MicroRNA miR-30a inhibits cisplatin resistance in ovarian cancer cells through autophagy

Abstract

We study whether microRNA miR-30a inhibits the autophagy through transforming growth factor (TGF)-β/Smad4 to generate cisplatin (DDP) resistance in ovarian cancer cells. The expression of miR-30a, Smad4, and TGF-β was detected in the serum of ovarian cancer patients and DDP-resistant cell lines (A2780) by quantitative real-time polymerase chain reaction (qRT-PCR). Computational search and western blotting were used to demonstrate the downstream target of miR-30a in ovarian cancer cells. Cell viability was measured with CCK8 assay. Apoptosis and autophagy of ovarian cancer cells were analyzed by flow cytometry and transmission electron microscopy, and the expressions of Beclin1 and LC3II protein were detected by western blotting. Expression of miR-30a was significantly decreased, while expressions of TGF-β and Smad4 mRNA were increased in serum of ovarian cancer patients after DDP chemotherapy as well as in DDP-resistant cells. Activation of autophagy contributed to DDP-resistance cells. Moreover, Bioinformatics software predicted Smad4 to be a target of miR-30a. Overexpression of miR-30a decreased the expression of Smad4 and TGF-β. Additionally, miR-30a-overexpressing inhibited DDP-induce autophagy and promoted DDP-resistant cell apoptosis. In conclusion, miR-30a mediates DDP resistance in ovarian cancer by inhibiting autophagy via the TGF-β/Smad4 pathway.

Keywords: Autophagy; Cisplatin; Ovarian cancer; Sensitivity; miR-30a.

Figure 1.

Expression of miR-30a, Smad4, and TGF-β in serum of ovarian cancer patients treated with DDP. A–C, qRT-PCR was used to detect the relative expression level of miR-30a (a) Smad4 (b) and TGF-β (c)

Figure 2.

Autophagy promotes DDP resistance in ovarian cancer cells. (a) CCK-8 assay was performed to detect the cell viability of A2780 and A2780-DDP cells. **P < 0.01 vs. A2780 group. (b) The protein expression of LC3I/II and Beclin1 were measured by western blotting; (c) CCK-8 assay was used to detect the cell viability; 3-MA, autophagy inhibitor; RAPA, autophagy activator rapamycin; **P < 0.01 vs. Blank control group; #P < 0.05 vs. 3-MA group; &P < 0.05 vs. RAPA group

Figure 3.

Expression of miR-30a, TGF-β, and Smad4 in DDP drug-resistant cells. (a) The miR-30a expression in A2780-DDP and A2780 cells was detected using qRT-PCR. (b) Western blotting was used to determine the protein expression of Smad4 and TGF-β in A2780 and A2780-DDP cells. (c) qRT-PCR was used to measure the mRNA expression of Smad4 and TGF-β. Compared with the blank control group, *P < 0.05 and **P < 0.01 vs. Control group; #P < 0.05 and ##P < 0.01 vs. DDP in A2780

Figure 4.

MiR-30a inhibits Smad4 and TGF-β expression in DDP drug-resistant cells. (a) TargetScan analysis indicated that there was a base complement site in miR-30a and Smad4 gene. (b) qRT-PCR detection of miR-30a expression after transfection with miR-30a mimics or mimics NC. **P < 0.01 vs. negative control group. (c) Western blotting was used to determine the protein expression of Smad4 and TGF-β in A2780 and A2780-DDP cells

Figure 5.

miR-30a suppressed autophagy by regulation of TGF-β/Smad4 signaling pathway. (a-b) MDC staining was detected by flow cytometry, **P < 0.01 vs. Blank control group; #P < 0.05 vs. DDP group; &P < 0.05 vs. miR-30a group. (c) Western blotting detected the expression of autophagy-related proteins (LC3II and Beclin1) and TGF-β/Smad4 pathway proteins

Figure 6.

Over-expression of miR-30a suppressed autophagy and promoted DDP-induced apoptosis by inhibition of TGFβ/Smad4 signaling pathway. (a-b) Cells apoptosis was detected by flow cytometry. **P < 0.01 vs. The first group; #P < 0.05 vs. DDP group. (c) Western blotting was used to detect the expression of TGF-β, LC3II, Beclin1, and Smadf4 in A2780 and A2780-DDP cells