Preliminary Study Of ABHD5 Regulating Arginine Methylation Of P53 In Colon Cancer Cells

Background: Colon cancer is one of the most common malignant tumors of the digestive tract,and its morbidity and mortality are increasing year by year.The occurrence of colon cancer is a complex process involving multi-gene participation,multi-stage and multi-factor effects,and the process of its occurrence and development is still unclear.Colon adenoma is currently considered to be the most precancerous lesion of colon cancer.The size,number and degree of adenoma are closely related to the incidence of colon cancer.95% of colon cancers are caused by adenoma carcinogenesis.The adenoma-cancer sequential theory has more and more evidence to support it.p53 is a very important tumor suppressor,which is mutated in about 50% of tumors.The decrease of p53 gene expression and transcriptional activity is an important driving factor for adenoma carcinogenesis,but the expression of p53 and transcriptional activity during adenoma carcinogenesis decline.It is not clear what kind of mechanism is regulated.Metabolic reprogramming is one of the important features of malignant tumors.A variety of metabolic related genes show changes in expression or activity during tumor development.ABHD5(abhydrolase domain containing 5)plays an important role in tumor suppressor gene in colon cancer.ABHD5 gene deletion can lead to decreased autophagy and promote tumor progression.Our study found that ABHD5 expression gradually decreased during adenoma carcinogenesis,accompanied by decreased p53 expression.We hypothesized that ABHD5 regulates p53 expression and transcriptional activity as an important driving factor for colon adenoma carcinogenesis,and initially explored ABHD5.The mechanism of interaction with p53.Objective1.Detection of correlation between ABHD5 and p53 in colon adenomas and adenocarcinoma.2.To explore the molecular mechanism of ABHD5 regulating p53 arginine methylation in colon cancer cells.Method:Immunohistochemical staining was used to detect the expression of ABHD5 and p53 in colon adenoma and normal tissues adjacent to adenoma.2.Establish a colony adenocarcinoma LS180 cell ABHD5 low expression cell line.Western blot was used to detect the expression of p53 protein,p53 arginine methylation(ARG333,335,337)in the control group and ABHD5 interference group,p21 RNA level was detected by RT-PCR,and cell proliferation ability was detected by soft agar colony assay.3.Establish a low expression cell line of colonic adenocarcinoma LS180 cells with ATGL.Western blot was used to detect the expression of p53 protein in the control group and ATGL interference group,p53 arginine methylation(ARG333,335,337)level,Western blot analysis The expression of PRMT5 protein in the ABHD5 interference group and protein immunoprecipitation(CO-IP)were used to detect the interaction between ABHD5 and PRMT5.4.Establish a cell line of colonic adenocarcinoma LS180 cells that overexpresses ABHD5.The binding between PRMT5 and Hsp90 in the control group and the ABHD5 interference group was detected by Western blot.The interaction between ABHD5 and Hsp90 was detected by protein immunoprecipitation(Co-IP).The distribution of ABHD5 and Hsp90 subcellular cells was detected by immunofluorescence.Results:1.Immunohistochemical staining showed that ABHD5 was lowly expressed in colon cancer and colon adenoma,and the expression was significantly lower than that in normal tissues.There was a positive correlation between ABHD5 and P53 expression in colon adenoma and colon cancer.ABHD5 deletion promoted the proliferation of colonic adenocarcinoma LS180 cells.RT-PCR and Western blot showed that p53 protein expression decreased and p21 mRNA level increased after knockdown of ABHD5.2.Western blot results showed that the level of p53 arginine methylation(ARG333,335,337)increased significantly after interfering with ABHD5 expression,and ABHD5 deletion promoted the expression of arginine methylase PRMT5,which was confirmed by RT-PCR and Western blot.The use of PRMT5 inhibitors or knockdown of PRMT5 expression in ABHD5 knockdown cell lines significantly reversed p53 protein levels and transcriptional activity in ABHD5-deficient cells;3.Overexpressing ABHD5(Q130P,E260K)can significantly reverse the expression of PRMT5.The expression of p53 protein and p53 mRNA indicate that ABHD5 does not depend on its classical metabolic pathway to regulate p53 expression and transcriptional activity.There is no binding between ABHD5 and PRMT5,which were found by co-immunoprecipitation.4.Co-immunoprecipitation demonstrated that ABHD5 competes with PRMT5 for binding to Hsp90.ABHD5 deletion leads to increased binding of PRMT5 and Hsp90,and decreased PRMT5 ubiquitination.Immunofluorescence results also confirmed that ABHD5 and Hsp90 are combined.Conclusion:1.Immunohistochemical staining showed that ABHD5 was positively correlated with P53 expression in colon adenoma and colon cancer.2.ABHD5 regulates p53 arginine methylation through a non-classical metabolic pathway.3.ABHD5 induces arginine methylation by regulating the expression of the methyltransferase PRMT5.