| Background Colorectal cancer(CRC)is a common disease and can cause cancer-related mortality worldwide.Although the rapid progress in molecular genetics of colorectal cancer give more opportunity to treat it,the prognosis is still poor because of chemotherapy resistance.Therefore,in order to improve the effectiveness of its treatment,further studies are urgently needed to help us having a good knowledge in the intrinsic molecular mechanism of the development and occurrence of colorectal cancer.Protein arginine methyltransferase(PRMTs)catalyzes the methylation of arginine residues of various proteins in cells,which is closely related to some key biological processes,such as intracellular signal transduction,gene expression and DNA recombination.As a type II PRMT,PRMT5 catalyzes symmetric dimethylation of histones and non-histones,and id be of significance in cell survival and proliferation.Many studies have shown that expression of PRMT5 is increased in multiple cancer cells,which directly affects development,occurrence and treatment of cancers.Despite the importance of PRMT5 in epigenetic regulation,the molecular mechanisms by which PRMT5 regulates the survival of cancer cells,including colorectal cancer,require further investigation.Metabolic reprogramming is one of the characteristic changes in cancer.In order to meet the needs of energy and biomacromolecule synthesis during malignant proliferation,cancer cells adjust their metabolic phenotype to abnormally activated aerobic glycolysis,namely the "Warburg effect".A large number of studies suggested that active glycolysis link to the self-renewal and differentiation of tumor stem cells,metastasis and recurrence,as well as chemotherapy resistance.Malignant proliferating cancer cells usually exhibit high glucose uptake rate,which is followed by a large accumulation of lactic acid,and accompanied by changes in the expression of metabolic enzymes in the glycolytic pathway.In addition,when the glycolysis function is inhibited,the proliferation and metastasis of cancer are decreased,and chemotherapy resistance can be reversed to a certain extent.Therefore,downregulation of glycolysis may effectively inhibit the progression and chemotherapy resistance of colorectal cancer,which is benefit for improving the prognosis of patients with colorectal cancer.Objectives 1.To investigate the expression of PRMT5 in colorectal cancer and its effect on the proliferation.2.To confirm the effect of PRMT5 on glycolytic metabolism of colorectal cancer cells.3.To explore the molecular mechanism by which PRMT5 promotes cell proliferation of colorectal cancer through regulating cell glycolysis.4.To explore the efficacy of PRMT5 inhibitor combined with 5-FU in the treatment of colorectal cancer.Methods 1.Expression and function of PRMT5 in colorectal cancer.We used TCGA datasets to evaluate the expression of PRMT5 in colorectal cancer,and both m RNA and protein level of PRMT5 in patient-derived colon cancer tissues and corresponding normal tissue were detected.We established stable PRMT5 knock down and overexpression colon cancer cell lines to investigate cell proliferation by MTT and clone formation assays;To determine whether the effect of PRMT5 on proliferation was dependent on its catalytic activity,two small molecule inhibitors of PRMT5,JNJ-64619178 and Tadalafil,were used,and cell proliferation was detected.2.Metabolic profiling changed by PRMT5 inhibitor.Analyses of metabolomics were performed using LC-MS/MS.Compound identification of metabolites was performed,and after normalized to total peak intensity,the processed data were analyzed by R package,where it was subjected to multivariate data analysis,including Pareto-scaled principal component analysis(PCA)and orthogonal partial least-squares discriminant analysis(OPLS-DA).The variable importance in the projection(VIP)value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification.Metabolites with the VIP value >1 and the P values less than 0.05 were considered as statistically significant.KEGG pathway analysis was used to identify the differential metabolic pathways.3.PRMT5 affects cell proliferation by activating glycolysis.We investigated the glucose uptake and