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

Abstract

The invention discloses a computer-aided drug screening method and system for screening new anti-colorectal cancer drugs, computer equipment and a storage medium, and creatively provides a method and system for screening new anti-colorectal cancer drugs based on PRMT5 and ALKBH5 or PRMT5 and CD276 for the field, computer equipment and a storage medium. The invention provides an efficient, accurate and rapid screening method for research and development of novel anti-colorectal cancer drugs, and has a wide application prospect in screening of the novel 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, and more specifically, 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] Worldwide, the incidence of colorectal cancer (CRC) ranks third among all types of tumors in both male and female patients, and its mortality rate is also among the top three. With the continued development of developing countries, the global incidence of colorectal cancer is expected to increase to 2.5 million new cases by 2035. The onset and pathological progression of colorectal cancer involve a series of genetic and epigenetic changes and are affected by the tumor immune microenvironment (TIME). Although genes, lifestyle, obesity and environmental factors are all associated with the onset of colorectal cancer, the exact reasons for the increase in its incidence and mortality are still not fully understood. With the deepening of biomedical and oncology research, the molecular mechanisms of colorectal cancer and the treatment methods of specific molecular targets are constantly being discovered. Exploring the molecular mechanisms of colorectal cancer and developing innovative treatment plans targeting specific molecular targets to prevent the malignant progression of colorectal cancer patients has received increasing attention.

[0003] In mammals, PRMTs are divided into three categories according to the form of arginine methylation modification. Type I includes PRMT1, PRMT2, PRMT3, PRMT4, PRMT6 and PRMT8, which can catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA). Type II includes PRMT5 and PRMT9, which catalyze the formation of MMA and symmetric dimethylarginine (SDMA), while type III PRMT7 is only known to catalyze the formation of MMA. Protein arginine methyltransferase 5 (PRMT5) is the main methyltransferase catalyzing SDMA. Symmetric dimethylation of proteins mediated by PRMT5 affects many biological processes, such as epigenetic control of gene expression, splicing regulation, circadian rhythm, DNA damage response, and germ cell development and pluripotency. Compared with PRMT5 homodimers, the PRMT5 / MEP50 complex shows higher affinity for SAM and target substrates, resulting in higher methylation activity of the hetero-octameric PRMT5 / MEP50 complex.

[0004] The tumor immune microenvironment is a collection of interactions between tumor cells and tumor-infiltrating immune cells. CD276 is a member of the immune checkpoint protein B7 family. It is highly expressed in tumor cells and activated tumor immune-infiltrating cells, helping tumor cells to evade the surveillance of cytotoxic T cells and natural killer cells (NK cells). It is closely related to the initiation, progression and response of tumor immunotherapy. It is considered to be an immune co-stimulatory or inhibitory factor that regulates the interaction between tumors and the immune system. Initial studies found that CD276 is a T cell stimulatory protein that can bind to an unknown counter-receptor to increase the production of IFN-γ during the activation of T cells. However, more and more studies have found that CD276 plays a negative regulatory role in human and mouse T cell immune responses by inhibiting T cell activation. Highly expressed CD276 can promote tumor progression through immune and non-immune mechanisms. Studies have shown that high expression of CD276 can activate JAK / STAT3, PI3K / AKT / mTOR, TLR4 / NF-KB and other pathways, and can promote the expression of IL8, 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 cancers with the highest morbidity and mortality rates worldwide. Existing treatments (such as surgery, chemotherapy, radiotherapy, etc.) have limited effects in some colorectal cancer patients and are often accompanied by serious side effects. The development of new drugs can target specific targets of colorectal cancer cells, improve the effectiveness of treatment, and reduce damage to normal cells. Therefore, there is an urgent need 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 art.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions: 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: Obtaining 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, 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 a candidate drug 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 of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and the ALKBH5 protein complex, or the spatial structure of the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and the CD276 protein complex, screening small molecule compounds with similar structures in a molecular database; The screened small molecule compounds are subjected to molecular docking with 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 the score of each small molecule compound, and the anti-colorectal cancer candidate drugs are obtained by sorting according to the score.

[0008] Further, 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; 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.

[0009] Furthermore, 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.

[0010] 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: 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 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 of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and the ALKBH5 protein complex, or the spatial structure of the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and the CD276 protein complex, screening small molecule compounds with similar structures in a molecular database; The screened small molecule compounds are subjected to molecular docking with 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 the score of each small molecule compound, and the anti-colorectal cancer candidate drugs are obtained by sorting according to the score.

[0011] Further, 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; 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.

[0012] Furthermore, 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.

