugs. The present invention innovatively provides a method, system, computer equipment and storage medium for screening new anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 in the field. The present invention provides an efficient, accurate and rapid screening method for the research and development of new anti-colorectal cancer drugs, and has broad application prospects in the screening of new anti-colorectal cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of computer-assisted drug screening, and specifically, relates to a computer-assisted drug screening method, system, computer equipment and storage medium for screening new anti-colorectal cancer drugs. More specifically, it relates to a screening method, system, computer equipment and storage medium for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276. Background Art

[0002] Globally, colorectal cancer (CRC) is the third most common type of cancer in both men and women, and its mortality rate ranks among the top three. With the continued development of developing countries, the global incidence of colorectal cancer is projected to increase to 2.5 million new cases by 2035. The pathogenesis and pathological progression of CRC involve a series of genetic and epigenetic changes and are influenced by the tumor immune microenvironment (TIME). Although genetic, lifestyle, obesity, and environmental factors are all associated with CRC, the precise causes of the increasing incidence and mortality are still unclear. With the advancement of biomedical and oncological research, the molecular mechanisms of CRC and therapies targeting specific molecular targets are constantly being discovered. Exploring the molecular mechanisms of CRC and developing innovative therapeutic strategies targeting specific molecular targets to prevent the malignant progression of CRC patients are gaining increasing attention.

[0003] In mammals, PRMTs are divided into three categories based on the form of arginine methylation modification. Type I, including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8, catalyzes the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA). Type II, including PRMT5 and PRMT9, catalyzes the formation of MMA and symmetric dimethylarginine (SDMA). Type III, including PRMT7, is known only to catalyze the formation of MMA. Protein arginine methyltransferase 5 (PRMT5) is the primary methyltransferase catalyzing SDMA. PRMT5-mediated symmetric dimethylation of proteins influences numerous biological processes, such as epigenetic control of gene expression, splicing regulation, circadian rhythms, DNA damage responses, and germ cell development and pluripotency. Compared to the PRMT5 homodimer, the PRMT5 / MEP50 complex exhibits a higher affinity for SAM and target substrates, resulting in a higher methylation activity of the heterooctameric PRMT5 / MEP50 complex.

[0004] The tumor immune microenvironment is the interplay between tumor cells and tumor-infiltrating immune cells. CD276, a member of the B7 family of immune checkpoint proteins, is highly expressed on tumor cells and activated tumor-infiltrating immune cells, helping them evade surveillance by cytotoxic T cells and natural killer (NK) cells. It is closely linked to the initiation, progression, and response to tumor immunotherapy. It is considered an immune co-stimulatory or inhibitory factor that regulates the interaction between tumors and the immune system. Initial studies identified CD276 as a T cell-stimulatory protein that binds to an unknown counter-receptor, thereby increasing IFN-γ production during T cell activation. However, increasing research has revealed that CD276 negatively regulates T cell immune responses in humans and mice by inhibiting T cell activation. Overexpression of CD276 can promote tumor progression through both immune and non-immune mechanisms. Studies have consistently shown that elevated CD276 expression can activate pathways such as JAK / STAT3, PI3K / AKT / mTOR, and TLR4 / NF-κB, and promote the expression of IL-8, MMP2 / 9, and STAT3. The immunosuppressive role of CD276 in the tumor microenvironment makes it an attractive immunotherapeutic target for cancer treatment.

[0005] Colorectal cancer is one of the most common cancers with the highest morbidity and mortality rates worldwide. Existing treatments (such as surgery, chemotherapy, and radiotherapy) have limited effectiveness in some colorectal cancer patients and are often accompanied by serious side effects. The development of new drugs can target specific colorectal cancer cells, improving the effectiveness of treatment and reducing damage to normal cells. Therefore, there is an urgent need in this field to develop an efficient screening method for new anti-colorectal cancer drugs. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a computer-assisted drug screening method, system, computer equipment and storage medium for screening new anti-colorectal cancer drugs in the field.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, the screening method comprising:

[0009] Obtain PRMT5 protein and ALKBH5 protein data, or PRMT5 protein and CD276 protein data;

[0010] Selecting the spatial structure of the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the PRMT5 protein and CD276 protein complex, and determining the binding site of the PRMT5 protein and ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and CD276 protein in the PRMT5 protein and CD276 protein complex as a binding site for a targeted drug; and using a computer-assisted drug screening method to obtain a candidate drug targeting the binding site;

[0011] The computer-assisted drug screening method comprises:

[0012] Obtaining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex;

[0013] Based on the spatial structure of the binding site between the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the binding site between the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex, screening a small molecule compound with a similar structure in a molecular database;

[0014] Molecular docking is performed on the screened small molecule compounds and ALKBH5 protein or CD276 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor ALKBH5 protein or CD276 protein to obtain a score for each small molecule compound, and the compounds are ranked according to the scores to obtain candidate anti-colorectal cancer drugs.

[0015] Furthermore, the method for determining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex comprises:

[0016] Construct n truncated expression mutants of PRMT5, where n is a natural number greater than or equal to 1;

[0017] Based on the n truncated expression mutants, immunoprecipitation experiments were performed to obtain the key regions for the interaction between PRMT5 and ALKBH5 or CD276;

[0018] The spatial structure of the key region for the interaction between PRMT5 and ALKBH5 is the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex;

[0019] The spatial structure of the key region for the interaction between PRMT5 and CD276 is the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex.

[0020] Furthermore, the screening method further comprises:

[0021] Obtaining colorectal cancer cell models and / or colorectal cancer animal models;

[0022] The colorectal cancer cell model and / or colorectal cancer animal model is treated with the screened anti-colorectal cancer candidate drug to verify the effectiveness of the anti-colorectal cancer candidate drug.

[0023] In a second aspect, the present invention provides a screening system for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, the screening system comprising:

[0024] A data acquisition unit, which acquires data of PRMT5 protein and ALKBH5 protein, or data of PRMT5 protein and CD276 protein;

[0025] a site determination unit, which selects the spatial structure of the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the PRMT5 protein and CD276 protein complex, and determines the binding site of the PRMT5 protein and ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and CD276 protein in the PRMT5 protein and CD276 protein complex as the binding site of the targeted drug;

[0026] a drug screening unit, which uses a computer-assisted drug screening method to obtain candidate drugs targeting the binding site;

[0027] The computer-assisted drug screening method comprises:

[0028] Obtaining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex;

[0029] Based on the spatial structure of the binding site between the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the binding site between the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex, screening a small molecule compound with a similar structure in a molecular database;

[0030] Molecular docking is performed on the screened small molecule compounds and ALKBH5 protein or CD276 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor ALKBH5 protein or CD276 protein to obtain a score for each small molecule compound, and the compounds are ranked according to the scores to obtain candidate anti-colorectal cancer drugs.

[0031] Furthermore, the method for determining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex comprises:

[0032] Construct n truncated expression mutants of PRMT5, where n is a natural number greater than or equal to 1;

[0033] Based on the n truncated expression mutants, immunoprecipitation experiments were performed to obtain the key regions for the interaction between PRMT5 and ALKBH5 or CD276;

[0034] The spatial structure of the key region for the interaction between PRMT5 and ALKBH5 is the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex;

[0035] The spatial structure of the key region for the interaction between PRMT5 and CD276 is the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex.

[0036] Furthermore, the screening system further comprises:

[0037] The drug effectiveness verification unit obtains a colorectal cancer cell model and / or a colorectal cancer animal model; and uses the screened anti-colorectal cancer candidate drug to treat the colorectal cancer cell model and / or the colorectal cancer animal model to verify the effectiveness of the anti-colorectal cancer candidate drug.

[0038] In a third aspect, the present invention provides a computer device for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, the computer device comprising:

[0039] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the screening method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 as described in the first aspect of the present invention is implemented.

[0040] In a fourth aspect, the present invention provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 as described in the first aspect of the present invention.

[0041] In a fifth aspect, the present invention provides an anti-colorectal cancer candidate drug obtained by screening the anti-colorectal cancer drug screening method based on PRMT5 and ALKBH5, or PRMT5 and CD276 according to the first aspect of the present invention.

[0042] In a sixth aspect, the present invention provides any of the following applications:

[0043] (1) Use of PRMT5 inhibitors in the preparation of drugs for the treatment of colorectal cancer;

[0044] (2) Use of a PRMT5 inhibitor and an anti-PD1 antibody in combination for the preparation of a drug for the treatment of colorectal cancer;

[0045] (3) Use of CD276 inhibitors in the preparation of drugs for the treatment of colorectal cancer;

[0046] (4) Use of a reagent for inhibiting the methylation level of the ALKBH5-R316 methylation site in the preparation of a drug for treating colorectal cancer;

[0047] (5) Use of a reagent for detecting the methylation level of the ALKBH5-R316 methylation site in the preparation of a diagnostic product or a predictive product for diagnosing colorectal cancer or predicting the prognosis of colorectal cancer.

