Fig. 4

circPLPP4 enhanced PIK3R1 expression by sponging miR-136 in EOC cells. A RNA-seq data of the top 20 up-regulated mRNAs in A2780 CDDP cells and A2780 CDDP-ASO#1 cells are showed as heatmaps. B Venn diagram demonstrated 4 genes that are putative miR-136 targets computationally predicted by four algorithms (miRanda, RNAhybrid, miRWalk and TargetScan) within the top 20 upregulated genes. C The expression of PIK3R1 was analyzed using RT-qPCR and western blot in A2780 CDDP, SKOV3 CDDP, A2780 and SKOV3 cells. D The expression of PIK3R1 was assessed using RT-qPCR and western blot in A2780 CDDP, SKOV3 CDDP, A2780 and SKOV3 cells. E Schematic of PIK3R1 3’UTR wild-type (WT) and mutant (Mut) luciferase reporter vectors is displayed (Top). The relative luciferase activities were examined in A2780CDDP cells co-transfected with miR-136 mimics or miR-NC and luciferase reporter vectors PIK3R1 3’UTR (WT) or PIK3R1 3’UTR (Mut) (Bottom). F, G The expression of PIK3R1 was detected by RT-qPCR in A2780 CDDP and SKOV3 CDDP cells were transfected with miR-126 mimic or co-transfected with the indicated vectors. H The expression levels of PIK3R1 were detected using RT-qPCR. A2780 cells were transfected with the indicated vectors and miR-136 mimics. I The expression levels of PIK3R1 were analyzed using RT-qPCR. A2780 CDDP cells were transfected with ASO-circPLPP4#1 alone or co-transfected the inhibitors. (J)The expression of PIK3R1, γ-H2AX, BRCA1, p-AKT, AKT and cleaved caspased 3 was examined by western blot. A2780 CDDP and SKOV3 CDDP cells were transfected with miR-136 mimic or co-transfected with the indicated vectors. K The IC50 was examined by the MTT assay. A2780CDDP cells were transfected with miR-136 mimic alone or cotransfected with the indicated vectors with CDDP treatment (5 μM) for 48 h. L The apoptosis percentages of A2780 CDDP cells transfected with miR-136 mimic alone or cotransfected with the indicated vectors upon CDDP exposure (5 μM) for 48 h. M The levels of PIK3R1 and apoptosis markers, γ-H2AX, BRCA1 and cleaved-caspased 3 were detected using western blotting in A2780 CDDP cells and SKOV3 CDDP cells transfected with ASO-circPLPP4#1alone or co-transfected with the inhibitor after CDDP treatment (5 μM). N Three-dimensional scatter plot of circPLPP4, miR-136 and PIK3R1 levels in 25 OC tissues. The results are displayed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns indicates no significance. Each error bar represents the mean ± SD of three independent experiments