Fig. 4

Downregulation of miR-509-3p transcription by COL11A1 increases DNMT1 stability. A The mRNA expression levels of COL11A1 and miR-509-3p in A2780CP70 and OVCAR-8 cells transfected with miR-509-3p mimics and in A2780 and OVCAR-3 cells transfected with the miR-509-3p inhibitor were evaluated using real-time RT-PCR. All experiments were performed in triplicate. B Upper panel: mRNA expression levels of miR-509-3p and COL11A1 in A2780CP70 and OVCAR-8 cells transfected with COL11A1 shRNA were evaluated using real-time RT-PCR. All experiments were performed in triplicate. * P < 0.05, ** P < 0.01, compared with shV. Lower panel: mRNA expression levels of miR-509-3p and COL11A1 in A2780 and OVCAR-3 cells transfected with the COL11A1 cDNA plasmid were evaluated using real-time RT-PCR. All experiments were performed in triplicate. * P < 0.05, compared with V. C A2780 cells were transfected with a COL11A1 cDNA plasmid and A2780CP70 cells were transfected with COL11A1 shRNA. COL11A1, DNMT1, DNMT3A, DNMT3B were evaluated using western blotting. β-actin was used as a loading control. D The binding activity of DNMT1, DNMT3A, and DNMT3B to the miR-509-3p promoter was evaluated using ChIP in COL11A1-overexpressing A2780 cells and COL11A1-knockdown A2780CP70 cells. Chromatin was isolated and immunoprecipitated using anti-DNMT1, -DNMT3A, and -DNMT3B antibodies, respectively. E The binding activity of DNMT1 to the miR-509-3p promoter was evaluated using ChIP in COL11A1-overexpressing A2780 cells treated with 1 or 10 µM 5-aza-2′-deoxycytidine for 5 days. Chromatin was isolated and immunoprecipitated using an anti-DNMT1 antibody. F A2780 cells were transfected with a COL11A1 cDNA plasmid and A2780CP70 cells were transfected with COL11A1 shRNA. The COL11A1, Akt, p-Akt (ser473), DNMT1, DNMT1 (ser84), DNMT1 (ser154), and p16 levels were evaluated using western blotting. β-actin was used as a loading control. G A2780 cells transfected with the COL11A1 cDNA plasmid and A2780CP70 cells transfected with COL11A1 shRNA and the corresponding cell lysates were immunoprecipitated using anti-DNMT1 antibodies. The resulting immunoprecipitates (IPs) were analyzed via immunoblotting (IB) using an anti-ubiquitin antibody. H Representative bisulfite sequencing of A2780 cells transfected with the COL11A1 cDNA plasmid and of A2780CP70 cells transfected with COL11A1 shRNA. I OVCAR-3 cells were transfected with a COL11A1 cDNA plasmid and OVCAR-8 cells were transfected with COL11A1 shRNA. The COL11A1, DNMT1, DNMT3A, DNMT3B, Akt, p-Akt (ser473), p-DNMT1 (ser84), p-DNMT1 (ser154), and p16 levels were evaluated using western blotting. β-actin was used as a loading control. J OVCAR-3 cells were transfected with a COL11A1 cDNA plasmid and OVCAR-8 cells were transfected with COL11A1 shRNA and the cell lysates were immunoprecipitated using anti-DNMT1 antibodies. The resulting IPs were analyzed via IB, using an anti-ubiquitin antibody. K The binding activity of DNMT1 to the miR-509-3p promoter was evaluated using ChIP in COL11A1-overexpressing OVCAR-3 cells and COL11A1-knockdown OVCAR-8 cells. Chromatin was isolated and immunoprecipitated using an anti-DNMT1 antibody. L Representative bisulfite sequencing of OVCAR-3 cells transfected with the COL11A1 cDNA plasmid and of OVCAR-8 cells transfected with COL11A1 shRNA