Effects Of MiR-34a Hypermethylation On The Expression Of ATG4B In Prostate Cancer

Objectives:The levels of methylation in the promoter region of miR-34a in prostate cancer tissue,normal prostate tissue and prostate cancer cell lines(PC-3,DU 145)and normal human prostate epithelial cells(RWPE-1)were detected by MSP or BSP.The expression of miR-34 and ATG4B in prostate cancer cell lines with/without inhibition of methylation were measured by QPCR.Dual luciferase reporter gene was used to detect the direct targeting relationship between miR-34a and ATG4B in prostate cancer cells.Methods:The first part:25 cases of prostate cancer tissues and normal tissues were collected,and the promoter methylation status of miR-34a was detected by MSP.QPCR was used to detect the expression level of miR-34a in cancer tissues and adjacent tissues.BSP was used to detect the promoter methylation status of miR-34a in prostate cancer cell lines(PC-3,DU145)and normal human prostatic epithelial cells(RWPE-1).QPCR was used to detect mRNA expression level of miR-34a and ATG4B in RWPE-1,PC-3 and DU145.The second part:The prostate cancer cell lines(PC-3 and DU145)were treated with methylation inhibitor 5'-azo deoxycytidine(10umol/L)for 48h.The mRNA of miR-34a and ATG4B were detected by QPCR.The prostate cancer cells in same lines are transfected by Has-miR-34a for 48h.QPCR was used to detect the expression level of miR-34a and ATG4B.WB was used to detect protein expression levels of ATG4B,Beclinl and LC3BII/I.Cell increment was detected by MTT.WB was used to detect protein expression levels of Caspase3,Caspase9,Bax and Bcl-2.And dual luciferase reporter gene was used to detect the direct targeting relationship between miR-34a and ATG4B in prostate cancer cells.Results:MSP showed that the degree of methylation of promoter region of miR-34a in PC-3 and DU 145 was significantly higher than that of RWPE-1,and similar results were detected in the prostate cancer tissues.Furthermore,we found that the expression of miR-34a in the prostate cancer tissue was significantly lower than that of the adjacent tissues of prostate cancer.In consistence with in vivo,the expression of miR-43 were decreased in prostate cancer cell PC-3 and DU145.And the mRNA expression of ATG4B in PC-3 and DU 145 was significantly higher than that of RWPE-1.After demethylation or miR-34a mimics transfection,the expression of miR-34a in PC-3 and DU145 was significantly increased,while the mRNA expression of ATG4B was significantly decreased.MTT showed that growth phase cells were significantly reduced in PC-3 and DU145 cells pretreated by demethylation or miR-34a mimics transfection.WB detection showed that in PC-3 and DU145 cell lines pretreated with miR-34a mimics the expression of apoptotic protein caspase 3,caspase 9 and Bax were significantly up-regulated,while the expression of autophagy related proteins ATG4B,Beclin-1 and LC3B II/I were obviously downregulated.The double luciferase reporter gene assay showed that the activity of luciferase was inhibited in both PC-3 and DU 145 cells,and there was no change in the activity of luciferase activity by miR-34a transfection.Conclusions:1.MiR-34a is epigenetically downregulated by DNA methylation in PCa tissue.2.The DNA methylation of miR-34a in prostate cancer cell lines PC-3 and DU 145 inhibited the expression of miR-34a and upregulated the expression of ATG4B.3.ATG4B is the target of miR-34a,and upregulation of miR-34a could reduce the expression of ATG4B and enhance the apoptosis of prostate cancer cells.