[0048] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0049] The present invention innovatively provides a novel screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, in the field. The present invention provides an efficient, accurate and rapid screening method for the research and development of new anti-colorectal cancer drugs, and has broad application prospects in the screening of new anti-colorectal cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0051] Figure 1: A schematic flow chart of a screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, provided in an embodiment of the present invention;

[0052] Figure 2 : Schematic diagram of a screening system for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, provided in an embodiment of the present invention;

[0053] Figure 3 : Schematic diagram of a computer device for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, provided in an embodiment of the present invention;

[0054] Figure 4 : PRMT5 affects the stability of ALKBH5 protein, among which, AC figure: Western blot and qRT-PCR experiments to detect the effect of GSK595 on the expression and transcription level of related m6A methyltransferase proteins in colorectal cancer cells HCT116, DLD1 and mouse colorectal cancer cells MC38; DG figure: Western blot and qRT-PCR experiments to detect the expression of ALKBH5 protein and mRNA; HI figure: Western blot experiment to detect the change of ALKBH5 protein half-life after treatment with protein synthesis inhibitor CHX (50μg / mL) by GSK595; JK figure: Western Blot analysis detected the effect of PRMT5 knockdown on the half-life of ALKBH5 protein after treatment with the protein synthesis inhibitor CHX (50 μg / mL) (***P<0.001, **P<0.01, *P<0.05); LM (Figure 1): Western blot analysis detected the effect of the proteasome inhibitor MG132 (10 μM / mL) on ALKBH5 protein degradation in HCT116 and MC38 cells overexpressing PRMT5 after 6 hours of treatment; NP (Figure 2): Co-IP and Western blot analysis detected the effects of PRMT5 expression and enzyme activity on the Ub ubiquitination level of ALKBH5.

[0055] Figure 5: PRMT5-mediated ALKBH5-R316 methylation enhances ALKBH5 ubiquitination and degradation, among which, AB: IP experiments detect the interaction between endogenous PRMT5 and ALKBH5; CD: Co-IP experiments detect the interaction between exogenous Flag-ALKBH5 and Myc-PRMT5; EF: IP experiments detect the MMA and SDMA levels of endogenous ALKBH5; G: GST pull-down experiments detect the binding of PRMT5 to ALKBH5 in vitro; H: Exomethylation experiments detect that PRMT5 directly mediates ALKBH5 methylation in vitro; IK: Western Blot analysis of the effects of PRMT5 expression and its enzyme activity on the SDMA modification of ALKBH5; Panel L: Co-IP assay to detect the SDMA methylation levels of various ALKBH5 mutants; Panel M: Mass spectrometry analysis of R316-ALKBH5 methylation; Panel N: Sequence alignment of ALKBH5 in mammals, with the ALKBH5-R316 site located in the protein sequence; Panel O: In vitro methylation assay to detect the SDMA of GST-ALKBH5-WT (292-395AA) and GST-ALKBH5-R316K (292-395AA); Panel PQ: Western blot analysis of the changes in the half-life of ALKBH5 protein after treatment with CHX (50 μg / mL) in HCT116 and MC38 cells overexpressing wild-type or mutant ALKBH5; Panel RS: Co-IP and Western blot analysis of the SDMA methylation levels of various ALKBH5 mutants; Panel B: Co-IP and Western blot analysis of the SDMA methylation levels of various ALKBH5 mutants; Panel C: Co-IP and Western blot analysis of the SDMA methylation levels of various ALKBH5 mutants; Panel D ... Blot analysis of the effects of ALKBH5 wild-type or mutant on the ubiquitination of ALKBH5 protein (***P<0.001, **P<0.01, *P<0.05);

[0056] Figure 6: PRMT5-mediated ALKBH5-R316 methylation enhances the binding of TRIM28 to ALKBH5 and promotes the ubiquitination and degradation of ALKBH5. AB: IP experiments detect the interaction between endogenous ALKBH5 and TRIM28; CD: Co-IP experiments detect the interaction between exogenous ALKBH5 and TRIM28; EF: Western blot and qRT-PCR experiments detect the expression of ALKBH5 protein and mRNA; GH: Western blot experiments detect the effect of TRIM28 on the protein half-life of ALKBH5 after CHX (50 μg / mL) treatment; IJ: Western blot experiments detect the effect of MG132 (10 μM / mL) treatment on the degradation of ALKBH5 protein in HCT116 and MC38 cells overexpressing TRIM28 after 6 hours (***P<0.001, **P<0.01, *P<0.05); K: Co-IP and Western Blot analysis of the effect of TRIM28 on the Ub ubiquitination level of ALKBH5; LN Figure: Co-IP and Western blot experiments to detect the effect of PRMT5-mediated ALKBH5 methylation on the binding of TRIM28 and ALKBH5; O Figure: Co-IP and Western blot experiments to detect the effect of PRMT5 on the binding of TRIM28 and ALKBH5 through methylation modification of ALKBH5-R316;

[0057] Figure 7 : PRMT5-mediated meR316-ALKBH5 enhances CD276 mRNA stability through m6A modification; Figure A: Distribution of genes with significant changes in m6A levels and gene expression levels in HCT116 cells treated with GSK595 compared with control cells; Figure B: Schematic diagram of screening analysis of regulated downstream genes; Figure C: Correlation between PRMT5 expression and CD276, DNAH17-AS1, POTEI, and SDCBP2-AS1 analyzed by TCGA database; Figure D: Pearson correlation coefficient test for the correlation between PRMT5 and CD276 protein expression levels; Figures EH: Western blots and qRT-PCR detection of CD276 protein and mRNA expression; Figures IJ: Effect of PRMT5 expression level or enzyme activity on CD276 Effect of mRNA abundance on CD276 expression (***P<0.001, **P<0.01, *P<0.05); Figure K: Peak distribution of mA in CD276 mRNA, mainly concentrated in CD276-3'UTR; Figure L: Schematic diagram of mA site mutation in CD276-3'UTR; Figure M: Relative luciferase activity of ALKBH5-overexpressing cells treated with wild-type or mutant CD276-'UTR reporter plasmids;

[0058] Figure 8 : ALKBH5-R316 methylation sites affect tumor immune evasion in colorectal cancer cells. Figure A: TIMER2.0 database analysis showed that ALKBH5 expression was positively correlated with CD8+ T cells, CD4+ T cells, and NK cells; Figure B: RTCA results showed that ALKBH5-R316K cells could resist anti-tumor T cell killing (***P<0.001, **P<0.01, *P<0.05); Figure C: Tumor dissection 3 weeks after subcutaneous injection of Vector / Alkbh5-WT / Alkbh5-R316K cells. Images; D: Tumor growth curves of each group; E: Tumor weight statistics of each group (***P<0.001, **P<0.01, *P<0.05); F: IHC staining of ALKBH5-R316 methylation, Ki-67, CD3, CD8a, and GZMB in subcutaneous tumor tissue sections; G: IHC staining scores of ALKBH5-R316 methylation, Ki-67, CD3, CD8a, and GZMB in subcutaneous tumor tissue sections (***P<0.001, **P<0.01, *P<0.05);

[0059] Figure 9 : PRMT5-mediated ALKBH5-R316 methylation is associated with poor prognosis in patients with colorectal cancer. Figure A: IHC staining of PRMT5 and ALKBH5-R316 methylation in colorectal cancer tissue microarray; Figure B: Pearson correlation coefficient test for the correlation between PRMT5 and ALKBH5-R316 methylation; Figure C: χ 2 The correlation between high ALKBH5-R316 methylation expression and PRMT5 expression, tumor size, and the presence or absence of lymph node metastasis was analyzed (***P<0.001, **P<0.01, *P<0.05); Figure F: IHC staining of meR316-ALKBH5 in colorectal cancer tissue microarray; Figure GH: Kaplan-Meier survival analysis showed that high ALKBH5-R316 methylation expression was positively correlated with the 5-year overall survival rate of colorectal cancer (P<0.001, log-rank test) and the 5-year disease-free survival rate of colorectal cancer patients (P<0.001, log-rank test);

[0060] Figure 10: The combination of PRMT5 inhibitor and anti-PD1 antibody significantly inhibited the progression of colorectal cancer. Among them, Figures AB: RTCA results showed that the reduction of PRMT5 expression level or enzyme activity promoted the killing effect of anti-tumor T cells (***P<0.001, **P<0.01, *P<0.05); Figure C: Schematic diagram of the mouse subcutaneous tumor treatment model; Figure D: Tumor anatomical image of the mouse subcutaneous tumor treatment model 2 weeks after treatment; Figure E: Tumor growth curves of each group; Figure F: Tumor weight statistics of each group ( ***P<0.001, **P<0.01, *P<0.05); G: IHC staining of ALKBH5-R316 methylation, Ki-67, CD3, CD8a and GZMB in tissue sections of subcutaneous tumors, and IHC staining scores of ALKBH5-R316 methylation, Ki-67, CD3, CD8a and GZMB in tissue sections of subcutaneous tumors (***P<0.001, **P<0.01, *P<0.05). DETAILED DESCRIPTION

[0061] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0062] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations are included in a specific order. However, it should be clearly understood that these operations may not be performed in the order in which they are presented herein or may be performed in parallel. The sequence numbers of the operations, such as S101, S102, etc., are merely used to distinguish different operations and do not represent any order of execution. In addition, these processes may include more or fewer operations, and these operations may be performed in sequence or in parallel.

