Fig. 3

circPLPP4 exerts its function by sponging miR-136. A Ago2-RNA RIP assay for circPLPP4 expression in the indicated cells. B Schematic illustration indicating potential target miRNAs of circPLPP4 as predicted by CircInteractome. C, D Gel electrophoresis and qRT-PCR were used to validated the specificity and efficiency of the circPLPP4 probe in A2780-CDDP and SKOV3-CDDP cells. E, F qRT-PCR analysis of the expression of thirteen potential target miRNAs in A2780-CDDP and SKOV3-CDDP cells. MiR-136 was stably pulled down by circPLPP4 in both A2780-CDDP and SKOV3-CDDP cells. G Biotinylated miRNA pull-down (WT or mut) and qRT-PCR assays indicating the levels of circPLPP4 in the indicated OC cells. GAPDH was acted as the negative control. H Schematic illustration of circPLPP4-wt and circPLPP4-mut luciferase reporter vectors; the binding ability between circPLPP4 and MiR-136 was measured by dual-luciferase reporter assay in A2780-CDDP and SKOV3-CDDP cells. I, J The luciferase activities of the circPLPP4 luciferase reporter vector (WT or mut) assessed after transfection with miR-136 mimics or mimic NC into A2780-CDDP and SKOV3-CDDP cells. K, L. FISH showing the expression of circPLPP4 and miR-136 in A2780-CDDP and A2780 cells. Nuclei were stained with DAPI. (M) Platinum-resistant or Platinum-sensitive OC tissues from patients. FISH scores of circPLPP4 and miR-136 were further evaluated in 30 Platinum-resistant and 19 Platinum-sensitive patient tissues. Nuclei were stained with DAPI. * 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