[0013] 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: 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 anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 described in the first aspect of the present invention is implemented.

[0014] 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.

[0015] In a fifth aspect, the present invention provides an anti-colorectal cancer candidate drug screened by 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.

[0016] In a sixth aspect, the present invention provides any of the following applications: (1) Use of PRMT5 inhibitors in the preparation of drugs for treating colorectal cancer; (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; (3) Use of CD276 inhibitors in the preparation of drugs for the treatment of colorectal cancer; (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; (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.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention innovatively provides a new screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 in the art. 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

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. 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.

[0020] Figure 1 : A schematic 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; Figure 2 : A 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; Figure 3 : A 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; Figure 4: PRMT5 affects the stability of ALKBH5 protein, including AC figure: Western blot and qRT-PCR experiments to detect the effect of GSK595 on the expression and transcription level of related m6A methylase 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 the half-life of ALKBH5 protein after treatment with protein synthesis inhibitor CHX (50μg / mL) by GSK595; JK figure: Western Blot experiments were used to detect the changes in the half-life of ALKBH5 protein after knocking down PRMT5 in CHX (50 μg / mL) (***P<0.001, **P<0.01, *P<0.05); LM: Western blot experiments were used to detect the effects of proteasome inhibitor MG132 (10 μM / mL) on the degradation of ALKBH5 protein in HCT116 and MC38 cells overexpressing PRMT5 after 6 hours of treatment; NP: Co-IP and Western blot were used to detect the effects of PRMT5 expression level and its enzyme activity level on the Ub ubiquitination level of ALKBH5; Figure 5: PRMT5-mediated ALKBH5-R316 methylation enhances ALKBH5 ubiquitination and degradation, where AB: IP experiments to detect the interaction between endogenous PRMT5 and ALKBH5; CD: Co-IP experiments to detect the interaction between exogenous Flag-ALKBH5 and Myc-PRMT5; EF: IP experiments to detect the MMA and SDMA levels of endogenous ALKBH5; G: GST pull-down experiments to detect the binding of PRMT5 to ALKBH5 in vitro; H: exomemethylation experiments to detect that PRMT5 directly mediates methylation of ALKBH5 in vitro; IK: Western Blot detection of PRMT5 expression level and its enzyme activity level affecting the SDMA modification level of ALKBH5; L: Co-IP experiment to detect the SDMA methylation level of each ALKBH5 mutant; M: Mass spectrometry analysis of R316-ALKBH5 methylation; N: Sequence alignment of ALKBH5 in mammals, ALKBH5-R316 site is located in the protein sequence; O: In vitro methylation experiment to detect SDMA of GST-ALKBH5-WT (292-395AA) and GST-ALKBH5-R316K (292-395AA); PQ: Western blot detection of changes in ALKBH5 protein half-life after CHX (50μg / mL) treatment of HCT116 and MC38 cells overexpressing ALKBH5 wild-type or mutants; RS: Co-IP and Western 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); Figure 6: PRMT5-mediated ALKBH5-R316 methylation enhances the binding of TRIM28 to ALKBH5 and promotes the ubiquitination and degradation of ALKBH5, where AB: IP experiments to detect the interaction between endogenous ALKBH5 and TRIM28; CD: Co-IP experiments to detect the interaction between exogenous ALKBH5 and TRIM28; EF: Western blot and qRT-PCR experiments to detect the expression of ALKBH5 protein and mRNA; GH: Western blot experiments to detect the effect of TRIM28 on the protein half-life of ALKBH5 after CHX (50μg / mL) treatment; IJ: Western blot experiments to detect the effect of MG132 (10μM / mL) treatment for 6 hours on the degradation of ALKBH5 protein in HCT116 and MC38 cells overexpressing TRIM28 (***P<0.001, **P<0.01, *P<0.05); K: Co-IP and Western Blot detection of the effect of TRIM28 on the Ub ubiquitination level of ALKBH5; LN Figure: Co-IP and Western blot experiments detected that PRMT5-mediated ALKBH5 methylation affects the binding of TRIM28 and ALKBH5; O Figure: Co-IP and Western blot experiments detected that PRMT5 affects the binding of TRIM28 and ALKBH5 through ALKBH5-R316 methylation modification; 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 and analysis of regulated downstream genes; Figure C: TCGA database analysis of the correlation between PRMT5 expression and CD276, DNAH17-AS1, POTEI, and SDCBP2-AS1; Figure D: Pearson correlation coefficient was used to test the correlation between PRMT5 and CD276 protein expression levels; Figure EH: Western blots and qRT-PCR detection of CD276 protein and mRNA expression; Figure IJ: Effect of PRMT5 expression level or enzyme activity on CD276 Effect of mRNA abundance (***P<0.001, **P<0.01, *P<0.05); K: The peak distribution of mA in CD276 mRNA is mainly concentrated in CD276-3`UTR; L: Schematic diagram of mA site mutation in CD276-3`UTR; M: Relative luciferase activity of ALKBH5 overexpressing cells treated with wild-type or mutant CD276-`UTR reporter plasmids; Figure 8 :ALKBH5-R316 methylation sites affect tumor immune evasion in colorectal cancer cells, among which, Figure A: TIMER2.0 database analysis showed that the expression of ALKBH5 was positively correlated with CD8+T, CD4+T, 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 Image; 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 tissue sections of subcutaneous tumors; G: 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); Fig. 9 : PRMT5-mediated ALKBH5-R316 methylation is associated with poor prognosis in patients with colorectal cancer, where A: IHC staining of PRMT5 and ALKBH5-R316 methylation in colorectal cancer tissue microarray; B: Pearson correlation coefficient was used to test the correlation between PRMT5 and ALKBH5-R316 methylation; C-E: χ 2 The high expression of ALKBH5-R316 methylation was analyzed and correlated with the expression of PRMT5, tumor size, and the proportion of lymph node metastasis (***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 expression of ALKBH5-R316 methylation was positively correlated with the 5-year overall survival rate of colorectal cancer (P<0.001, log-rank test); and positively correlated with the 5-year disease-free survival rate of colorectal cancer patients (P<0.001, log-rank test); Fig.10: The combined treatment of PRMT5 inhibitor and anti-PD1 antibody significantly inhibited the progression of colorectal cancer, among which, Figure 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 mouse subcutaneous tumor treatment model; Figure D: Tumor anatomical image after 2 weeks of mouse subcutaneous tumor treatment model; Figure E: Tumor growth curve of each group; Figure F: Statistics of tumor weight 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