[0063] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0064] Figure 1 1 is a flow chart of a screening method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, provided in an embodiment of the present invention. Specifically, the screening method comprises the following steps:

[0065] S101: Obtain PRMT5 protein and ALKBH5 protein data, or PRMT5 protein and CD276 protein data;

[0066] In one embodiment, the PRMT5 refers to protein arginase methyltransferase 5 (PRMT5), a type II arginine methyltransferase, which is an epigenetic target with great clinical potential. It regulates many physiological functions in mammalian cells by symmetrically dimethylating arginine residues on histones and non-histone proteins. The PRMT5 (protein arginine methyltransferase 5 [Homo sapiens (human)]) has a Gene ID of 10419 in NCBI, and its corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0067] In one embodiment, ALKBH5 refers to human demethylase Alk B homolog 5 (AlkB homolog 5), an RNA demethylase that catalyzes the reversal of RNA methylation, i.e., removing methylation from mRNA, thereby stabilizing mRNA and enhancing DNA repair. The NCBI Gene ID for ALKBH5 (alkB homolog 5, RNA demethylase [Homo sapiens (human)]) is 54890, and detailed information (including sequence information) is available at https: / / www.ncbi.nlm.nih.gov / gene / .

[0068] In one embodiment, the CD276 (also known as B7-H3) is a type I transmembrane glycoprotein and a member of the B7 superfamily. It is not expressed in monocytes, granulocytes, and normal human tissues, but is expressed on some dendritic cells, T cells, natural killer (NK) cells, and B cells. It is a newly discovered immune checkpoint in recent years. This target can not only promote the immune escape of tumor cells by suppressing the immune system, but also mediate tumor metastasis, drug resistance, and angiogenesis through some non-immune pathways, thereby promoting cancer progression. The CD276 (CD276 molecule [Homo sapiens (human)]) has a Gene ID of 80381 in NCBI, and its corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0069] In one embodiment, the present invention demonstrates that PRMT5 affects the stability of ALKBH5 protein, specifically as follows:

[0070] First, the present invention demonstrates that PRMT5 regulates the m6A modification level in colorectal cancer cells through ALKBH5. Since m6A modification is mainly catalyzed by writers (methyltransferases): METTL3, METTL14, and METTL16, and demethylated by erasers (demethyltransferases): FTO and ALKBH5, Western blot was used to detect the effect of the PRMT5 inhibitor GSK595 on the expression levels of m6A methylation modification-related enzymes in colorectal cancer cells. The results showed that the METTL3, METTL14, METTL16, and FTO protein levels in colorectal cancer cells HCT116, DLD1, and mouse colon cancer cells MC38 were not affected after treatment with GSK595 ( Figure 4 Figure A), however, ALKBH5 protein levels were significantly increased ( Figure 4 ), qRT-PCR experiments further revealed that ALKBH5 mRNA levels in colorectal cancer cells HCT116, DLD1, and mouse colon cancer cells MC38 were not affected by GSK595 treatment ( Figure 4 Figure C in the figure).

[0071] Secondly, the present invention demonstrates that changes in PRMT5 expression levels affect ALKBH5 protein levels. The above experiments show that changes in PRMT5 enzyme activity can affect ALKBH5 protein levels. We further examined whether changes in PRMT5 expression levels in colorectal cancer cells HCT116, DLD1, and mouse colorectal cancer cells MC38 affect ALKBH5 protein levels. Western blot and qRT-PCR results showed that overexpression of PRMT5 can reduce ALKBH5 protein levels in colorectal cancer cells HCT116, DLD1, and mouse colorectal cancer cells MC38 ( Figure 4 D in Figure ), while the transcription level did not change significantly ( Figure 4 Figure E in the figure), knockdown of PRMT5 promoted the increase of ALKBH5 protein level ( Figure 4 F in Figure ), while the transcription level also did not change significantly ( Figure 4 Figure G in Figure 1).

[0072] Again, the present invention proves that changes in PRMT5 enzyme activity or expression levels affect the stability of ALKBH5 protein. There are many types of protein degradation pathways, among which the most important pathway is degradation through the ubiquitin-proteasome pathway. In order to further study the effect of PRMT5 enzyme activity and PRMT5 expression level on the half-life of ALKBH5 protein, the present invention uses colorectal cancer cells HCT116 and mouse colorectal cancer cells MC38 treated with GSK595 and stable colorectal cancer cells HCT116 and mouse colorectal cancer cells MC38 with knockdown of PRMT5. After treatment with CHX (actinomycin, inhibiting protein synthesis), the results of Western Blot experiments showed that in HCT116 and MC38 cells treated with the PRMT5 inhibitor GSK595, the half-life of ALKBH5 was significantly prolonged compared with the control group ( Figure 4 Figure H in Figure 4 Figure I). In HCT116 and MC38 cells with PRMT5 interference, the half-life of ALKBH5 was significantly prolonged compared with the control group ( Figure 4 Figure J in Figure 4 Figure K in Figure 1).

[0073] Since the loss of PRMT5 enzyme activity or reduced expression level can prolong the stability of ALKBH5 protein, the proteasome inhibitor MG132 (inhibiting protein ubiquitination degradation) was then used to treat HCT116 and MC38 cells overexpressing PRMT5 and control cells, and the changes in ALKBH5 protein were detected by Western blot. The results showed that the expression of ALKBH5 protein in HCT116 and MC38 cells overexpressing PRMT5 treated with MG132 was significantly increased ( Figure 4 In the L graph, Figure 4 M in the figure), which can inhibit the reduction of ALKBH5 expression caused by PRMT5 overexpression, further verifying that PRMT5 can degrade ALKBH5 through the ubiquitin proteasome pathway.

[0074] Finally, the present invention proves that PRMT5 mediates the ubiquitination degradation of ALKBH5. The above results suggest that PRMT5 can affect the protein stability of ALKBH5. Therefore, the present invention uses HCT116 cells that overexpress PRMT5, knock down PRMT5, and treat with the PRMT5 inhibitor GSK595 to detect the effect of PRMT5 on the ubiquitination level of ALKBH5 by immunoprecipitation and Western blot experiments. The results show that the Ub ubiquitination level of ALKBH5 increases after overexpression of PRMT5 ( Figure 4 Figure N in the figure), the Ub ubiquitination level of ALKBH5 was reduced after knockdown of PRMT5 ( Figure 4O in the figure), the Ub ubiquitination level of ALKBH5 was reduced after treatment with the PRMT5 inhibitor GSK595 ( Figure 4 (Figure P in the figure).

[0075] In one embodiment, the present invention demonstrates that PRMT5-mediated ALKBH5-R316 methylation enhances ALKBH5 ubiquitination and degradation, specifically as follows:

[0076] First, the present invention proves that PRMT5 can bind to ALKBH5 and undergo symmetrical dimethylation modification. The present invention found that PRMT5 and ALKBH5 exist in combination through mass spectrometry detection. To further study the interaction mechanism between PRMT5 and ALKBH5 proteins, the present invention extracted HCT116 and MC38 cell proteins for endogenous immunoprecipitation experiments (IP) and found that there is an interaction between PRMT5 and ALKBH5 proteins ( Figure 5 Figure A in Figure 5 Subsequently, by co-transfecting Flag-ALKBH5 and Myc-PRMT5 overexpression plasmids into HCT116 and MC38 cells, the interaction between ALKBH5 and PRMT5 proteins was verified by exogenous immunoprecipitation (Co-IP) using the corresponding tag antibodies ( Figure 5 Figure C in Figure 5 To investigate whether ALKBH5 can undergo arginine methylation modification, endogenous immunoprecipitation (IP) experiments were performed in HCT116 and MC38 cells using ALKBH5 antibodies, and detection was performed using monomethylation antibody (MMA) and symmetric dimethylation antibody (SDMA). The results showed that ALKBH5 protein can undergo MMA and SDMA modification ( Figure 5 Figure E in Figure 5 Figure F in the figure).

