Figure 2
miR-491-5p directly targets BCL-XL and may regulate additional targets involved in miRNA-mediated cell death in IGROV1-R10 cells. (a) IGROV1-R10 ovarian chemoresistant cell line was transfected with indicated RNAi (20 nM). BCL-XL and MCL1 protein expression levels were determined by western blot 72 h after transfection. The relative densitometry values (calculated relative to β-actin) were determined using ImageJ software and are shown on the bottom of each western blot. (b) Relative quantification in real-time PCR of BCL-XL mRNA levels in IGROV1-R10 cells after transfection with 20 nM indicated miRNA mimics or with siRNA targeting BCL-XL (siRNA-BCL-XL). Data are normalized to GAPDH mRNA levels as an endogenous control. Results are expressed as mean±S.D. of three independent experiments. (c) Schematic representation of 3′-UTR BCL-XL mRNA with two predicted binding sites of hsa-miR-491-5p. Luciferase reporter constructs containing human BCL-XL 3′-UTR wild-type (WT) or mutant deletion of putative binding site 1, site 2 or both sites for miR-491-5p were transfected into SKOV3 cells concomitantly with miR-491-5p or the control miRNA (miR-Ctrl). Luciferase assays were carried out 24 h after transfection. Firefly luciferase activity was normalized to renilla luciferase. Data are presented as mean±S.D. of three independent experiments. (d) Real-time curve monitoring growth of IGROV1-R10 with the xCELLigence System. Cells were grown for 24 h before transfection with indicated RNAi (miRNA or siRNA). CI was recorded every 2 h for a time span of 96 h. The experiment was performed three times in triplicates; shown is mean±S.D. on representative experiment. (e) IGROV1-R10 cells were transfected with indicated RNAi. Seventy-two hours after transfection, cells were subjected to flow cytometry to determine the percentage of sub-G1 population. For each condition, the percentage of sub-G1 population, representative of three independents experiments, is indicated. (f) Protein expression levels of PARP (native and cleaved forms) were determined by western blot 72 h after transfection of IGROV1-R10 cells with indicated RNAi