[0021] 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.

[0022] In some of the processes described in the specification and claims of the present invention and the above-mentioned drawings, multiple operations appearing in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this document or may be executed in parallel. The serial numbers of the operations, such as S101, S102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel.

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0024] Figure 1 The present invention provides a method for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276. Specifically, the screening method comprises the following steps: S101: Obtain PRMT5 protein and ALKBH5 protein data, or PRMT5 protein and CD276 protein data; 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, regulating many physiological functions in mammalian cells by symmetrically dimethylating arginine residues on histones and non-histones. The Gene ID of the PRMT5 (protein arginine methyltransferase 5 [Homo sapiens (human)]) in NCBI is 10419, and its corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0025] In one embodiment, the ALKBH5 refers to human demethylase Alk B homologous protein 5 (AlkB homolog5), an RNA demethylase that catalyzes the reversal reaction of RNA methylation modification, i.e., removes methylation modification from mRNA, thereby stabilizing mRNA and enhancing DNA repair ability. The Gene ID of the ALKBH5 (alkB homolog 5, RNA demethylase [Homo sapiens (human)]) in NCBI is 54890, and its corresponding detailed information (including sequence information, etc.) can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0026] In one embodiment, the CD276 (also known as B7-H3) is a type I transmembrane glycoprotein, a member of the B7 superfamily, not expressed in monocytes, granulocytes and normal human tissues, but expressed in 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 inhibiting 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 / .

[0027] In one embodiment, the present invention demonstrates that PRMT5 affects the stability of ALKBH5 protein, specifically as follows: First, the present invention proves that PRMT5 regulates the m6A modification level in colorectal cancer cells through ALKBH5. Since m6A modification is mainly catalyzed by Writer (methyltransferase): METTL3, METTL14, METTL16, and demethylated by Eraser (demethyltransferase): FTO and ALKBH5, Western blot was used to detect the effect of PRMT5 inhibitor GSK595 on the expression level 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 GSK595 treatment ( Figure 4 However, the ALKBH5 protein level was significantly increased ( Figure 4 qRT-PCR experiments further revealed that the 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).

[0028] Secondly, the present invention proves 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 detected 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), knockdown of PRMT5 promoted the increase of ALKBH5 protein level ( Figure 4 F), while the transcription level did not change significantly ( Figure 4 Figure G in the figure).

[0029] 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 show that in HCT116 and MC38 cells treated with the PRMT5 inhibitor GSK595, the half-life of ALKBH5 is significantly prolonged compared with the control group ( Figure 4 Figure H in Figure 4 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 1.

