Fig 2: Invasive phenotypes of epithelial ovarian carcinoma cells are regulated by miR-335. (A) Messenger RNA (mRNA) expression levels of collagen type XI alpha 1 (COL11A1) and miR-335 in A2780CP70 and OVCAR-8 cells transfected with miR-335 mimic, as well as A2780 and OVCAR-3 cells transfected with miR-335 inhibitor, were evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). All experiments were performed in triplicate. (B) The cell growth of A2780CP70 and OVCAR-8 cells transfected with miR-335 mimic and A2780 and OVCAR-3 cells transfected with miR-335 inhibitor for 48 h. Cell viability was assessed by 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. All experiments were performed in triplicate; ** p < 0.01, *** p < 0.001, compared with control. (C) pCMV6-AC-GFP-COL11A1 and miR-335/NC were co-transfected into A2780CP70 and OVCAR-8 cell lines. The experimental results of MTT displayed that the cell proliferation rate was obviously decreased after transfection with the mimic of miR-335, while the overexpression of COL11A1 turned over the apparent inhibition of cell proliferation by miR-335. NC: negative control; ** p < 0.01, *** p < 0.001. (D) The invasive ability of A2780CP70 and OVCAR-8 cells transfected with miR-335 mimic and A2780, as well as OVCAR-3 cells transfected with miR-335 inhibitor for 48 h. All data represent the mean ± SD of three separate experiments; ** p < 0.01, compared with control. (E) Upper panel: the protein expression of COL11A1, Ets-1, and MMP3 in A2780 cells transfected with miR-335 inhibitor, as well as A2780CP70 cells transfected with miR-335 mimic, were evaluated by western blot. β-Actin is used as a protein loading control. Lower panel: MMP3 activity is evaluated in A2780 cells transfected with miR-335 inhibitor, as well as A2780CP70 cells transfected with miR-335 mimic (Figure S2A). All experiments were performed in triplicate.

Fig 3: SC66 increases the sensitivity of mouse xenografts to anticancer drugs.a A2780/COL11A1 ovarian cancer xenografts treated with only SC66 at doses of 5 mg/kg and 15 mg/kg or combined with 3 mg/kg cisplatin. * P < 0.05, on Day 23, SC66 15 mg/kg + CDDP 3 mg/kg vs. CDDP alone (P = 0.021) or varying doses of SC66 (5 mg/kg, P = 0.021; 15 mg/kg, P = 0.043). b A2780/COL11A1 ovarian cancer xenografts treated with only SC66 at doses of 5 and 15 mg/kg or combined with 12 mg/kg paclitaxel. CDDP: cisplatin; PAC: paclitaxel. Tumor size (mean ± SD): control group 6648.38 ± 2258.12 mm3; 5 mg/kg SC66 group 3883.70 ± 843.91 mm3; 15 mg/kg SC66 group 1006.93 ± 819.18 mm3; CDDP group 2691.58 ± 1756.27 mm3; 5 mg/kg SC66 + 3 mg/kg CDDP group 995.83 ± 519.68 mm3; 15 mg/kg SC66 + 3 mg/kg CDDP group 256.30 ± 90.52 mm3; PAC group 5086.15 ± 2816.66 mm3; 5 mg/kg SC66 + 12 mg/kg PAC group 1143.63 ± 291.24 mm3; 15 mg/kg SC66 + 12 mg/kg PAC group 964.13 ± 853.27 mm3. c Representative IHC photos of p-Akt, Akt, and COL11A1 in ovarian tumor samples from mice treated with SC66 or vehicle controls

Fig 4: SC66 inhibits Akt signaling and expression of COL11A1, TWIST1, and Mcl-1 in ovarian cancer cells.a The protein expression levels of p-Akt, Akt, and COL11A1 in a panel of eight ovarian cancer cell lines were evaluated by western blotting. β-Actin protein was used as an internal loading control. All experiments were performed in triplicate. b The half maximal inhibitory concentration (IC50) value (mean ± SD) of SC66 in ovarian cancer cells was measured using the MTT assay. c, d The protein expression levels of p-Akt, Akt, p-p70S6K, p70S6K, p-4EBP1, 4EBP1, COL11A1, TWIST1, and Mcl-1 in cells treated with different concentrations of SC66 for 24 h were evaluated by western blotting. β-Actin was used as an internal loading control. All experiments were performed in triplicate

Fig 5: miR-509–3p regulates cell proliferation, invasion, and chemoresistance through SUMO-3.(A) Left panel: the putative miR-509–3p binding site in its 3'-UTR contains mutant wt-SUMO-3 and the corresponding mut-SUMO-3 (red star). Right panel: A2780CP70 cells were co-transfected with the wt-SUMO-3/mut-SUMO-3 vector and miR-509–3p NC/miR-509–3p mimics. Compared with that of the control group, the luciferase activity of the wt-SUMO-3 reporter gene was significantly reduced by miR-509–3p mimic transfection. In cells co-transfected with the miR-509–3p and mut-SUMO-3 reporter genes, the reporter gene activity did not significantly decrease. (B) The protein expression of SUMO-3 in A2780CP70 and OVCAR-8 cells transfected with miR-509–3p mimics, as well as that in A2780 and OVCAR-3 cells transfected with the miR-509–3p inhibitor, were evaluated using western blotting. ß-actin was using as a loading control. (C) Upper panel: the protein expression of COL11A1 and SUMO-3 in A2780/COL11A1 cells transfected with miR-509–3p mimics, as well as that of A2780CP70/shCOL11A1 cells transfected with the miR-509–3p inhibitor, were evaluated using western blotting. ß-actin was used as a loading control. Lower panel: the mRNA expression levels of miR-509–3p in A2780/COL11A1 cells transfected with miR-509–3p mimics, as well as those of A2780CP70/shCOL11A1 cells transfected with the miR-509–3p inhibitor were evaluated using real-time RT-PCR. All experiments were performed in triplicate. (D) A2780CP70 cells were co-transfected with pCMV3-ORF-SUMO-3 and miR-509–3p/NC. The MTT results showed that the cell proliferation rate was obviously decreased after transfection with the miR-509–3p mimics, while the overexpression of SUMO-3 reversed the apparent cell proliferation inhibition induced by miR-509–3p. NC: negative control; *** P < 0.001. (E) A2780CP70 cells were co-transfected with pCMV3-ORF-SUMO-3 and miR-509–3p/NC, and then their invasive ability was evaluated. All data represent the mean ± SD of three separate experiments; ** P < 0.01, *** P < 0.001, compared with the control. (F) A2780CP70 cells were co-transfected with pCMV3-ORF-SUMO-3 and miR-509–3p/NC, and then treated with different concentrations of cisplatin for 48 h. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. All experiments were performed in triplicate. (G) A model illustrating the hypothetical role of miR-509–3p regulation in ovarian cancer cells.