METTL14 Mediates MAPK Signaling Pathway M6A Methylation And Mechanism Research In The Progression Of Prostate Cancer

Objective:In vivo and in vitro experiments were conducted to detect the expression level of methyltransferase protein 14(METTL14)in prostate cancer tissues and cells,and to explore the role of METTL14 in castration sensitive prostate cancer(CSPC)to castration resistant prostate cancer(CRPC)mechanism of action.Methods:The results of Me RIP and RNA double-sequencing were analyzed by pathway enrichment to compare different genes and screen MAPK signal transduction pathways.The expression of METTL14 protein at the tissue level of CSPC and CRPC was detected by immunohistochemical method,and the quantitative analysis of the tissue level of prostate cancer was performed with Image J software.The expression levels of METTL14 m RNA and protein in CSPC and CRPC cell lines were detected by QPCR and Western Blot experiments and compared and analyzed.Use small interfering RNA(si-RNA)and lentiviral transfection to construct a prostate cancer cell line that stably knocks down METTL14.QPCR and Western Blot experiments are performed to further verify the efficiency of knocking down the METTL14 gene.Through a series of in vitro cell experiments,such as MTT experiments,plate cloning experiments,and transwell experiments verified the effects of knocking down the METTL14 gene on the proliferation,invasion and viability of human prostate cancer cells.QPCR and Western Blot experiments were performed on human prostate cancer cells to verify the differential gene expression of MAPK pathway.The subcutaneous tissues of nude mice’s abdomen were planted with C4-2cells that knocked down the METL14 gene through lentiviral transfection as the experimental group,and C4-2 cells were planted as the blank control group,each of the two groups of experimental nude mice has 5,10 nude mice formed tumors within2 weeks of seeding tumors.The tumor size was accurately measured at 2,3,4,5,6,and 7 weeks respectively and compared volume every stage.The two groups of nude mice were harvested in 8 weeks and do Western Blot experiment,verify differential gene expression and understand the relationship between METTL14 and m6 A and the target genes RAS,MEK,explore the potential pathogenesis of the occurrence and development of CRPC.Results:The results of immunohistochemistry proved that the protein level of METTL14 was higher in CRPC tissues than CSPC,the difference between CSPC and CRPC was statistically significant(P<0.001).The m RNA and protein levels of METTL14 were highly expressed in various prostate cancer cell lines,and the expression was higher in CRPC than in CSPC.Knockdown of the CRPC cell line of METL14 found that the overall level of m6 A in prostate cancer cells was reduced,and cancer cell proliferation,invasion and viability were significantly reduced.At the same time,the expression of RAS,RAF,MEK,MAPK m RNA and protein in the MAPK signaling pathway was significantly reduced,and the expression levels of phosphorylated RAF,MEK and MAPK proteins also decreased significantly.The C4-2 cell line that knocked down the METTL14 gene was planted in nude mice.After tumor formation,the tumor volume was found to be significantly smaller than that of the non-knockdown group.Through immunohistochemical experiments on tumors in nude mice,it was found that after knocking down the METTL14 gene,the levels of RAS,MEK2 proteins were significantly lower than those in the control group.Conclusion:METTL14 is highly expressed in prostate cancer tissues and cells,and is higher in CRPC than in CSPC.METTL14 can affect the proliferation,invasion and viability of prostate cancer cells.Knockdown of METTL14 gene will weaken the proliferation and invasion ability of CRPC cells and reduce cancer cells viability.In CRPC,METTL14 mediates the change of m6 A,knockdown of METL14 gene in CRPC cells affect the m RNA and protein expression levels of RAS,RAF,MEK and MAPK,and decrease the expression levels of phosphorylated RAF,MEK and MAPK,thereby restrain the activation of the MAPK pathway as a whole.METTL14 regulates the changes of RAS,MEK2 in the MAPK signaling pathway mediated by m6 A.It is an important mechanism for the conversion of CSPC to CRPC,which can be used as a clinical diagnostic indicator of the progress of CSPC to CRPC.