The Tumor Promotor Role And Molecular Mechanism Of M~6A Reader YTHDF2 In Prostate Cancer

Background:Prostate cancer is the most common male cancer in European and American countries.With the changes of the lifestyle and the popularization of prostate cancer antigen early detection,the incidence rate of prostate cancer is increasing and has ranked as the 6th common male cancer in China in recent years.The bad life quality and prognosis of castration resistant prostate cancer and metastatic prostate cancer pose a serious threat to the male health,which has been a tough issue in urology all the time.It seems crucial for the earlier diagnosis and effective therapy to investigate the specific mechanism of prostate cancer carcinogenesis and tumor progression.N6-methyladenosine(m6A)is the most abundant modification in mRNA of humans.M6A is a revisable process where it can be methylated by writers,exerted by readers and removed by erasers.Emerging evidence has supported the fact that m6A is comprehensively involved in various diseases especially cancers.As a crucial reader,YTHDF2 usually mediates the degradation of m6A-modified mRNA in m6A-dependent way.However,the function and mechanisms of m6A especially YTHDF2 in prostate cancer still remain elusive.Objectives:To uncover the expression pattern of YTHDF2 and METTL3 in prostate cancer and analyze the association between the expression level and overall survival rate;To investigate the specific molecular mechanism induced by YTHDF2 in prostate cancer.To identify the expression pattern,biofunction and molecular mechanism of YTHDF2 targetsMethods:The Bioinformatics analysis with TCGA and Oncomine databases was conducted to investigate the expression pattern of both YTHDF2 and METTL3 in prostate cancer and uncover the association between the expression and overall survival rate with Kaplan-Meier method.Endogenous expression silencing of YTHDF2 and METTL3 was established with lentivirus-based shRNA technique.RNA dot blot assay was used to evaluate the total m6A level alteration induced by YTHDF2 and METTL3 expression.The in vivo and in vitro biofunctional assays were used to investigate the biofunction of YTHDF2 and METTL3 in prostate cancer.MeRIP sequencing and bioinformatics analysis were used to screen the targets of YTHDF2 which further confirmed with RIP-RT-qPCR,RT-qPCR and western blot.In addition,MeRIP-RT-qPCR and demethylation regent treatment were conducted to further confirm the regulation mediated by YTHDF2 is m6A-dependent.The TCGA and Oncomine databases were used to identify the expression pattern of YTHDF2 target LHPP,and the association between the expression level and overall survival rate with Kaplan-Meier method.In addition,cellular biofunctional assays and western blot assay were also performed to evaluate the biofunction induced by LHPP and the downstream pathway.Results:The upregulated YTHDF2 and METTL3 in prostate cancer predicted a worse overall survival rate and the expression level was associated with Gleason score,T stage,lymph node metastasis.Knock-down of YTHDF2 elevated the total m6A level while knocking down METTL3 decreased the total m6A level.Knock-down of YTHDF2 or METTL3 markedly inhibited the proliferation and migration of prostate cancer in vivo and in vitro,and the inhibited phosphorylated AKT was also observed.LHPP and NKX3-1 were identified as the direct targets of both YTHDF2 and METTL3.YTHDF2 directly bound to the m6A modification sites of LHPP and NKX3-1 to mediate the mRNA degradation.Knock-down of YTHDF2 or METTL3 significantly induced the expression of LHPP and NKX3-1 at both mRNA and protein levels.In addition,knock-down of METTL3 significantly inhibited the m6A level of LHPP and NKX3-1,and demethylation treatment consistently upregulated the expression of LHPP and NKX3-1 at mRNA level Overexpression of LHPP presented the consistent phenotypes and AKT phosphorylation inhibition with knock-down of YTHDF2 or METTL3.Phosphorylated AKT was consequently confirmed as the common downstream of METTL3/YTHDF2/LHPP/NKX3-1 to induce tumor proliferation and migrationConclusion:Both upregulated YTHDF2 and METTL3 predict worse overall survival rate in prostate cancer.We propose a novel regulatory mechanism in which YTHDF2 mediates the mRNA degradation of the tumor suppressors LHPP and NKX3-1 in m6A-dependent way to promote AKT phosphorylation-induced tumor progression in prostate cancer.We hope our findings may provide new concepts of prostate cancer biology.