[0030] 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 The L graph in Figure 4 M in the figure), which can inhibit the decrease in ALKBH5 expression caused by PRMT5 overexpression, further verifying that PRMT5 can degrade ALKBH5 through the ubiquitin proteasome pathway.

[0031] 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 are treated 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 N in Figure ), the Ub ubiquitination level of ALKBH5 was reduced after knocking down PRMT5 ( Figure 4The Ub ubiquitination level of ALKBH5 was reduced after treatment with the PRMT5 inhibitor GSK595 ( Figure 4 Figure P in the figure).

[0032] In one embodiment, the present invention demonstrates that PRMT5-mediated ALKBH5-R316 methylation enhances ALKBH5 ubiquitination degradation, specifically as follows: First, the present invention proves that PRMT5 can bind to ALKBH5 and undergo symmetrical dimethylation modification. The present invention found that PRMT5 and ALKBH5 bind to each other 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, an exogenous immunoprecipitation experiment (Co-IP) was performed using the corresponding tag antibodies to verify the interaction between ALKBH5 and PRMT5 proteins ( Figure 5 Figure C in Figure 5 In order to explore whether ALKBH5 can undergo arginine methylation modification, endogenous immunoprecipitation (IP) experiments were performed with ALKBH5 antibody in HCT116 and MC38 cells, respectively, and detection was performed with 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).

[0033] 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 of ALKBH5, considering that PRMT5 can be self-methylated, 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 constructs GST-ALKBH5 and GST-ALKBH5 (292-395aa) truncated plasmids, thereby distinguishing GST-ALKBH5 from PRMT5 in terms of protein molecular weight. Subsequently, after inducing protein expression, the protein is 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 conducts a GST pull-down experiment, and the results show 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).

[0034] Again, the present invention proves that changes in PRMT5 expression level 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 Figure 2), the SDMA modification level of ALKBH5 was reduced after knocking down PRMT5 ( Figure 5 The SDMA modification level of ALKBH5 was reduced after treatment with the PRMT5 inhibitor GSK595 ( Figure 5 Figure 1.

[0035] Again, the present invention proves that arginine 316 (R316) is the main site for PRMT5-mediated methylation modification of ALKBH5. In order to explore the specific sites of ALKBH5 arginine methylation modification by PRMT5, we obtained methylated ALKBH5 protein through in vitro methylation reaction, and then performed mass spectrometry identification. The results showed that arginine residues (R) at sites 249, 250, 269, 283, 316, 357, and 359 may be methylated by PRMT5 (Table 1). Table 1 ALKBH5 methylation modification sites No Site Sequence 1 R249 RVSEPVLSLPVRR 2 R250 RRGSVTVLSGYAADEITHCIRPQDIKE 3 R269 RGSVTVLSGYAADEITHCIRPQDIKE 4 R283 RAVIILRK 5 R316 RLSGNNRDPALKPKR 6 R357 RSVLLPTHRR 7 R359 RRGSFSSENYWRK

[0036] In order to clarify 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 in HEK293T cells, Co-IP experimental detection found that compared with other mutant groups, the ALKBH5-R316K mutation significantly reduced the PRMT5-mediated ALKBH5SDMA modification level ( 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 indicates 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 the figure). Subsequently, the present invention constructed a GST-R316K-ALKBH5 (292-395aa) mutant plasmid, induced protein expression, and obtained GST-ALKBH5-WT (292-395aa) and GST-ALKBH5-R316K (292-395aa) proteins by purifying the proteins. The present invention conducted an in vitro methylation experiment using HMT Assay, and the results showed that the SDMA modification of GST-R316K-ALKBH5 (292-395aa) was significantly inhibited ( Figure 5 The above data indicate that ALKBH5 is symmetrically dimethylated at R316 by PRMT5 (meR316-ALKBH5, meALKBH5).

[0037] Finally, the present invention proves 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. In view of the fact 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 found 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, 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 was higher than the ALKBH5 mutant overexpression group in ALKBH5 ubiquitination level ( Figure 5 The R graph in Figure 5 S Figure in Figure 5). These results suggest that PRMT5-mediated ALKBH5-R316 methylation reduces the protein stability of ALKBH5 by enhancing ALKBH5 ubiquitination and degradation. In summary, PRMT5-mediated ALKBH5-R316 methylation plays a key role in regulating the stability of ALKBH5.

