Aberrantly Expressed MicroRNAs Involved In Prostate Cancer And Preliminary Study On Effective Mechanism Of MiRNA-182

Prostate cancer (PCa) continues to be one of the biggest health problems for the aging male. PCa has become one of the most common malignancies in men worldwide, with strongly varying rates of tumor progression and responses to treatment. If the tumor is confined to the prostate, patients can be treated by surgical removal of the tumor or by radiation, with high efficacy. By contrast, therapy for unconfined tumors still represents a major problem. Therefore, better understanding the pathogenesis of PCa and exploring novel intervention targets for PCa are urgently demanding tasks. MicroRNAs (miRNAs) are a group of small (approximately20-22nucleotides) endogenous noncoding RNAs. Mature miRNAs negatively regulate their target genes through imperfect complementary sequence pairing to the3’untranslated region (UTR) of target genes resulting in either mRNA degradation or translational repression. Numerous studies have demonstrated that aberrant expression of miRNAs is closely associated with proliferation, invasion, metastasis and the prognosis of various cancers, including PCa. However, the relationship between miRNAs and PCa has only started to be elucidated in recent years. More miRNAs should be selected and studied in order to better understanding the progression of PCa.Part I Screen the aberrantly expressed miRNAs involved in PCaObjective To screen the aberrantly expressed miRNAs between PCa and BPH.Methods Five PCa tissues and Three benign prostatic hyperplasia tissues were collected between January2010and December2012. We first screened the miRNAs related to PCa by miRNA microarrays. Real-time RT-PCR was used for the confirmation of some differentially expressed miRNAs.Results In PCa tissues, five miRNAs (miR-345, miR-145, miR-221, miR-27b and miR-378) were down-regulated and twenty-two miRNAs were up-regulated. And miRNA-182was up-regulated significantly.Conclusion As the most significant differentially expressed miRNA between PCa and BPH tissues, miR-182was chosen for further study. Part Ⅱ The function of aberrantly expressed miRNAs in PCaObjective To investigate the function of miRNAs in PCa.Methods We transfected miRNA mimics or inhibitros into PC-3PCa cells. MiRNA expression level was detected by real-time RT-PCR after transfection with mimics or inhibitors in PC-3cells. We further investigated the effect of miR-182on proliferation using flow cytometry. To investigate the possible regulative involvement of miR-182, early apoptosis of PC-3cells were detected after transfection, and the change of the invasion capability of PC-3cells were detected by Transwell.Results miR-182expression was markedly increased in four common PCa cell lines tested (PC-3, DU145,22Rv1and LNCaP), compared to normal prostate epithelial RWPE-1cell, indicating that miR-182is upregulated in PCa cell lines and PC-3has the highest expression level. Then PC-3cells were chosen for further study. MiR-182expression level was detected by real-time RT-PCR after transfection with mimics or inhibitors in PC-3cells. Results showed that mimics or inhibitors were efficiently transfected and the expression level is highest in miR-182mimics group and lowest in miR-182inhibitor group. Using MTT assays, we observed that the growth rate of miR-182overexpressing cells was dramatically increased, compared to miR-182mimics-NC-transfected cells (P<0.05). On the contrary, after downregulation of miR-182by an inhiobitor, the growth rate of PC-3cells was dramatically decreased compared to miR-182inhibitor-NC-transfected cells (P<0.05). MiR-182-overexpressing PC-3cells had a significantly lower percentage of cells in the G0/G1phase and increased percentage of cells in the S phase, compared to miR-182mimics-NC-transfected cells. On the contrary, after downregulation of miR-182by an inhiobitor, PC-3cells had a significantly higher percentage of cells in the G0/G1phase and decreased percentage of cells in the S phase, compared to miR-182inhibitor-NC-transfected cells. The early apoptosis rate of miR-182overexpressing cells was dramatically decreased, compared to miR-182mimics-NC-transfected cells. On the contrary, after downregulation of miR-182by an inhiobitor, the early apoptosis rate of PC-3cells was dramatically increased compared to miR-182inhibitor-NC-transfected cells. Overexpression of miR-182significantly increased the invasive potential of PC-3cells when transfected with miR-182mimics compared with control cells in Transwell assay with Matrigel, and cells transfected with miR-182inhibitor resulted in a significantly decresed invasive potential.Conclusion Ectopic overexpression of miR-182significantly promotes the proliferation, increases the invasion, promotes the G1/S cell cycle transition and reduces early apotosis of PC-3cells.Part Ⅲ The investigation of miRNA targets in PCaObjective To predict and confirm the putative miRNA targets of miRNA-182.Methods The putative miRNA targets were predicted using the TargetScan, miRBase, and PicTar algorithms. Western blotting was performed to determine the protein expression. To confirm the function of the putative miRNA, we synthesized the double-stranded sequence which included the miRNA binding site and cloned into the luciferase reporter plasmid.Results We revealed that the putative miR-182target was NDRG1pridicted by using algorithms miRBase, TargetScan and PicTar. Western Blotting results showed that overexpression of miR-182in PC-3cells significantly decreased the protein expression levels of NDRG1after transfection with miR-182mimics, compared to miR-182mimics-NC-transfected cells. On the contrary, after downregulation of miR-182by an inhiobitor, the protein expression levels of NDRGlwas dramatically increased compared to miR-182inhibitor-NC-transfected cells. We synthesized the double-stranded sequence which included the miR-182binding site and cloned into the luciferase reporter plasmid. Luciferase activity of the pmirGLO/NDRG1-UTR was dramatically inhibited by overexpression of miR-182with cotransfection with miR-182mimics and significantly increased by downregulation of miR-182with cotransfection with miR-182inhibitors in PC-3cells, compared to the control plasmids.Conclusion MiR-182could downregulate expression of NDRG1by directly targeting the NDRG13’-UTR. This data indicates that miR-182plays an essential role in the regulation of PCa cell proliferation and may function as an onco-miRNA. MiR-182may sever as a novel therapeutic target for the treatment of PCa.