evaluated ECAR and OCR using a biological energy analyzer to investigate the energy status of colorectal cancer.In order to clarify that the pro-proliferation effect of PRMT5 on colorectal cancer cells is dependent on glycolysis,we treated the control group and PRMT5 overexpressed colon cancer cells with glycolytic inhibitor 2-DG respectively,then analyzed the cell proliferation.4.Explore the molecular mechanism through which PRMT5 regulates cell proliferation of colorectal cancer.First,we used real-time quantitative polymerase chain reaction(PCR)to investigate the m RNA level of glycolytic enzymes to identify a possible key target of PRMT5;Then,PRMT5 inhibitor JNJ-64619178 was used to verify the protein expression of this metabolic enzyme in various colon cancer cell lines.Further,TCGA database was used to analyze the expression of this enzyme in colorectal cancer.And knock down of this enzyme by si RNA was performed to investigate its effect on the proliferation of colorectal cancer.Moreover,to analyze the contribution of the enzyme in PRMT5-induced cell proliferation,si RNA was used to knockdown the expression of LDHA in control group and PRMT5 overexpressed colon cancer cells,respectively.Finally,the transcriptional regulation of PRMT5 on the enzyme was detected by dual luciferase reporter assay and Ch IP assay.5.Evaluate the effectiveness of combined therapy.In vitro,HCT116 and SW480 cells was treated by PRMT5 inhibitors and 5-FU alone or combination.MTT test was conducted to detect cell proliferation;The efficacy of combined treatment of PRMT5 inhibitor and 5-FU was verified in vivo using a subcutaneous cancer model in nude mice,and the expression of glycolytic enzymes in the tumor was detected.Results 1.PRMT5 was overexpressed and promoted cell proliferation in colorectal cancer.PRMT5 were significantly higher in colorectal cancer tissues than their normal counterpart.In MTT tests and clone formation assays,knockdown of PRMT5 inhibited cell proliferation in colorectal cancer,while overexpression of PRMT5 promoted cell proliferation;JNJ-64619178 and Tadalafil both inhibited cell proliferation of colorectal cancers.2.PRMT5 affected metabolic profiling in colorectal cancer.Totally,we identified 246 metabolites,among which 29 metabolites were differential in Tadalafil group when compared with control group.And the most significant metabolic pathways were alanine,aspartate and glutamate metabolism,GABAergic synapse,biosynthesis of amino acids and oxidative phosphorylation pathway.3.PRMT5 promoted cell proliferation by activating glycolytic metabolism.PRMT5 increased glucose uptake in colorectal cancer.And PRMT5 knock down or inhibition decreased ECAR levels in colorectal cancer cell,while OCR level increased;Glycolytic inhibitor 2-DG can eliminate the pro-proliferation effect of PRMT5 in colorectal cancer.4.PRMT5 transcriptional regulated the expression of LDHA in colorectal cancer cells.PRMT5 knockdown or use of PRMT5 inhibitors can induce changes in the expression of glycolytic enzymes,among which LDHA expression was varied most.TCGA database analysis showed that LDHA was highly expressed in colorectal cancer cells.Although LDHA knockdown had no significant effect on the cell proliferation,in cells overexpressed PRMT5,cell proliferation was significantly reduced.Dual luciferase reporter assay showed that overexpression of PRMT5 could promote LDHA transcription.And Ch IP assay verified that PRMT5 inhibition could decrease the enrichment of LDHA promoter.5.PRMT5 inhibitors increased the cell sensitivity to 5-FU.MTT assay showed that Tadalafil could promote the proliferation-inhibition effect of 5-FU in vitro;In vivo experiment,combination of Tadalafil and 5-FU could effectively inhibit tumor growth and reduce the expression of LDHA.Conclusion In conclusion,we demonstrated that PRMT5 promotes cell proliferation through activating glycolysis in colorectal cancer.Mechanically,PRMT5 elevates glycolysis through transcriptional activation of LDHA.Furthermore,combination of PRMT5 inhibitor and 5-FU can significantly improve the efficacy of chemotherapy in colorectal cancer,suggesting that PRMT5 may be an effective therapeutic target for colorectal cancer. |