[0038] In one embodiment, the present invention demonstrates that PRMT5-mediated ALKBH5-R316 methylation enhances the binding of TRIM28 to ALKBH5 and promotes ALKBH5 ubiquitination degradation, specifically as follows: First, the present invention proves that TRIM28 can bind to ALKBH5. The present invention searches for potential E3 ubiquitin ligases that cause ALKBH5 ubiquitination degradation through mass spectrometry detection, and finds that ALKBH5 and TRM28 may bind to each other. The present invention extracts HCT116 cell and MC38 cell proteins and performs endogenous immunoprecipitation experiments (IP) to find that there is an interaction between ALKBH5 and TRIM28 proteins ( Figure 6 Figure 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 corresponding tag antibodies were used to perform exogenous immunoprecipitation experiments (Co-IP) to verify the interaction between ALKBH5 and TRIM28 proteins ( Figure 6 Figure C in Figure 6 Figure D in the figure).

[0039] Secondly, the present invention proves that TRIM28 affects the expression of ALKBH5 protein. In order 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).

[0040] 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 overexpressed TRIM28. After CHX treatment, the results of Western Blot experiments showed that the protein half-life of ALKBH5 in HCT116 and MC38 cells that overexpressed TRIM28 was significantly shorter than that in the control group ( Figure 6 The G graph in Figure 6 Subsequently, the proteasome inhibitor MG132 was used to treat HCT116 and MC38 cells overexpressing TRIM28 and the control group 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 In the I diagram, Figure 6 This further verified that the E3 ubiquitin ligase TRIM28 can degrade ALKBH5 through the ubiquitin proteasome pathway.

[0041] Again, the present invention proves that TRIM28 promotes the ubiquitination 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 1.

[0042] 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 detects the effect of PRMT5-mediated ALKBH5-R316 methylation on the binding of TRIM28 to ALKBH5 by Co-IP and Western blot experiments. The results show that meR316-ALKBH5 increases after overexpression of PRMT5, that is, the SDMA modification level of ALKBH5 increases, and at the same time, the binding of TRIM28 to ALKBH5 increases ( Figure 6 L in Figure ), after knocking down PRMT5, 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 M in the figure), 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 the figure). Subsequently, the present invention co-transfected PRMT5 plasmid and Flag-ALKBH5-WT and Flag-ALKBH5-R316K in HCT116 to perform Co-IP experiments. Western blot results showed that meR316-ALKBH5 was significantly reduced in cells overexpressing R316K-ALKBH5, that is, the 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 suggest that PRMT5-mediated ALKBH5-R316 methylation reduces ALKBH5 stability and depends on recruiting TRIM28 to ubiquitinate and degrade ALKBH5.

[0043] In one embodiment, the present invention demonstrates that PRMT5-mediated meR316-ALKBH5 enhances CD276 mRNA stability through m6A modification, specifically as follows: 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 meR316-ALKBH5 through m6A modification mediated by PRMT5, HCT116 cells treated with the PRMT5 inhibitor GSK595 were detected using m6A MeRIP-seq and RNA-seq. After treatment with GSK595, 2408 genes showed reduced m6A modification and 12 genes showed reduced 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 2). 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 C in Figure 2, R=0.12, P=0.025). Finally, the present invention used anti-PRMT5 and anti-CD276 antibodies to perform IHC detection on the colorectal cancer tissue combination chip (including 84 cases of rectal adenocarcinoma tissues), and found through linear correlation analysis that the expression levels of PRMT5 and CD276 were positively correlated (correlation coefficient was 0.3598, P values ​​were <0.0001) ( Figure 7 The above experimental results confirmed that CD276 is a target gene regulated by ALKBH5 through m6A modification mediated by PRMT5.

[0044] Secondly, the present invention proves that changes in PRMT5 expression level or enzyme activity affect the expression of CD276 at the transcriptional level and protein level. After HCT116 cells were treated with PRMT5-specific inhibitor GSK595, qRT-PCR experiments revealed that CD276 was significantly reduced at the mRNA level ( Figure 7 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 CD276 mRNA levels were significantly increased ( Figure 7 Western blot analysis revealed that the protein level of CD276 was elevated ( Figure 7 Figure H in the figure).

[0045] Again, the present invention proves 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 At the same time, after overexpression of PRMT5 in HCT116 cells, actinomycin D (an inhibitor of RNA transcription synthesis) was added, and the mRNA half-life of CD276 was significantly increased by qRT-PCR ( Figure 7 Figure J in Figure 1).

