Fig. 4

MiR-634 regulates genes involved in cell cycle regulation and the Ras-MAPK pathway. Ovarian cancer cell lines (A2780, A2780 DDP) and patient derived tumor cells (culture #3) were transiently transfected with scrambled control (-) or miR-634 mimic (+). 48 h after transfection protein lysates were analyzed for the expression of putative miR-634 target genes by Western blotting (A, B) or RT-PCR (C). a Transfection with miR-634 reduces Cyclin D1 protein levels in the A2780 cell lines and in primary ovarian cancer cell cultures. β-actin is used as a loading control. b MiR-634 overexpression reduces protein levels of GRB2, ERK2, RSK1 and RSK2, components of the Ras-MAPK pathway in the A2780 and A2780 DDP cell lines and primary ovarian cancer cell cultures. β-actin is used as a loading control. c MiR-634 overexpression lowers mRNA levels of CCND1, GRB2, ERK2, RSK1 and RSK2 in the A2780 DDP cell lines 48 h after transfection. Expression was normalized to HPRT and GAPDH expression. Depicted are average values ± SD (n = 2). The mRNA level in cells transfected with scrambled mimic is set at 100 %. d MiR-634 binds directly to elements of the 3’UTR of CCND1, GRB2, ERK2 and RSK2. A2780 DDP cells were transfected with a scrambled mimic or a miR-634 mimic. After 8 h, the same cells were transfected with a Renilla luciferase reporter construct (psiCHECK™-2) containing a region of 500 bp surrounding the predicted target sites (Additional file 1: Figure S7), or a luciferase construct in which the miR-634 binding sites were mutated. Since RSK2 contains both a canonical and a non-canonical miR-634 binding site, one construct was generated that contained a mutation in the canonical binding site (RSK2_2 mut 1) and another construct that contained mutations in both the canonical and non-canonical site (RSK2_2 mut 2). The Renilla luciferase activity was measured and normalized using the Firefly luciferase activity. For each construct, the relative luciferase activity of cells transfected with the scrambled mimic was set at 1. Depicted are the average values ± SD (n = 3). * p < 0.05, ** p < 0.01