MiR-137 Regulates The Resistance Of Prostate Cancer Cell Line To Bicalutamide By Targeting TRIM24 And Its Epigenetic Mechanism

Objective:To investigate the regulatory effect of miR-137 on resistance of prostate cancer cell line to bicalutamide by targeting TRIM24 and its epigenetic mechanism.Methods: Bicalutamide-resistant cell model was established in LNCaP prostate cancer cell line,and the model was verified by MTT test.The miRNAs of wild-type LNCaP prostate cancer cell line(parental cell)and bicalutamide-resistant LNCaP prostate cancer cell line(resistant cell)were detected by microarray,and the differential expression of miRNAs in both parent and resistant cells was compared.miR-137,which was down-regulatedthe most,was selected as the target miRNA for study,and the effect of miR-137 on the sensitivity to bicalutamide in prostate cancer was detected.The potential target genes of miR-137 were predicted by miRNA target prediction softwares and gene function analysis.The expression level of TRIM24,a potential target gene of miR-137,was detected in the parental and resistant prostate cancer cells.Then the effect of miR-137 on the expression of TRIM24 in the resistant prostate cancer cells was detected.The direct interaction between miR-137 and TRIM24 was confirmed by double luciferase reporter gene experiment.The effect of TRIM24 gene silencing on the expression of TRIM24 in prostate cancer cells and the sensitivity of bicalutamide was detected.Overexpression of TRIM24 blocked the sensitization of miR-137 to bicalutamide in prostate cancer cells.The correlation between miR-137 and TRIM24 mRNA level in various prostate cancer cell lines and the expression level and correlation of miR-137 and TRIM24 mRNA in prostate cancer tissues were detected and analyzed.The effects of EZH2 silencing on the expression of miR-137 and TRIM24 in resistant prostate cancer cells were detected.Bioinformatics analysis of miR-137 was carried out to find methylation sites,and the binding of DNMT1,DNMT3 b and MeCP2 with miR-137 gene promoter were detected by chromatin immunoprecipitation(Ch Ip)assay.The effect of MeCP2 silencing on the expression of miR-137 and TRIM24 in resistant prostate cancer cells was detected.The effect of 5-Aza-Cd R,a methylated inhibitor,on the expression of miR-137 and TRIM24,and the binding of DNMT3B,MeCP2 and miR-137 gene were detected.Results:Bicalutamide-resistant cell model was established successfully.The results of miRNAs microarray analysis showed that there were many kinds of miRNAs with differential expression in parental cells and resistant prostate cancer cells,and miR-137 was the most down-regulated.Overexpression of miR-137 can improve the sensitivity of prostate cancer to bicalutamide.Through miRNA target prediction software and gene function analysis,it is found that TRIM24 gene may be a potential target gene of miR-137.Compared with parental cells,mRNA and protein levels of TRIM24 in resistant prostate cancer cells were significantly up-regulated,while miR-137 could down-regulate mRNA and protein levels of TRIM24 in resistant prostate cancer cells.In prostate cancer cells,miR-137 directly targeted the3’-UTR region of trim24 and dow-regulated the expression of TRIM24.Silencing TRIM24 gene can significantly reduce the mRNA,protein levels of TRIM24 in prostate cancer cellsand improve the sensitivity of prostate cancer cells to bicalutamide.Overexpression of TRIM24 blocked the sensitization of miR-137 to bicalutamide.miR-137 was negatively correlated with mRNA level of TRIM24 in LNCaP,PC3 and ARCa P cell lines.Compared with paracancerous tissues,prostate cancer tissues had signifanctly lower mRNA expression level of miR-137 and signifianctly higher mRNA expression level of TRIM24 with negative correlation.Silencing EZH2 had no effect on the mRNA expression of miR-137 and TRIM24 in resistant prostate cancer cells.Bioinformatics analysis of miR-137 showed that three hypermethylation sites were found near the transcription start site(-276-+187).The results of Ch Ip experiment showed that DNMT1 did not bind to three methylated sites,while DNMT3 B and MeCP2 bound to three methylated sites.After MeCP2 was silenced,the mRNA and protein levels of miR-137 were significantly up-regulated,and the mRNA and protein levels of TRIM24 were significantly down-regulated.After treatment of resistant prostate cancer cells with5-Aza-Cd R,the mRNA and protein levels of miR-137 were significantly up-regulated,wihel the mRNA and protein levels of TRIM24 were significantly down-regulated.And the binding of DNMT3 B,MeCP2 and miR-137 gene were significantly reduced.Conclusion: miR-137 regulates bicalutamide resistance in prostate cancer by directly targeting TRIM24,and DNMT3 B and MeCP2 are involved in the epigenetic regulation of miR-137 in prostate cancer cells.Silencing MeCP2 can up-regulate miR-137 and down-regulate TRIM24 expression at the same time.