[0077] Secondly, the present invention proves that PRMT5 binds to ALKBH5 in vitro and directly mediates methylation modification of ALKBH5. In order to clarify whether PRMT5 can directly mediate the SDMA modification formation of ALKBH5, considering that PRMT5 can self-methylate, and because the molecular weight of GST-ALKBH5 and PRMT5 proteins are similar, it is impossible to identify which protein at the same molecular weight position is methylated during in vitro methylation experiments. Therefore, the present invention constructed GST-ALKBH5 and GST-ALKBH5 (292-395aa) truncated plasmids to distinguish GST-ALKBH5 from PRMT5 in terms of protein molecular weight. Subsequently, after inducing protein expression, the protein was purified to obtain GST-ALKBH5 protein for GST pull-down experiments and GST-ALKBH5 (292-395aa) protein for in vitro methylation experiments. First, the present invention carried out a GST pull-down experiment, and the results showed that ALKBH5 can directly bind to PRMT5 in vitro ( Figure 5 Then, the present invention conducted an in vitro methylation experiment using HMT Assay, and the results showed that ALKBH5 could be methylated by PRMT5 in vitro ( Figure 5 Figure H in the figure).

[0078] Again, the present invention proves that changes in PRMT5 expression levels or enzyme activity affect the methylation modification of ALKBH5 protein. The present invention uses HCT116 cells transfected with Flag-ALKBH5 overexpression plasmid, and simultaneously overexpresses PRMT5, knocks down PRMT5, and treats with PRMT5 inhibitor GSK595. The effect of PRMT5 on the SDMA modification level of ALKBH5 is detected by immunoprecipitation and Western blot experiments. The results show that the SDMA modification level of ALKBH5 increases after overexpression of PRMT5 ( Figure 5 Figure I in the figure), the SDMA modification level of ALKBH5 was reduced after knockdown of PRMT5 ( Figure 5 J in Figure 3), the SDMA modification level of ALKBH5 was reduced after treatment with the PRMT5 inhibitor GSK595 ( Figure 5 Figure K in Figure 1).

[0079] Furthermore, the present invention demonstrates that arginine 316 (R316) is the primary site for PRMT5-mediated methylation of ALKBH5. To explore the specific sites of ALKBH5 arginine methylation by PRMT5, we obtained methylated ALKBH5 protein through in vitro methylation reactions and then performed mass spectrometry analysis. The results showed that arginine residues (R) at positions 249, 250, 269, 283, 316, 357, and 359 were likely methylated by PRMT5 (Table 1).

[0080] Table 1 ALKBH5 methylation modification sites

[0081] No Site Sequence 1 R249 RVSEPVLSLPVRR 2 R250 RRGSVTVLSGYAADEITHCIRPQDIKE 3 R269 RGSVTVLSGYAADEITHCIRPQDIKE 4 R283 RAVIILRK 5 R316 RLSGNNRDPALKPKR 6 R357 RSVLLPTHRR 7 R359 RRGSFSSENYWRK

[0082] In order to identify the main methylation sites in ALKBH5, the present invention constructed Flag-ALKBH5-R249K, Flag-ALKBH5-R250K, Flag-ALKBH5-R269K, Flag-ALKBH5-R283K, Flag-ALKBH5-R316K, Flag-ALKBH5-R357K and Flag-ALKBH5-R359K mutants. After co-transfection of each mutant plasmid and PRMT5 overexpression plasmid into HEK293T cells, Co-IP assay revealed that the ALKBH5-R316K mutation significantly reduced the PRMT5-mediated ALKBH5SDMA modification level compared with other mutant groups ( Figure 5 Therefore, the subsequent research of the present invention focused on the R316 site of ALKBH5. Since PRMT5 catalyzes the generation of symmetric dimethylarginine (SDMA) from arginine residues, this suggests that PRMT5 mediates the formation of ALKBH5-R316 SDMA ( Figure 5 In addition, the R316 methylation site on ALKBH5 is highly conserved in mammals ( Figure 5 N in FIG). Subsequently, the present invention constructed a GST-R316K-ALKBH5 (292-395aa) mutant plasmid, induced protein expression, and purified the proteins to obtain GST-ALKBH5-WT (292-395aa) and GST-ALKBH5-R316K (292-395aa) proteins, respectively. The present invention conducted an in vitro methylation experiment using HMT Assay, and the results showed that SDMA modification of GST-R316K-ALKBH5 (292-395aa) was significantly inhibited ( Figure 5 Together, these data indicate that ALKBH5 is symmetrically dimethylated at R316 by PRMT5 (meR316-ALKBH5, meALKBH5).

[0083] Finally, the present invention demonstrates that PRMT5-mediated ALKBH5-R316 methylation modification reduces the protein stability of ALKBH5 and enhances the ubiquitination degradation of ALKBH5. In order to further explore whether PRMT5-mediated ALKBH5-R316 methylation can affect the protein stability of ALKBH5. Given that the amino acid at the same position in the mouse ALKBH5 protein sequence is one more than that in the human ALKBH5 protein sequence, the present invention constructed human Flag-ALKBH5-WT and Flag-ALKBH5-R316K plasmids, as well as mouse Flag-Alkbh5-WT and Flag-Alkh5-R317K plasmids. They were transfected into HCT116 and MC38 cells, respectively, and after treating the above cells with CHX, Western blot detection showed that the half-life of the ALKBH5 wild-type overexpression group was significantly shorter than that of the ALKBH5 mutant overexpression group ( Figure 5 In the P picture, Figure 5 Subsequently, the present invention co-transfected PRMT5 plasmid and Flag-ALKBH5-WT, Flag-ALKBH5-R316K, Flag-Alkbh5-WT, and Flag-Alkbh5-R317K plasmids into HCT116 and MC38 cells, respectively, and then performed IP-Flag experiments to detect the differences in ALKBH5 protein ubiquitination. Western blot results showed that in HCT116 and MC38 cells, the ALKBH5 wild-type overexpression group had an increased ALKBH5 ubiquitination level compared to the ALKBH5 mutant overexpression group ( Figure 5 The R graph in Figure 5 (Figure S in Figure 5). These results suggest that PRMT5-mediated ALKBH5-R316 methylation reduces ALKBH5 protein stability by enhancing ALKBH5 ubiquitination and degradation. In summary, PRMT5-mediated ALKBH5-R316 methylation plays a key role in regulating ALKBH5 stability.

[0084] In one embodiment, the present invention demonstrates that PRMT5-mediated ALKBH5-R316 methylation enhances the binding of TRIM28 to ALKBH5 and promotes ALKBH5 ubiquitination and degradation, specifically as follows:

[0085] First, the present invention proves that TRIM28 can bind to ALKBH5. The present invention uses mass spectrometry to search for potential E3 ubiquitin ligases that lead to ALKBH5 ubiquitination and degradation, and finds that ALKBH5 and TRM28 may bind to each other. The present invention extracts proteins from HCT116 cells and MC38 cells and performs endogenous immunoprecipitation (IP) experiments to find that there is an interaction between ALKBH5 and TRIM28 proteins ( Figure 6Figure A in Figure 6 Subsequently, HCT116 cells were co-transfected with Flag-ALKBH5 and Myc-TRIM28 overexpression plasmids, and MC38 cells were co-transfected with Flag-Alkbh5 and His-Trim28 overexpression plasmids. The interaction between ALKBH5 and TRIM28 proteins was verified by exogenous immunoprecipitation (Co-IP) using the corresponding tag antibodies ( Figure 6 Figure C in Figure 6 Figure D in the figure).

[0086] Secondly, the present invention proves that TRIM28 affects the expression of ALKBH5 protein. To explore whether TRIM28 has an effect on ALKBH5, Western blot and qRT-PCR results showed that overexpression of TRIM28 can reduce the level of ALKBH5 protein in colorectal cancer cells HCT116 and mouse colorectal cancer cells MC38 ( Figure 6 E in Figure ), while the transcription level did not change significantly ( Figure 6 Figure F in the figure).

[0087] Again, the present invention proves that TRIM28 affects the protein stability of ALKBH5. In order to further study the effect of TRIM28 expression level on the half-life of ALKBH5, the present invention used colorectal cancer cells HCT116 and mouse colorectal cancer cells MC38 that overexpress TRIM28. After CHX treatment, the results of Western Blot experiments showed that the protein half-life of ALKBH5 in HCT116 and MC38 cells that overexpress TRIM28 was significantly shortened compared with the control group ( Figure 6 G diagram in Figure 6 Subsequently, the proteasome inhibitor MG132 was used to treat HCT116 and MC38 cells overexpressing TRIM28 and the control cells, and the changes in ALKBH5 protein were detected by Western blot. The results showed that the expression of ALKBH5 protein in HCT116 and MC38 cells overexpressing TRIM28 treated with MG132 was significantly increased ( Figure 6 Figure I in the figure, Figure 6 This further confirmed that the E3 ubiquitin ligase TRIM28 can degrade ALKBH5 through the ubiquitin-proteasome pathway.

