Icant raise in the quantity of cells in the G0-G1 phase of the cell cycle when compared with miR-CON. This suggests that miR-3607 overexpression induces a G0-G1 arrest in PCa cell lines.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptMol Cancer Ther. Author manuscript; readily available in PMC 2015 July 01.Saini et al.PagemiR-3607 overexpression induces apoptosis in prostate cancer cell linesAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptWe measured apoptosis in control (mock or miR-CON transfected) and miR-3607tranfected cells by flow cytometric analysis of Annexin-V-FITC-7-AAD stained PC3/ Du145/LNCaP cells (Figure 2D). It was observed that the typical Thrombopoietin Receptor custom synthesis Apoptotic cell fractions (Early apoptotic + Apoptotic) were substantially increased upon miR-3607 overexpression in comparison with miR-CON/mock transfected cells with a concomitant decrease in the viable cell population. This suggests that miR-3607 induces apoptosis in PCa cell lines. Overexpression of miR-3607 expression reduces invasiveness of prostate cancer cell lines We performed transwell migration and invasion assays in handle (mock or miR-CON transfected) and miR-3607-tranfected PC3/Du145/LNCaP PCa cell lines (Figure three). These assays showed that overexpression of miR-3607 significantly decreased the invasiveness (Figure 3A) and migratory abilities (Figure 3B) of all of the PCa cell lines tested. miR-3607 knockdown increases invasiveness and proliferation of regular immortalized prostate epithelial cell lines Inside a reciprocal method, we knocked down miR-3607 expression in typical immortalized prostate epithelial cell lines (RWPE1 and PWR1E) working with miRVANA anti-miRNA inhibitor (Ambion) followed by functional assays (Figure 4). Basal level of miR-3607 expression in these typical immortalized prostate epithelial cell lines is greater than that of PC3 and Du145 (Fig. S2). miR-3607 knockdown was confirmed by RT-PCR (Figure 4A). Our final results suggest that knockdown of miR-3607 enhanced the proliferation, invasiveness and motility of non-transformed epithelial cells (Figure 4B ). Cell cycle analysis showed a considerable improve in G2-M phase upon miR-3607 inhibition (Figure 4E). These outcomes help a tumor-suppresseive part for miR-3607 in PCa. miR-3607 straight targets SRC family members of kinases in prostate cancer In silico analysis identified that SRC family members kinases LYN and SRC are putative miR-3607 targets. LYN possesses 1 possible miR-3607 binding site inside its 3-UTR while SRC has two possible miR-3607 binding websites (Figure 5A). Though other miRNAs are predicted to target SRC/LYN, the potential Bacterial review capability of miR-3607 to simultaneously bind to 3 UTRs of both SFK members of the family makes it exceptional. To validate these SRC kinases as target genes for miR-3607, we performed Western blot analysis for these kinases in PC3 cells that had been either mock transfected or transfected with miR-3607/miR-CON (Figure 5B). Interestingly, miR-3607 overexpression led to decreased protein levels of LYN and SRC. Further, we investigated no matter whether these nonreceptor tyrosine kinases are direct functional targets of miR-3607 in PCa. We transiently transfected PC3 cells using the control/LYN/SRC 3UTR luciferase reporter plasmids together with miR-3607 precursor/miR-CON (Figure 5C). miR-3607 overexpression led to substantial decreases in LYN/SRC luciferase reporter activity as in comparison with miR-CON/mock transfected cells suggesting that miR-3607 directly represses these genes.Mol Cancer Ther. Author manu.
