The Tumor-suppressive Effects Of MicroRNA-608 In Human Prostate Cancer And The Related Mechanisms

ObjectiveThis research aimed to investigate the functions of miR-608 in prostate cancer(PCa)and reveal the relevant mechanisms.Methods(1)MicroRNA tailing quantitative real-time PCR(qRT-PCR)was adopted to measure the expression of miR-608 in PCa cell line DU145 and PC3,as well as in normal prostate cell line RWPE-1.The miR-608 expression pattern in PCa tissues and paired peritumoral tissues was determined by chromogenic in situ hybridization(CISH)staining of prostate cancer tissue microarray(TMA).(2)Bisulfite sequencing PCR(BSP)was employed to assess the methylation status of the CpG island near the transcription start site(TSS)of miR-608 gene in PCa cells.Further,5-aza-2'-deoxycytidine DNA methylase inhibitor was used to treat PCa cells,and the expression of miR-608 in PCa cell lines was quantified before and after 5-aza treatment.(3)Lipofectamine 2000 was utilized for transfecting miR-608 mimic into PCa cell lines.Cell viability assay,colony formation assay,flow cytometry cell cycle assay,flow cytometry apoptosis and active caspase-3 assay,cell wound healing assay and Transwell migration assay were performed to study the functions of miR-608 in PCa cell line DU 145 and PC3.Besides,PC3 cell subcutaneous tumorigenesis assay followed by intratumoral injection with miR-608/NC mimic was conducted to study the effects of miR-608 in PCa cells in vivo.(4)qRT-PCR,western blot analysis,and dual-luciferase reporter assays were conducted to ascertain the target of miR-608 in PCa.Cell function assays and rescue experiments using target siRNA and miR-608 inhibitor were performed to further verify the target of miR-608 in PCa.In addition,immunohistochemistry staining of PCa TMA was used to assess the expression pattern of miR-608 target protein in PCa tissues and paired peritumoral tissues.Results(1)MiR-608 was down-regulated in PCa cell lines and tissues compared with normal prostate cell line and peritumoral tissues,which was partly attributed to the methylation of CpG islands near the transcription start site(TSS)of miR-608 gene.(2)Intracellular over-expression of miR-608 inhibited PC3 cell subcutaneous tumorigenesis in vivo,and suppressed PCa cell proliferation,G2/M transition and migration in vitro,which was independent of EMT-associated mechanisms.Then RAC2,a GTPase previously considered to be hematopoiesis-specific but now discovered for the first time to exist and play important tumorigenic roles in PCa,was verified to mediate the effects of miR-608 through RAC2/PAK4/LIMK1/cofilin pathway.MiR-608 also promoted the apoptosis of PCa cells through BCL2L1/caspase-3 pathway by targeting the 3'-UTR of BCL2L1.(3)PAK4,the downstream effector of RAC2,was targeted by miR-608 at the mRNA coding sequence(CDS)instead of the canonical 3 '-UTR.(4)Knocking-down RAC2,PAK4 or BCL2L1 with siRNAs reproduced the anti-proliferative,mitosis-obstructive,anti-migratory and pro-apoptotic effects of miR-608 in PCa cells,which could be attenuated by down-regulating miR-608 with miR-608 inhibitor.RAC2 and BCL2L1 were up-regulated in PCa tissues compared with peritumoral tissues,which provided their diagnostic potentials in PCa.ConclusionMiR-608 is down-regulated in PCa cell lines and tissues,and acts as a PCa tumor suppressor.It suppresses the proliferation,induces G2/M arrest,and inhibits the migration of PCa cells by targeting the 3'-UTRs of RAC2 and BCL2L1,and the CDS of PAK4.The inhibitory effects of miR-608 in PCa are dependent on RAC2/PAK4/LIMK1/cofilin and BCL2L1/caspase-3 signaling pathways.