[0088] Again, the present invention proves that TRIM28 promotes the ubiquitination and degradation of ALKBH5. The present invention uses HCT116 cells overexpressing TRIM28 to detect the effect of TRIM28 on the ubiquitination level of ALKBH5 by immunoprecipitation and Western blot experiments. The results show that the Ub ubiquitination level of ALKBH5 increases after overexpression of TRIM28 ( Figure 6 Figure K in Figure 1).

[0089] Finally, the present invention proves that PRMT5-mediated ALKBH5-R316 methylation can enhance the binding of TRIM28 to ALKBH5. The present invention uses HCT116 cells transfected with Flag-ALKBH5 overexpression plasmid, overexpresses PRMT5, knocks down PRMT5, and treats with PRMT5 inhibitor GSK595, and uses Co-IP and Western blot experiments to detect the effect of PRMT5-mediated ALKBH5-R316 methylation on the binding of TRIM28 to ALKBH5. The results show that after overexpression of PRMT5, meR316-ALKBH5 increases, that is, the SDMA modification level of ALKBH5 increases. At the same time, the binding of TRIM28 to ALKBH5 increases ( Figure 6 L in Figure 1), knocking down PRMT5 reduced meR316-ALKBH5, that is, the SDMA modification level of ALKBH5 was reduced, and the binding of TRIM28 to ALKBH5 was reduced ( Figure 6 M in Figure 3), after treatment with the PRMT5 inhibitor GSK595, meR316-ALKBH5 was reduced, that is, the SDMA modification level of ALKBH5 was reduced, and the binding of TRIM28 to ALKBH5 was reduced ( Figure 6 N in Figure 1). Subsequently, the present invention co-transfected HCT116 cells with PRMT5 plasmid, Flag-ALKBH5-WT, and Flag-ALKBH5-R316K to perform Co-IP experiments. Western blot results showed that meR316-ALKBH5 was significantly reduced in cells overexpressing R316K-ALKBH5, that is, SDMA modification of ALKBH5 was reduced. At the same time, compared with cells overexpressing ALKBH5-WT, ALKBH5-R316K inhibited the binding between TRIM28 and ALKBH5 ( Figure 6 O). Taken together, these data indicate that PRMT5-mediated ALKBH5-R316 methylation reduces ALKBH5 stability and relies on recruiting TRIM28 for ALKBH5 ubiquitination and degradation.

[0090] In one embodiment, the present invention demonstrates that PRMT5-mediated meR316-ALKBH5 enhances CD276 mRNA stability through m6A modification, specifically as follows:

[0091] First, the present invention demonstrates that CD276 is a target gene regulated by ALKBH5 through m6A modification mediated by PRMT5. To further explore the target genes regulated by PRMT5-mediated meR316-ALKBH5 through m6A modification, m6A MeRIP-seq and RNA-seq were used to detect HCT116 cells treated with the PRMT5 inhibitor GSK595. After GSK595 treatment, 2408 genes showed reduced m6A modification and 12 genes showed decreased transcription ( Figure 7 The present invention compared the results of meRIP-seq and RNA-seq and identified 6 overlapping genes with reduced m6A methylation and down-regulated ( Figure 7 Figure B in Figure 1). Subsequently, the GDPIA (http: / / gepia.cancer-pku.cn / ) database was used for analysis. In TCGA tumor samples, only CD276 expression was positively correlated with PRMT5 at the transcriptional level and was statistically significant ( Figure 7 Finally, the present invention used anti-PRMT5 and anti-CD276 antibodies to perform IHC detection on a colorectal cancer tissue combination chip (including 84 cases of rectal adenocarcinoma tissues). Linear correlation analysis showed that the expression levels of PRMT5 and CD276 were positively correlated (correlation coefficient was 0.3598, P values ​​were <0.0001) ( Figure 7 (D in Figure ). These experimental results confirm that CD276 is a target gene regulated by ALKBH5 through PRMT5-mediated m6A modification.

[0092] Secondly, the present invention demonstrates that changes in PRMT5 expression levels or enzyme activity affect CD276 expression at the transcriptional and protein levels. After HCT116 cells were treated with the PRMT5-specific inhibitor GSK595, qRT-PCR experiments revealed that CD276 mRNA levels were significantly reduced ( Figure 7 E in the figure), Western blot analysis revealed that the protein level of CD276 was decreased ( Figure 7 At the same time, after overexpression of PRMT5 in HCT116 cells, qRT-PCR experiments revealed that the mRNA level of CD276 was significantly increased ( Figure 7 F in Figure ), Western blot analysis revealed that the protein level of CD276 was elevated ( Figure 7 Figure H in the figure).

[0093] Again, the present invention demonstrates that changes in PRMT5 expression levels or enzyme activity affect the half-life of CD276 mRNA. After HCT116 cells were treated with the PRMT5-specific inhibitor GSK595, actinomycin D was added, and qRT-PCR experiments revealed that the mRNA half-life of CD276 was significantly reduced ( Figure 7 Figure I). At the same time, after overexpressing PRMT5 in HCT116 cells, actinomycin D (an inhibitor of RNA transcription synthesis) was added, and qRT-PCR experiments showed that the mRNA half-life of CD276 was significantly increased ( Figure 7 Figure J in Figure 3).

[0094] Finally, the present invention demonstrates that ALKBH5 regulates CD276 through m6A modification. MeRIP-seq data analysis revealed that the abundance of m6A modification in CD276 mRNA was mainly concentrated in the 3'UTR region. Compared with the control group, the abundance of m6A modification in the CD276-3'UTR region decreased in the GSK595-treated group ( Figure 7 To identify the detailed ALKBH5 binding sites in CD276-3'UTR, we performed m6 modification site analysis using the SRAMP database (http: / / www.cuilab.cn / sramp). Two potential sites with high confidence were selected based on the scores, and wild-type WT, mutant Mut1, mutant Mut2, and co-mutant Mut1+Mut2 reporter genes were constructed ( Figure 7 L in Figure 1). Dual-luciferase reporter gene experiments revealed that luciferase activity decreased in the group co-transfected with the wild-type CD276-3'UTR-WT reporter plasmid and the ALKBH5 overexpression plasmid, while luciferase expression recovered in the group co-transfected with the mutant CD276-3'UTR-Mut1 reporter plasmid and the ALKBH5 overexpression plasmid. Luciferase expression decreased or partially decreased in the group co-transfected with the mutant CD276-3'UTR-Mut2 or the co-mutant CD276-3'UTR-Mut1+Mut2 and the ALKBH5 overexpression plasmid ( Figure 7 The above experimental results indicate that ALKBH5 modifies the CD276-3'UTR region through m6A, thereby affecting the metabolic level of CD276 mRNA.

[0095] In one embodiment, the present invention demonstrates that the ALKBH5-R316 methylation site affects tumor immune evasion in colorectal cancer cells, specifically as follows:

[0096] First, the present invention demonstrates that ALKBH5 expression is positively correlated with immune cell infiltration. In order to explore the relationship between the expression level of ALKBH5 and the abundance of tumor-infiltrating immune cells (TIICs) in colorectal cancer, the TIMER2.0 (http: / / timer.comp-genomics.org / timer / ) database was used for analysis. The results showed that ALKBH5 was positively correlated with CD8+T cells (Rho=0.29, P=1.03e-06), CD4+T cells (Rho=0.127, P=3.59e-02), and NK cells (Rho=0.26, P=1.24e-05) in colon cancer (COAD). It was also observed that ALKBH5 was positively correlated with CD8+T cells (Rho=0.154, P=1.47e-01), CD4+T cells (Rho=0.262, P=1.27e-02), and NK cells (Rho=0.148, P=1.64e-01) in rectal cancer (READ). Figure 8 These results support that the expression level of ALKBH5 plays a key role in the abundant presence of tumor-infiltrating immune cells in the colorectal cancer tumor microenvironment.

[0097] Secondly, the present invention demonstrates that in vitro phenotypic experiments exploring the methylation site of ALKBH5-R316 can promote T cells to kill tumors. RTCA detection found that when the same T cell effector-target ratio was given to HCT116 cells and T cells were co-incubated with effector cells (effector-target ratio 1:1), the number of surviving cells in the group co-incubated with overexpressed ALKBH5-WT and T cells was less than that in the group co-incubated with overexpressed ALKBH5-R316K and T cells, and the number of surviving cells was significantly suppressed as the co-incubation time prolonged ( Figure 8 However, the difference in the number of surviving cells between the Vector-overexpressing group and the T cell-co-cultured group was small ( Figure 8 (B) The results confirmed that the ALKBH5-R316 methylation site can promote T cells to kill tumors.

