Dicer affects cisplatin‑mediated apoptosis in epithelial ovarian cancer cells

Abstract

Dicer is an essential enzyme that processes micro (mi)-RNA precursors into mature miRNAs, and serves a critical role in cancer development and progression by regulating gene expression. However, the role of Dicer in cisplatin‑mediated apoptosis and chemotherapy resistance in epithelial ovarian cancer (EOC) cells is poorly understood. In the present study, Dicer was expressed at low levels in cisplatin‑resistant A2780 cells when compared with parental cells. In addition, knocking down Dicer using short hairpin RNA decreased the sensitivity of A2780 and CAOV3 cells to cisplatin. Furthermore, downregulating Dicer significantly inhibited cisplatin‑induced apoptosis in ovarian cancer cells, and decreased the levels of proteins involved in apoptosis signaling pathways, including P73, P63, P53, caspase‑9 and caspase‑3. These findings indicated that Dicer may be a promising target for overcoming drug resistance in ovarian cancer.

Figure 1.

Dicer expression levels are decreased in A2780/DDP cells. (A) Western blots presenting the Dicer protein levels in A2780 cells following treatment with 20 µM cisplatin for 0, 12 and 24 h. (B) Cell survival following cisplatin treatment was significantly higher in A2780/DDP cells when compared with A2780 cells, as determined by MTT assays. (C) The cisplatin IC₅₀ was significantly increased in A2780/DDP cells when compared with that in A2780 cells following the MTT assays. (D) The relative level of Dicer mRNA was decreased in A2780/DDP cells when compared with A2780 cells, as determined by reverse transcription-quantitative polymerase chain reaction. Dicer expression was normalized to β-actin. (E) Dicer protein expression was downregulated in A2780/DDP cells when compared with A2780 cells according to western blot analyses. *P<0.05, **P<0.01 and ***P<0.001, vs. A2780/as indicated.

Figure 2.

Dicer knockdown increases cisplatin resistance in ovarian cancer cells. (A) The expression levels of Dicer mRNA and (B) protein in A2780 and CAOV3 cells treated with Dicer-targeted shRNA (shDicer) or scrambled shRNA (shNC) and in parental control cells was determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses, respectively. **P<0.01, as indicated. The relative viability of (C) A2780/shDicer vs. A2780/shNC and that of (D) CAOV3/shDicer vs. CAOV3/shNC were measured by MTT assays after treating cells with the indicated concentrations of cisplatin. (E and F) The IC₅₀ for cisplatin in the ovarian cancer cell lines, (E) A2780 and (F) CAOV3 are shown. ***P<0.001, as indicated. IC₅₀, half-maximal inhibitory concentration; shRNA, short hairpin RNA; NC, negative control.

Figure 3.

Dicer knockdown inhibits cisplatin-induced cell apoptosis in ovarian cancer cells. (A) Representative fluorescence images of A2780 and CAOV3 parental control, shNC and shDicer cells stained with Hoechst 33342 (blue) following cisplatin treatment. The yellow arrows indicate apoptotic cells with nuclear fragmentation. Scale bars, 50 µm. (B) The percentages of apoptotic cells were quantified by immunofluorescence analyses. (C) Apoptotic parental control, shNC and shDicer cells of the A2780 and CAOV3 cell lines were assessed using flow cytometry with Annexin V and PI double staining following cisplatin treatment. (D and E) The percentages of apoptotic (D) A2780 and (E) CAOV3 cells were quantified by flow cytometric analyses. Data are presented as the mean ± standard deviation. *P<0.05 and ***P<0.001, as indicated. shRNA, short hairpin RNA; NC, negative control; PI, propidium iodide.

Figure 4.

Dicer knockdown decreases the expression levels of apoptosis-associated proteins induced by cisplatin in ovarian cancer cells. Western blot analyses demonstrated that the expression levels of proteins involved in apoptosis signaling pathways, including P73, P63, P53, caspase-9 and caspase-3, were decreased in shDicer cells when compared with shNC cells following cisplatin treatment in the A2780 and CAOV3 cell lines. Western blot analyses also demonstrated that P21 protein expression was not affected by Dicer downregulation in A2780 and CAOV3 cells, while it was decreased following cisplatin treatment. The results were normalized to the expression of the reference protein β-actin. shRNA, short hairpin RNA; NC, negative control.