[0046] Finally, the present invention proves that ALKBH5 regulates CD276 through m6A modification. MeRIP-seq data analysis found 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 In order to identify the detailed ALKBH5 binding sites in CD276-3`UTR, we used the SRAMP database (http: / / www.cuilab.cn / sramp) to analyze the m6 modification sites, selected two potential sites with high confidence according to the score, and constructed wild-type WT, mutant Mut1, mutant Mut2 and co-mutant Mut1+Mut2 reporter genes ( Figure 7 L in Figure 1). The dual luciferase reporter gene experiment revealed that the luciferase activity was reduced in the group co-transfected with the wild-type CD276-3`UTR-WT reporter plasmid and over-expressed ALKBH5 plasmid, while the luciferase expression was restored in the group co-transfected with the mutant CD276-3`UTR-Mut1 reporter plasmid and over-expressed ALKBH5 plasmid. The luciferase expression was reduced or partially reduced in the group co-transfected with the mutant CD276-3`UTR-Mut2 or the co-mutant CD276-3`UTR-Mut1+Mut2 and over-expressed ALKBH5 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.

[0047] In one embodiment, the present invention demonstrates that the ALKBH5-R316 methylation site affects tumor immune escape in colorectal cancer cells, specifically as follows: First, the present invention proves that ALKBH5 expression and immune cell infiltration are positively correlated. 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). At the same time, it was 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 presence of a large number of tumor-infiltrating immune cells in the colorectal cancer tumor microenvironment.

[0048] Secondly, the present invention proves that in vitro phenotypic experiments to explore the methylation sites of ALKBH5-R316 can promote T cells to kill tumors. Through RTCA detection, it was found that when the same T cell effector-target ratio was given to co-incubate T cells in HCT116 cells (effector-target ratio 1:1), the number of surviving cells in the co-incubation group with overexpression of ALKBH5-WT plus T cells was less than that in the co-incubation group with overexpression of ALKBH5-R316K plus T cells, and the number of surviving cells was significantly inhibited as the co-incubation time was prolonged ( Figure 8 However, the difference in the number of surviving cells between the Vector-overexpressing group and the T-cell-co-cultured group and the ALKBH5-R316K-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.

[0049] Finally, the present invention proves 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 cells MC38, and then used female C57BL / 6 mice to subcutaneously inject the above three cells to construct a subcutaneous tumor model of colorectal cancer (the mice were randomly divided into 3 groups, n = 5, 5 × 10 5Cell volume). In 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, and 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 8 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).

[0050] The methylation level of ALKBH5-R316 in tumor tissue sections was detected by immunohistochemistry (IHC) staining, which confirmed that the present invention detected high levels of ALKBH5 methylation only 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 In the F graph, 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.

[0051] In one embodiment, the present invention demonstrates that PRMT5-mediated ALKBH5-R316 methylation is associated with poor prognosis of colorectal cancer patients, specifically as follows: First, the present invention proves that PRMT5 and ALKBH5-R316 methylation are positively correlated in colorectal cancer. In order to evaluate the clinical significance of our research, the present invention used anti-PRMT5 and anti-ALKBH5-R316 methylation antibodies to perform IHC detection 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 ( Fig. 9 A in the figure). The present invention found through linear correlation analysis that there was a positive correlation between the expression levels of PRMT5 and ALKBH5-R316 methylation (meALKBH5) (the correlation coefficients were 0.4365, and the P values ​​were <0.0001) ( Fig. 9 Figure B in the figure).

[0052] 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% ( Fig. 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% ( Fig. 9 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% ( Fig. 9 Figure E in the figure).

[0053] Finally, the present invention proves that ALKBH5-R316 methylation can be used as an independent prognostic factor for colorectal cancer. ALKBH5-R316 methylation immunohistochemical staining was performed on tissue chips 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 ( Fig. 9 Figure F in Figure 2). 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 ( Fig. 9 G in Figure ) and disease-free survival ( Fig. 9 H in Figure ).

[0054] 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 cases of colorectal cancer showed that the expression of ALKBH5-R316 methylation (P < 0.001), TNM stage (P < 0.001), tumor size (P < 0.001), and differentiation degree (P < 0.001) were significantly correlated with the 5-year overall survival rate and 5-year disease-free survival rate of patients in univariate regression analysis; the expression of ALKBH5-R316 methylation (P < 0.05) was significantly correlated with the 5-year overall survival rate and 5-year disease-free survival rate in multivariate regression analysis. The above results strongly support that the high expression of ALKBH5-R316 methylation is closely related to the poor prognosis of colorectal cancer patients and can be used as an independent prognostic factor to determine the prognosis of breast cancer. These results confirm our hypothesis that PRMT5-mediated ALKBH5-R316 methylation inhibits the immune infiltration of colorectal cancer cells by reducing the stability of ALKBH5.

[0055] In one embodiment, the present invention demonstrates that the combined treatment of PRMT5 inhibitors and anti-PD1 antibodies significantly inhibits the progression of colorectal cancer, specifically as follows: First, the present invention demonstrates that reduction in PRMT5 expression level or enzyme activity promotes anti-tumor T cell killing.