[0098] Finally, the present invention demonstrates that in vivo experiments exploring the ALKBH5-R316 methylation site can inhibit tumor progression. The present invention used lentivirus to construct Vector, Alkbh5-WT and Alkbh5-R317K stable cell lines in mouse colorectal cancer cell line MC38. Subsequently, female C57BL / 6 mice were subcutaneously injected with the three cells to construct colorectal cancer subcutaneous tumor models (mice were randomly divided into 3 groups, n = 5, 5 × 10 5In vivo experiments further explored the effect of PRMT5-mediated ALKBH5-R316 methylation on the progression of colorectal cancer. The weight and subcutaneous tumor volume of mice were measured every three days for 20 consecutive days. Tumor growth curves were drawn to detect the progression of subcutaneous tumors in each group of mice. The results showed that the weight of mice in each experimental group was stable ( Figure 8 C in the figure), the subcutaneous tumor growth in the Alkbh5-WT group was significantly reduced compared with the Alkbh5-R317K group and the Vextor group ( Figure 8 D in the figure), the tumors formed were smaller ( Figure 8 Figure C in the figure), the tumor weight is relatively light ( Figure 8 Figure E in the figure).

[0099] Immunohistochemistry (IHC) staining was used to detect the methylation level of ALKBH5-R316 in tumor tissue sections, confirming that the present invention only detected high levels of ALKBH5 methylation in mice injected with Alkbh5-WT cells ( Figure 8 Secondly, the number of Ki-67 positive cells in the Alkbh5-WT group was significantly lower than that in the Alkbh5-WT group (Figure F, first row, meALKBH5). Figure 8 Finally, compared with the Alkbh5-R317K group and the Vextor group, the number of cells positive for tumor immune infiltration-related markers CD3, CD8a, and GZMB in the Alkbh5-WT group was significantly higher ( Figure 8 Figure F in the figure, Figure 8 Taken together, these experimental results indicate that the ALKBH5-R316 methylation site plays a key role in promoting T cell killing of tumors.

[0100] In one embodiment, the present invention demonstrates that PRMT5-mediated ALKBH5-R316 methylation is associated with poor prognosis in patients with colorectal cancer, specifically as follows:

[0101] First, the present invention demonstrates that PRMT5 and ALKBH5-R316 methylation are positively correlated in colorectal cancer. To evaluate the clinical significance of our research, the present invention used anti-PRMT5 and anti-ALKBH5-R316 methylation antibodies to perform IHC on a colorectal cancer tissue combination chip containing 84 rectal adenocarcinoma tissues. After statistical analysis, the present invention found that in colorectal cancer tumors with high expression of PRMT5, ALKBH5-R316 methylation (meALKBH5) was also highly expressed ( Figure 9 Figure A in the figure). The present invention found that the expression levels of PRMT5 and ALKBH5-R316 methylation (meALKBH5) were positively correlated through linear correlation analysis (correlation coefficients were 0.4365, P values ​​were <0.0001) ( Figure 9 Figure B in the figure).

[0102] Secondly, the present invention demonstrates the correlation analysis between high expression of ALKBH5-R316 methylation and clinical characteristics of colorectal cancer patients. Statistical analysis found that in colorectal cancer tissues with high expression of PRMT5, high expression of ALKBH5-R316 methylation (meALKBH5) accounted for 61%, while in colorectal cancer tissues with low expression of PRMT5, high expression of ALKBH5-R316 methylation (meALKBH5) accounted for only 15% ( Figure 9 In colorectal cancer tissues with a tumor diameter greater than 5 cm, ALKBH5-R316 methylation (meALKBH5) high expression accounted for 58%, while in colorectal cancer tissues with a tumor diameter less than 5 cm, ALKBH5-R316 methylation (meALKBH5) high expression accounted for only 45% ( Figure 9 Figure D). In colorectal cancer tissues with lymph node metastasis, ALKBH5-R316 methylation (meALKBH5) high expression also accounted for 58%, while in colorectal cancer tissues without lymph node metastasis, ALKBH5-R316 methylation (meALKBH5) high expression accounted for only 33% ( Figure 9 Figure E in the figure).

[0103] Finally, the present invention demonstrates that ALKBH5-R316 methylation can be used as an independent prognostic factor for colorectal cancer. ALKBH5-R316 methylation immunohistochemical staining was performed on tissue microarrays of colorectal cancer patients with complete clinical data (a total of 212 cases), and the expression of ALKBH5-R316 methylation was scored and graded. IRS 0-4 was defined as low expression of ALKBH5-R316 methylation, and IRS 6-12 was defined as high expression of ALKBH5-R316 methylation. Figure 9 Figure F in Figure 3). Kaplan-Meier survival analysis was used to compare the 5-year overall survival rate (n=212, 60-month follow-up) and disease-free survival rate (n=138, 60-month follow-up) of colorectal cancer patients with high and low ALKBH5-R316 methylation expression. The results showed that colorectal cancer patients with high ALKBH5-R316 methylation expression had a poor 5-year overall survival rate ( Figure 9 G in Figure ) and disease-free survival ( Figure 9 H in Figure ).

[0104] However, statistical analysis found that the expression level of ALKBH5-R316 methylation had no significant correlation with age (P=0.725) and gender (P=0.705); but was significantly correlated with TNM stage (P<0.0001), invasion depth (P<0.0001), lymph node metastasis (P<0.0001), distant metastasis (P=0.008), and tumor size (P<0.0001). Cox regression analysis of 212 colorectal cancer cases showed that ALKBH5-R316 methylation expression (P < 0.001), TNM stage (P < 0.001), tumor size (P < 0.001), and differentiation (P < 0.001) were significantly associated with 5-year overall survival and 5-year disease-free survival in univariate regression analysis. Multivariate regression analysis also showed that ALKBH5-R316 methylation expression (P < 0.05) was significantly associated with 5-year overall survival and 5-year disease-free survival. These results strongly support the idea that high ALKBH5-R316 methylation expression is closely associated with poor prognosis in colorectal cancer patients and can serve as an independent prognostic factor for breast cancer prognosis. These results confirm our hypothesis that PRMT5-mediated ALKBH5-R316 methylation inhibits immune infiltration in colorectal cancer cells by reducing ALKBH5 stability.

[0105] In one embodiment, the present invention demonstrates that the combination of PRMT5 inhibitors and anti-PD1 antibodies significantly inhibits the progression of colorectal cancer, specifically as follows:

[0106] First, the present invention demonstrates that reduction of PRMT5 expression level or enzyme activity promotes anti-tumor T cell killing.

[0107] The present invention uses HCT116 cells with stable knockdown of PRMT5. Through RTCA detection, it is found that when T cells are co-incubated with the same T cell effector-target ratio (effector-target ratio 1:1), the number of surviving cells in the cells with stable knockdown of shPRMT5 and T cells is less than that in the group co-incubated with stably transfected shCtrl cells, and the number of surviving cells is significantly inhibited as the co-incubation time prolongs ( Figure 10 Figure A in Figure 1). Similarly, when HCT116 cells treated with the PRMT5 inhibitor GSK595 were co-incubated with T cells at the same T cell effector-target ratio (1:1), the number of surviving cells in the GSK595-treated and T cell-co-incubated group was less than that in the DMSO-plus-T cell co-incubation group, and the number of surviving cells was significantly inhibited as the co-incubation time prolonged ( Figure 10 These results suggest that reduced PRMT5 expression or enzyme activity can promote anti-tumor T cell killing.

[0108] Then, the present invention demonstrated that the combination of PRMT5 inhibitors and anti-PD1 antibodies significantly inhibited the progression of colorectal cancer. The present invention used female C57BL / 6 mice to inoculate mouse colorectal cancer cells MC38 (5×10 5 7 days later, the subcutaneous tumor size reached 50 mm. 3 Afterwards, the tumor-bearing mice were randomly divided into 4 groups, with 6 mice in each group, and the PBS+IgG, GSK595+IgG, PBS+PD-1Ab, and GSK595+PD-1Ab treatment model groups were established. According to the weight of the mice, PBS or GSK595 were intraperitoneally injected at 5 mg / kg daily, and IgG or PD-1Ab were intraperitoneally injected at 200 μg every 3 days for treatment ( Figure 10 The tumor size of each group of mice was measured every 3 days, and the data were recorded for two weeks. The tumor growth curve was drawn to detect the progression of subcutaneous tumors in each group of mice. The results showed that the weight of mice in each experimental group was stable ( Figure 10 Figure D in the figure shows that compared with the PBS+IgG group, the tumor growth of the GSK595+IgG group and the PBS+PD-1Ab group was inhibited, and the tumor growth inhibition in the GSK595+PD-1Ab group was the most significant ( Figure 10 Compared with the PBS+IgG group, the tumor weights of the GSK595+IgG group and the PBS+PD-1Ab group were reduced, and the tumor weight reduction in the GSK595+PD-1Ab group was the most significant ( Figure 10 The above results suggest that GSK595 combined with PD-1Ab can effectively inhibit the growth of colorectal cancer cells in mice.

