MiR-221/222 promote chemoresistance to cisplatin in ovarian cancer cells by targeting PTEN/PI3K/AKT signaling pathway

Abstract

Cisplatin resistance is one of the main limitations in the treatment of ovarian cancer, and its mechanism has not been fully understood. The objectives of this study were to determine the role of miR-221/222 and its underlying mechanism in chemoresistance of ovarian cancer. We demonstrated that miR-221/222 expression levels were higher in A2780/CP cells compared with A2780 S cells. An in vitro cell viability assay showed that downregulation of miR-221/222 sensitized A2780/CP cells to cisplatin-induced cytotoxicity. Moreover, we found that knockdown of miR-221/222 by its specific inhibitors promoted the cisplatin-induced apoptosis in A2780/CP cells. Using bioinformatic analysis and luciferase reporter assay, miR-221/222 were found to directly target PTEN. Moreover, knockdown of miR-221/222 in A2780/CP cells significantly upregulated PTEN and downregulated PI3KCA and p-Akt expression. In conclusion, our results demonstrated that miR-221/222 induced cisplatin resistance by targeting PTEN mediated PI3K/Akt pathway in A2780/CP cells, suggesting that miR-221/222/PTEN/PI3K/Akt may be a promising prognostic and therapeutic target to overcome cisplatin resistance and treat ovarian cancer in the future.

Keywords: Apoptosis; Cisplatin; MiR-221/222; Ovarian cancer; PTEN.

Fig. 1

Expression of miR-221/222 and PTEN in A2780 cells. a Growth inhibition rates of cisplatin for A2780 S and A2780/CP as determined by an MTT assay. Cisplatin treatment decreased ovarian cancer cells growth significantly in a dose-dependent manner. The comparisons of IC₅₀ indicated that A2780 S cells were more sensitive to cisplatin (IC₅₀—5.1 µM) than A2780/CP cells (IC₅₀—10 µM) (**p < 0.01). b RT-PCR results showed that A2780/CP cells had higher expression of miR-221/222 compared with A2780 S cells with the mean increase of 3.5 and 3.25 folds, respectively, compared with A2780 S cells. U6 was used as loading control. Expression of PTEN significantly decreased in A2780/CP cells compared to control cells. GAPDH was used as loading control. Data were expressed as mean and the SD from three independent experiments. (*p < 0.05, ***p < 0.001) (ANOVA)

Fig. 2

Efficient transfection of miR-221/222 inhibitors in A2780/CP cells a A2780/CP cells were transfected with miR-221 inhibitor, miR-222 inhibitor or scrambled oligonucleotide. A fluorescence microscope and flow cytometry analysis were used to detect the transfection efficiency. As shown in Figs. a and b, a majority of the cells were transfected after 48 h. c Relative expression of miR-221/222 was evaluated by RT-PCR. Expression of miR-221/222 was decreased in A2780/CP-transfected cells versus control cells. The expression of miR-221/222 was at the lowest level 48 h after transfection, indicating effective transfection of miR-221/222 inhibitors (***p < 0.001, ****p < 0.001) (ANOVA)

Fig. 3

Inhibition of miR-221 and miR-222 increases sensitivity of A2780/CP cells to cisplatin. A2780/CP cells were treated with increasing concentrations of cisplatin after being transfected with miR-221/222 inhibitors. After 48 h, MTT assays were performed to assess chemoresistance. Cisplatin-treated group was considered control. We observed that cell viability significantly decreased after cisplatin treatment in A2780/CP-miR-221/222-inhibitor cells compared with control group. Moreover, downregulation of miR-221 and miR-222 induced more significant reduction of cell viability under various concentrations of cisplatin treatment in A2780/CP cells (p < 0.001, ANOVA)

Fig. 4

Inhibition of miR-221/222 promote cisplatin-induced apoptosis of A2780/CP cells. A2780 cells were transfected with miR-221 and/or miR-222 inhibitor and analyzed 48 h later, and apoptosis was measured by annexin V/PI staining. The results showed that transfection of miR-221 or miR-222 inhibitor in A2780/CP cells increased the ratio of apoptotic cells significantly. Interestingly, a strong increase of apoptotic rate was detected in cells transfected with miR-221 and miR-222 inhibitors (p < 0.0001, ANOVA)

Fig. 5

PTEN is a direct target of miR-221/222 in A2780/CP cells. a The prediction of the binding between miR-221/222 and PTEN by miRTarBase. b Predicted miR-221/222-target interaction network according to strong evidence. c Western blot analysis showed that miR-221/222 inhibition led to significant increase of PTEN in A2780/CP cells. Co-transfection of miR-221 and miR-222 inhibitors into A2780/CP cells considerably upregulated PTEN and downregulated PIK3CA and p-AKT expression compared to other groups. d The luciferase activity of A2780/CP cells was detected after the co-transfection of pGL3-control vector or pGL3-PTEN 3′UTR vector with miR-221 and/or miR-222 inhibitor. There were significant differences in luciferase activity among the groups. Data represent the mean and the SD from three independent experiments (**p < 0.01, ***p < 0.001). (ANOVA)