[0056] The present invention uses HCT116 cells with stable knockdown of PRMT5, and through RTCA detection, it is found that when the same T cell effector-target ratio (effector-target ratio 1:1) is given to co-incubate T cells, compared with the stable transfection shCtrl plus T cell co-incubation group, the number of surviving cells in the stable knockdown shPRMT5 plus T cell co-incubation group is less, and as the co-incubation time is prolonged, the number of surviving cells is significantly inhibited ( Fig.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), compared with the DMSO plus T cell co-incubation group, the number of surviving cells in the GSK595-treated cells co-incubated with T cells was less, and as the co-incubation time increased, the number of surviving cells was significantly inhibited ( Fig.10 The above results suggest that reduced PRMT5 expression or enzyme activity can promote anti-tumor T cell killing.

[0057] Then, the present invention demonstrates that the combination of PRMT5 inhibitor and anti-PD1 antibody significantly inhibits the progression of colorectal cancer. The present invention uses female C57BL / 6 mice to inoculate mouse colorectal cancer cells MC38 (5×10 5 The subcutaneous injection of cells was used to construct a subcutaneous tumor model of colorectal cancer. After 7 days, 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 was intraperitoneally injected at 5 mg / kg daily, and IgG or PD-1Ab was intraperitoneally injected at 200 μg every 3 days for treatment ( Fig.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 ( Fig.10 Figure D in Figure 2 shows that compared with the PBS+IgG group, the tumor growth in the GSK595+IgG group and the PBS+PD-1Ab group was inhibited, and the inhibition of tumor growth in the GSK595+PD-1Ab group was the most obvious ( Fig.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 ( Fig.10 The above results suggest that GSK595 combined with PD-1Ab can effectively inhibit the growth of colorectal cancer cells in mice.

[0058] 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 ( Fig.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 ( Fig.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 group and the PBS+PD-1Ab group increased, and the number of cells positive for tumor immune infiltration-related markers CD3, CD8a, and GZMB in the GSK595+PD-1Ab group increased most significantly ( Fig.10 F in Figure ), and the difference was statistically significant ( Fig.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.

[0059] In this study, the inventors of the present invention integrated MeRIP-seq and RNA-seq sequencing analysis to find potential target genes in colorectal cancer cells treated with PRMT5 inhibitors and knockdown PRMT5. Through screening, it was found that CD276 may be regulated by m6A modification. Subsequently, experiments showed that CD276 was positively correlated with PRMT5 at both the transcriptional level and the protein level, proving the interaction between ALKBH5 protein and CD276 mRNA. Through the analysis of MeRIP-seq, it was found that the m6A modification on the 3'UTR region of CD276 mRNA in PRMT5-mediated meR316-ALKBH5 was significantly increased, further confirming that PRMT5-mediated meR316-ALKBH5 can increase the stability of CD276 mRNA and promote its protein level increase, thereby giving full play to the inherent biological function of CD276 to affect the progression of colorectal cancer.

[0060] Subsequently, the inventors of the present invention used tumor tissue specimens from patients with colorectal cancer to analyze the correlation between meR316-ALKBH5 expression levels and clinical pathological characteristics through immunohistochemical experiments. The data showed that high meR316-ALKBH5 levels were positively correlated with TNM, invasion depth, lymph node metastasis and tumor size in CRC patients. In addition, the results of immunohistochemical 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 in patients with colorectal cancer, and reveal the relationship between meR316-ALKBH5 and clinical malignant outcomes in patients with colorectal cancer.

[0061] 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), which enhanced the binding of ALKBH5 to TRIM28 and led 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 patients with colorectal cancer. Subsequently, this study demonstrated that PRMT5-mediated meR316-ALKBH5 increased CD276 mRNA stability through m6A modification, thereby enhancing colorectal cancer tumor immune escape in vitro and in vivo. Finally, this study demonstrated that the combination of PRMT5 inhibitors and anti-PD1 antibodies significantly curbed the progression of colorectal cancer. The results of this study provide a theoretical basis for exploring colorectal cancer treatment strategies targeting the PRMT5-meR316-ALKBH5-CD276 signaling axis.

[0062] In one embodiment, the present invention demonstrates that there is an obvious binding between PRMT5 protein and ALKBH5 protein, and 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. The screening method can be used to effectively and quickly screen new anti-colorectal cancer drugs, and has broad application prospects.

[0063] 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. The screening method can be used to effectively and quickly screen new anti-colorectal cancer drugs, and has broad application prospects.