[0109] Immunohistochemistry (IHC) staining was used to detect the methylation level of ALKBH5-R316 in tumor tissue sections, confirming that GSK595 can inhibit the methylation of ALKBH5-R316 ( Figure 10 Secondly, compared with the PBS+IgG group, the number of Ki-67-positive cells in the tumors of the GSK595+IgG group and the PBS+PD-1Ab group was significantly reduced, and the number of Ki-67-positive cells in the tumors of the GSK595+PD-1Ab group decreased most significantly ( Figure 10 Finally, compared with the PBS+IgG group, the number of cells positive for tumor immune infiltration-related markers CD3, CD8a, and GZMB in the GSK595+IgG and PBS+PD-1Ab groups increased, and the increase in the number of cells positive for tumor immune infiltration-related markers CD3, CD8a, and GZMB in the GSK595+PD-1Ab group was most significant ( Figure 10 F in Figure ), and the difference was statistically significant ( Figure 10In summary, these experimental results indicate that ALKBH5-R316 methylation plays a key role in promoting T cell killing of tumors, and GSK595 combined with PD-1Ab can effectively inhibit the progression of colorectal cancer cells in mice.

[0110] In this study, the inventors integrated MeRIP-seq and RNA-seq sequencing analysis to identify potential target genes in colorectal cancer cells treated with PRMT5 inhibitors and PRMT5 knockdown. Screening revealed that CD276 may be regulated by m6A modification. Subsequently, experiments revealed that CD276 was positively correlated with PRMT5 at both the transcriptional and protein levels, demonstrating an interaction between ALKBH5 protein and CD276 mRNA. MeRIP-seq analysis revealed a significant increase in m6A modification in the 3'UTR region of CD276 mRNA in PRMT5-mediated meR316-ALKBH5, further confirming that PRMT5-mediated meR316-ALKBH5 can increase the stability of CD276 mRNA and promote its protein level, thereby fully exerting the inherent biological function of CD276 to affect the progression of colorectal cancer.

[0111] Subsequently, the inventors of the present invention used tumor tissue specimens from colorectal cancer patients to analyze the correlation between meR316-ALKBH5 expression levels and clinical pathological characteristics through immunohistochemistry experiments. The data showed that high meR316-ALKBH5 levels were positively correlated with TNM, invasion depth, lymph node metastasis and tumor size of CRC patients. In addition, the results of immunohistochemistry experiments showed that high expression of meR316-ALKBH5 was positively correlated not only with tumor size but also with lymph node metastasis. Finally, the inventors of the present invention demonstrated that colorectal cancer patients with high expression of meR316-ALKBH5 were significantly associated with relatively poor 5-year overall survival and disease-free survival. These results strongly support the correlation between high expression of meR316-ALKBH5 and clinical pathological characteristics of colorectal cancer patients, and reveal the relationship between meR316-ALKBH5 and clinical malignant outcomes in colorectal cancer patients.

[0112] In this study, the inventors of the present invention found that treating colorectal cancer cells with the small molecule inhibitor GSK3326595 (GSK595) of PRMT5 or knocking down PRMT5 (shPRMT5) can cause changes in methylation modification of ALKBH5-R316, thereby epigenetically regulating the mRNA stability of CD276 through m6A modification. It was also found that ALKBH5 was symmetrically dimethylated by PRMT5 at R316 (meR316-ALKBH5), enhancing the binding of ALKBH5 to TRIM28 and leading to ubiquitination and degradation of ALKBH5. In addition, this study revealed that ALKBH5-R316 is essential for ALKBH5-mediated colorectal cancer tumor immune escape. This study also found a significant correlation between meR316-ALKBH5 and poor prognosis in colorectal cancer patients. Subsequently, this study demonstrated that PRMT5-mediated meR316-ALKBH5 increases CD276 mRNA stability through m6A modification, thereby enhancing colorectal cancer tumor immune escape in vitro and in vivo. Finally, this study demonstrated that combination therapy with a PRMT5 inhibitor and an anti-PD-1 antibody significantly inhibited colorectal cancer progression. These findings provide a theoretical basis for exploring colorectal cancer treatment strategies targeting the PRMT5-meR316-ALKBH5-CD276 signaling axis.

[0113] In one embodiment, the present invention demonstrates that there is a significant binding between PRMT5 protein and ALKBH5 protein, and that inhibiting PRMT5 can inhibit the expression of ALKBH5, thereby inhibiting the occurrence and development of colorectal cancer. Based on this, the present invention has developed for the first time a screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5. This screening method can be used to effectively and rapidly screen new anti-colorectal cancer drugs, and has broad application prospects.

[0114] In one embodiment, the present invention demonstrates that the expression levels of PRMT5 and CD276 are significantly positively correlated, and PRMT5 can promote the expression of CD276, thereby enhancing the immune escape of colorectal cancer; while inhibiting PRMT5 can inhibit the expression of CD276, thereby inhibiting the immune escape of colorectal cancer, and can be used in the treatment of colorectal cancer. Based on this, the present invention has developed for the first time a screening method for anti-colorectal cancer drugs based on PRMT5 and CD276. This screening method can be used to effectively and rapidly screen new anti-colorectal cancer drugs, and has broad application prospects.

[0115] S102: selecting the spatial structure of the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the PRMT5 protein and CD276 protein complex, and determining the binding site of the PRMT5 protein and ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and CD276 protein in the PRMT5 protein and CD276 protein complex as a binding site for a targeted drug;

[0116] In one embodiment, the method for determining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex comprises: constructing n truncated expression mutants of PRMT5, where n is a natural number greater than or equal to 1;

[0117] Based on the n truncated expression mutants, immunoprecipitation experiments were performed to obtain the key regions for the interaction between PRMT5 and ALKBH5 or CD276;

[0118] The spatial structure of the key region for the interaction between PRMT5 and ALKBH5 is the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex;

[0119] The spatial structure of the key region for the interaction between PRMT5 and CD276 is the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex.

[0120] In one embodiment, the binding site can be obtained by conventional methods well known to those skilled in the art, and the present invention has no particular limitation on the specific method for determining the binding site.

[0121] S103: using a computer-assisted drug screening method to obtain a candidate drug targeting the binding site;

[0122] The computer-assisted drug screening method comprises:

[0123] Obtaining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex;

[0124] Based on the spatial structure of the binding site between the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the binding site between the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex, screening a small molecule compound with a similar structure in a molecular database;

[0125] Molecular docking is performed on the screened small molecule compounds and ALKBH5 protein or CD276 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor ALKBH5 protein or CD276 protein to obtain a score for each small molecule compound, and the compounds are ranked according to the scores to obtain candidate anti-colorectal cancer drugs.

[0126] In one embodiment, the screening method further comprises:

[0127] Obtaining colorectal cancer cell models and / or colorectal cancer animal models;

[0128] The colorectal cancer cell model and / or colorectal cancer animal model is treated with the screened anti-colorectal cancer candidate drug to verify the effectiveness of the anti-colorectal cancer candidate drug.

[0129] In one embodiment, the small molecule compound includes siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic small molecule compound. It should be noted that the present invention has no particular limitation on the specific type of the small molecule compound, and any small molecule compound that may be used in the computer-assisted drug screening method provided by the present invention and may produce a corresponding effect (inhibiting ALKBH5 or CD276 expression) falls within the scope of protection of the present invention.

[0130] In one embodiment, in molecular docking, affinity refers to how tightly a molecule binds to a receptor. A high affinity means a more stable binding, while a low affinity means a less stable binding. Affinity is typically calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0131] To assess affinity, scoring systems or methods are often used to quantify the interaction between a molecule and a receptor. These scoring methods are based on different algorithms and physical models and can reflect the binding energy, interaction type, and affinity between the molecule and the receptor.

[0132] In one embodiment, molecular docking is performed between the screened small molecule compounds and ALKBH5 protein or CD276 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor ALKBH5 protein or CD276 protein to obtain a score for each small molecule compound, and the compounds are sorted according to the scores to obtain candidate anti-colorectal cancer drugs. Small molecule compounds with higher affinity or binding energy targeting the receptor ALKBH5 protein or CD276 protein are preferably selected as candidate anti-colorectal cancer drugs.

