The Inhibition Effect Of MiR-193A-3p In Prostate Cancer Cells By Targeting CCND1

Background:Prostate cancer (PCa) is one of the most common malignancy in male. According to National Cancer Institute, PCa has the highest incidence among all the urological tumors ever since2008. So far there is still lack of profound understanding to the oncogenesis and biological progression of PCa. Besides the common epidemialogical and genetic causes, many complicated molecular incidences are the vital reasons of oncogenesis, local progression and metastasis. microRNAs (i.e. miRNA) are small (-22nt), non-coding RNA, they funtion as modulator in various critical biopathway. Tumorigenesis in numerous cancers always related to aberrant miRNA expression profile, especially in PCa.Objective:In this study, RT-PCR was utilized to investigate the expression of miR-193a-3p in two different PCa cell lines (DU-145and PC3). Then a series of funtional study was conducted right after the transfection of miR-193a-3p. Subsequently bioinformatic tool was used to screen the potential target for miR-193a-3p. At last, verifying the mechanism of miR-193a-3p controlling its downstream target gene.Method:1. Compare whether PCa cell lines (DU-145, PC3) and normal prostate cell line (RWPE-1) has a difference in miR-193a-3p expression.2. Construct miR-193a-3p overexpression model by transfecting in vitro miR-193a-3p mimic into all the cell lines. Evaluate the functional profile via cell viability analysis (CCK-8), flow cytometry analysis, and colony formation assay, etc.3. Predict the target genes of miR-193a-3p with bioinformatical tool (TargetScan). Validate the predicted target with RT-PCR or Western blot on both transcriptional and translational level. Meanwile dual-luciferase reporter assay are conducted to confirm the sequential relavance of miR-193a-3p and the target gene. Verify the mechanism of miR-193a-3p controlling its downstream target gene by RNAi and plasmid-overexpression assay.Result:1. miR-193a-3p shows a relatively low expression level in PCa cell lines, the expression rate is46.3%and19.0%in DU-145and PC3, respectively.2. Gain-of-function study of miR-193a-3p via transfecting mimic into all the cell lines. Cell viability repression was observed significantly after transfection. In PC3cell lines,25nM of mimic transfection may decline cell viability by13%(48hours after transfection) and24%(72hours of transfection).In DU-145those numbers come to17%and63%at48and72hours, respectively. Flow cytometry showed that miR-193a-3p mimic can induce G1phase arrest significantly in both cell lines. Comparing to control group (NC), the proportion of G1cell counts in DU-145and PC3are54.89%to77.36%and61.80%to81.57%, respectively (p<0.05). In colony formation assay, miR-193a-3p may also inhibit the growth and formation of cell colony. In DU-145and PC3cells, the colony formation rates are59.1%(p<0.05) and31.6%(p<0.05), respectively.3. According to TargetScan, miR-193a-3p may have the target gene such as Cyclin D1, MMP-9or CADM1, etc. After a confimation by RT-PCR, Cyclin D1expression showed an obvious decrease in both DU-145and PC3cells, while neither MMP-9nor CADM-1showed the same trend. Further dual-luciferase report experiment confirmed Cyclin D1is the direct target of miR-193a-3p in prostate cancer cell lines. Additional RNAi and rescue experiments validate the identical role of miR-193a-3p in cell cycle and cell proliferation.Conclusion:1. miR-193a-3p has a lower expression level in PCa cell lines.2. Forced overexpression of miR-193a-3p can induce G1phase arrest in DU-145and PC3cells, inhibiting cell proliferation.3. Cyclin D1is the target gene of miR-193a-3p. By inhibiting the expression of Cyclin D1, miR-193a-3p can arrest the cell cycle at G1phase.