[0064] 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 the binding site of the targeted drug; 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; 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.

[0065] 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.

[0066] S103: using a computer-assisted drug screening method to obtain a candidate drug 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 of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and the ALKBH5 protein complex, or the spatial structure of the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and the CD276 protein complex, screening small molecule compounds with similar structures in a molecular database; The screened small molecule compounds are subjected to molecular docking with 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 the score of each small molecule compound, and the anti-colorectal cancer candidate drugs are obtained by sorting according to the score.

[0067] In one embodiment, 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.

[0068] 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 protection scope of the present invention.

[0069] In one embodiment, in molecular docking, affinity refers to how tightly a molecule binds to a receptor. A high affinity means that the binding is more stable, while a low affinity means that the binding is less stable. Affinity is usually calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0070] In order to evaluate affinity, some scoring systems or scoring methods are usually used to quantify the interaction between molecules and receptors. These scoring methods are based on different algorithms and physical models, which can reflect the binding energy, interaction type and affinity between molecules and receptors.

[0071] 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 anti-colorectal cancer candidate drugs are obtained by sorting according to the scores. Small molecule compounds with higher affinity or binding energy targeting the receptor ALKBH5 protein or CD276 protein are preferably selected as anti-colorectal cancer candidate drugs.

[0072] 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, protein-nucleic acid docking, which are all well-known to those skilled in the art.

[0073] 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 often used for drug virtual screening.

[0074] 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: 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 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 of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and the ALKBH5 protein complex, or the spatial structure of the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and the CD276 protein complex, screening small molecule compounds with similar structures in a molecular database; The screened small molecule compounds are subjected to molecular docking with 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 the score of each small molecule compound, and the anti-colorectal cancer candidate drugs are obtained by sorting according to the score.

[0075] 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; 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.

[0076] In one embodiment, 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.

[0077] Figure 3 : is a schematic diagram of a computer device for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, provided by an embodiment of the present invention. Specifically, the computer device includes: 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 anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 described in the first aspect of the present invention is implemented.

[0078] An embodiment of the present invention further provides a computer-readable storage medium having a computer program stored thereon. When the computer program is executed by a processor, 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 is implemented.

[0079] The embodiment of the present invention also provides an anti-colorectal cancer candidate drug screened 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.

[0080] The embodiments of the present invention also provide any of the following applications: (1) Use of PRMT5 inhibitors in the preparation of drugs for treating colorectal cancer; (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; (3) Use of CD276 inhibitors in the preparation of drugs for the treatment of colorectal cancer; (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; (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.

[0081] The verification results of this verification example show that assigning inherent weights to indications can moderately improve the performance of the method relative to the default settings. Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working 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.

[0082] In the several embodiments provided in the present 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 only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, 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.

[0083] The units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they may be located in one place, or they may be distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the scheme of this embodiment. In addition, the functional units in the various embodiments of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated units may be implemented in the form of hardware or in the form of software functional units.

[0084] A person of ordinary skill in the art can 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, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0085] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related 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.

[0086] 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 a general technician in this field, according to the ideas of the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276, characterized in that: The screening method comprises: Obtaining 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 of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and the ALKBH5 protein complex, or the spatial structure of the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and the CD276 protein complex, screening small molecule compounds with similar structures in a molecular database; The screened small molecule compounds are subjected to molecular docking with 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 the score of each small molecule compound, and the anti-colorectal cancer candidate drugs are obtained by sorting according to the score.

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 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; 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.

3. 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.

4. A screening system for screening 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 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 of the PRMT5 protein and the ALKBH5 protein in the PRMT5 protein and the ALKBH5 protein complex, or the spatial structure of the binding site of the PRMT5 protein and the CD276 protein in the PRMT5 protein and the CD276 protein complex, screening small molecule compounds with similar structures in a molecular database; The screened small molecule compounds are subjected to molecular docking with 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 the score of each small molecule compound, and the anti-colorectal cancer candidate drugs are obtained by sorting according to the score.

5. The screening system for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 according to claim 4, characterized in that: 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; 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.

6. The screening system for screening anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 according to claim 4, 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.

7. 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 as described in any one of claims 1-3 is implemented.

8. 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, the screening method for anti-colorectal cancer drugs based on PRMT5 and ALKBH5, or PRMT5 and CD276 as described in any one of claims 1 to 3 is implemented.

9. 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 any one of claims 1 to 3.

10. Any of the following applications: (1) Use of PRMT5 inhibitors in the preparation of drugs for treating colorectal cancer; (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; (3) Use of CD276 inhibitors in the preparation of drugs for the treatment of colorectal cancer; (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; (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.

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