[0133] In one embodiment, the modes of drug screening using computer-assisted drug screening technology include one or more of the following: protein-small molecule docking, protein-protein docking, and protein-nucleic acid docking, all of which are well known to those skilled in the art.

[0134] In one embodiment, the molecular libraries used in drug virtual screening include: ZINC, PubChem, DrugBank, ChEMBL, ChemDB, HMDB, BindingDB, SMPDB, ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M and Interbioscreen, etc. These databases are also commonly used in drug virtual screening.

[0135] Figure 2 Schematic diagram of a screening system for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, provided in an embodiment of the present invention. Specifically, the screening system includes:

[0136] A data acquisition unit, which acquires data of PRMT5 protein and ALKBH5 protein, or data of PRMT5 protein and CD276 protein;

[0137] a site determination unit, which selects the spatial structure of the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the PRMT5 protein and CD276 protein complex, and determines the binding site of the PRMT5 protein and ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and CD276 protein in the PRMT5 protein and CD276 protein complex as the binding site of the targeted drug;

[0138] a drug screening unit, which uses a computer-assisted drug screening method to obtain candidate drugs targeting the binding site;

[0139] The computer-assisted drug screening method comprises:

[0140] Obtaining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex;

[0141] Based on the spatial structure of the binding site between the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the binding site between the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex, screening a small molecule compound with a similar structure in a molecular database;

[0142] Molecular docking is performed on the screened small molecule compounds and ALKBH5 protein or CD276 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor ALKBH5 protein or CD276 protein to obtain a score for each small molecule compound, and the compounds are ranked according to the scores to obtain candidate anti-colorectal cancer drugs.

[0143] In one embodiment, the method for determining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex comprises:

[0144] Construct n truncated expression mutants of PRMT5, where n is a natural number greater than or equal to 1;

[0145] Based on the n truncated expression mutants, immunoprecipitation experiments were performed to obtain the key regions for the interaction between PRMT5 and ALKBH5 or CD276;

[0146] The spatial structure of the key region for the interaction between PRMT5 and ALKBH5 is the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex;

[0147] The spatial structure of the key region for the interaction between PRMT5 and CD276 is the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex.

[0148] In one embodiment, the screening system further comprises:

[0149] The drug effectiveness verification unit obtains a colorectal cancer cell model and / or a colorectal cancer animal model; and uses the screened anti-colorectal cancer candidate drug to treat the colorectal cancer cell model and / or the colorectal cancer animal model to verify the effectiveness of the anti-colorectal cancer candidate drug.

[0150] Figure 3 Schematic diagram of a computer device for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, provided in an embodiment of the present invention. Specifically, the computer device includes:

[0151] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the screening method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 as described in the first aspect of the present invention is implemented.

[0152] An embodiment of the present invention also provides a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, it implements the screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 as described in the first aspect of the present invention.

[0153] The embodiment of the present invention also provides an anti-colorectal cancer candidate drug obtained by screening according to the screening method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 as described in the first aspect of the present invention.

[0154] The present invention also provides any of the following applications:

[0155] (1) Use of PRMT5 inhibitors in the preparation of drugs for the treatment of colorectal cancer;

[0156] (2) Use of a PRMT5 inhibitor and an anti-PD1 antibody in combination for the preparation of a drug for the treatment of colorectal cancer;

[0157] (3) Use of CD276 inhibitors in the preparation of drugs for the treatment of colorectal cancer;

[0158] (4) Use of a reagent for inhibiting the methylation level of the ALKBH5-R316 methylation site in the preparation of a drug for treating colorectal cancer;

[0159] (5) Use of a reagent for detecting the methylation level of the ALKBH5-R316 methylation site in the preparation of a diagnostic product or a predictive product for diagnosing colorectal cancer or predicting the prognosis of colorectal cancer.

[0160] The validation results of this validation example demonstrate that assigning inherent weights to indications can moderately improve the performance of the method compared to the default settings. Those skilled in the art will clearly understand that, for ease and brevity of description, the specific operating processes of the systems, devices, and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0161] In the several embodiments provided in this application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are merely schematic. For example, the division of the units is merely a logical function division. In actual implementation, there may be other division methods, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0162] The units described as separate components may or may not be physically separate, and the components displayed as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the present embodiment. In addition, the functional units in the various embodiments of the present invention may be integrated into a single processing unit, or each unit may exist physically separately, or two or more units may be integrated into a single unit. The above-mentioned integrated units may be implemented in the form of hardware or in the form of software functional units.

[0163] Those skilled in the art will understand that all or part of the steps in the various methods of the above embodiments can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium, which may include: read-only memory (ROM), random access memory (RAM), disk or optical disk, etc.

[0164] Those skilled in the art will understand that all or part of the steps in the above-mentioned embodiment method can be implemented by instructing the relevant hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc.

[0165] The above is a detailed introduction to the computer-assisted drug screening method, system, computer equipment and storage medium for screening new anti-colorectal cancer drugs provided by the present invention. For those skilled in the art, based on the ideas of the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, characterized in that: The screening method comprises: Obtain PRMT5 protein and ALKBH5 protein data, or PRMT5 protein and CD276 protein data; Selecting the spatial structure of the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the PRMT5 protein and CD276 protein complex, and determining the binding site of the PRMT5 protein and ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and CD276 protein in the PRMT5 protein and CD276 protein complex as the binding site of the targeted drug; Using a computer-assisted drug screening method to obtain candidate drugs targeting the binding site; The computer-assisted drug screening method comprises: Obtaining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex; Based on the spatial structure of the binding site between the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the binding site between the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex, screening a small molecule compound with a similar structure in a molecular database; Performing molecular docking on the screened small molecule compounds and ALKBH5 protein or CD276 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor ALKBH5 protein or CD276 protein to obtain a score for each small molecule compound, and ranking them according to the score to obtain candidate anti-colorectal cancer drugs; The method for determining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex comprises: Construct n truncated expression mutants of PRMT5, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, immunoprecipitation experiments were performed to obtain the key regions for the interaction between PRMT5 and ALKBH5 or CD276; The spatial structure of the key region for the interaction between PRMT5 and ALKBH5 is the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex; The spatial structure of the key region for the interaction between PRMT5 and CD276 is the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex.

2. The method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 according to claim 1, characterized in that: The screening method further comprises: Obtaining colorectal cancer cell models and / or colorectal cancer animal models; The colorectal cancer cell model and / or colorectal cancer animal model is treated with the screened anti-colorectal cancer candidate drug to verify the effectiveness of the anti-colorectal cancer candidate drug.

3. A screening system for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, characterized in that: The screening system comprises: A data acquisition unit, which acquires data of PRMT5 protein and ALKBH5 protein, or data of PRMT5 protein and CD276 protein; a site determination unit, which selects the spatial structure of the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the PRMT5 protein and CD276 protein complex, and determines the binding site of the PRMT5 protein and ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and CD276 protein in the PRMT5 protein and CD276 protein complex as the binding site of the targeted drug; a drug screening unit, which uses a computer-assisted drug screening method to obtain candidate drugs targeting the binding site; The computer-assisted drug screening method comprises: Obtaining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex; Based on the spatial structure of the binding site between the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the spatial structure of the binding site between the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex, screening a small molecule compound with a similar structure in a molecular database; Performing molecular docking on the screened small molecule compounds and ALKBH5 protein or CD276 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor ALKBH5 protein or CD276 protein to obtain a score for each small molecule compound, and ranking them according to the score to obtain candidate anti-colorectal cancer drugs; The method for determining the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex, or the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex comprises: Construct n truncated expression mutants of PRMT5, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, immunoprecipitation experiments were performed to obtain the key regions for the interaction between PRMT5 and ALKBH5 or CD276; The spatial structure of the key region for the interaction between PRMT5 and ALKBH5 is the binding site of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and ALKBH5 protein complex; The spatial structure of the key region for the interaction between PRMT5 and CD276 is the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and CD276 protein complex.

4. The screening system for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 according to claim 3, characterized in that: The screening system further comprises: The drug effectiveness verification unit obtains a colorectal cancer cell model and / or a colorectal cancer animal model, and uses the screened anti-colorectal cancer candidate drug to treat the colorectal cancer cell model and / or the colorectal cancer animal model to verify the effectiveness of the anti-colorectal cancer candidate drug.

5. A computer device for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, characterized in that: The computer device comprises: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the screening method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 according to claim 1 or 2 is implemented.

6. A computer-readable storage medium, characterized in that The computer-readable storage medium stores a computer program, and when the computer program is executed by the processor, it implements the screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 according to claim 1 or 2.

7. An anti-colorectal cancer candidate drug obtained by screening according to the screening method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 according to claim 1 or 2.

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