Figure 4.
Genes in cell cycle regulation are direct targets of miR-31. A, proliferation assay of LNCaP cells transfected with miR-31 or miR-NC (n = 6, * p < 0.001). B, colony formation analysis of VCaP cells overexpressing miR-31 or vector alone (n = 3). C, cell cycle analysis of LNCaP cells transfected with miR-31 or miR-NC by FACS (n = 3). D, caspase 3/7 activity in LNCaP cells transfected with miR-31 or miR-NC (n = 6). E, expression of genes involved in cell cycle in LNCaP cells transfected with miR-31 or miR-NC, evaluated by qPCR (n = 3). F, immunoblot of E2F1 with lysates from LNCaP cells transfected with miR-31 or miR-NC (top). Schematic graph illustrates the miR-31 MRE within the 3′UTR of E2F1 (bottom). G, luciferase activity of LNCaP cells co-transfected with reporter constructs containing WT or mutant (mt) E2F1 3′UTR or vector alone (v) with either miR-31 or miR-NC (n = 3, **p < 0.01). H, expression levels of indicated proteins from LNCaP cells transfected with miR-31 or miR-NC by immunoblot. I, luciferase activity of LNCaP cells transfected with reporter constructs containing 3′UTRs of CDK1, E2F2, EXO1, FOXM1, or MCM2 in conjunction with miR-31 or miR-NC (n = 3, *p < 0.05, **p < 0.01). J–K, luciferase activity of LNCaP cells transfected with reporter constructs containing WT or mutant MREs of E2F2 and FOXM1 in conjunction with miR-31 or miR-NC (n = 3, *p < 0.05, **p < 0.01). All bar graphs are shown with mean ± SEM.