Fig. 7

PVT1 modulates ULK1 expression by sponging miR-20a-5p. a The venn diagram of common microRNAs targeting PVT1 3’UTR and ULK1 3’UTR. Three microRNAs were identified, including miR-20a-5p, miR-302a-3p and miR-17-5p; b PVT1 biotin pull down assay was performed. The level of target microRNAs in the pull down of biotin-labelled PVT1 or negative control was investigated and quantified by qRT-PCR in HPAF-II cells; c Western blot analysis of ULK1 expression in HPAF-II cells with or without overexpression (upper) or suppression (lower) of target microRNAs (miR-20a-5p, miR-302a-3p, miR-17-5p). Interestingly, overexpression of miR-20a-5p suppressed ULK1 protein expression most. While inhibition of miR-20a-5p promoted ULK1 protein expression effectively; d RIP assay and qRT-PCR were conducted to detect the enrichment of PVT1 and miR-20a-5p by using AGO2 antibody in HPAF-II cells. Non-immune IgG acted as an internal control. U6 was used as a non-specific control. Enhanced enrichment of PVT1 and miR-20a-5p with AGO2 antibody was observed compared with IgG; e Bioinformatics prediction of miR-20a-5p binding sites in PVT1 3’UTR sequence using Starbase or ULK1 3’UTR sequence using TargetScan was presented; f Dual-luciferase assays showed diminished luciferase activity was observed when co-transfection of psiCHECK-ULK1-WT and miR-20a-5p occurred in HEK 293 T and HPAF-II cells; g Luciferase reporter assay implied that decreased luciferase activity occurred in HEK 293 T and HPAF-II cells co-transfected with psiCHECK-PVT1-WT and miR-20a-5p compared with luciferase reporter with mutant type of PVT1 and miR-20a-5p; h, i qRT-PCR analysis of miR-20a-5p with augment (h) of PVT1 expression in Capan-2 and MIA PaCa-2 cells or attenuation (i) of PVT1 expression in SW1990 and HPAF-II cells; j Overexpression of miR-20a-5p made no contribution to the level of PVT1 expression. Data are presented as the mean ± S.D. (n = 3). ^* P < 0.05, ^** P < 0.01 by Student’s t-test