microRNA-199a is able to reverse cisplatin resistance in human ovarian cancer cells through the inhibition of mammalian target of rapamycin

Abstract

microRNAs (miRNAs/miRs) may have a crucial function in tumor metastasis through the regulation of a plethora of signaling pathways. Increasing evidence has shown that miR-199a is important in regulating the tumor metastasis of ovarian cancer, although the precise biological function of miR-199a is unclear at present. In the current study, it was observed that the expression levels of miR-199a were higher in OV2008 cells compared with C13* cells. However, lower levels of mammalian target of rapamycin (mTOR) protein were detected by western blotting in the OV2008 cells compared with the C13* cells. The miR-199a levels were increased in the C13* cells using miR-199a mimics and the mTOR levels were observed to decrease. This may have resulted in a reversal of cisplatin resistance in the C13* cells. To test this hypothesis, the Renilla luciferase reporter gene system was used to analyze the mTOR levels. The results indicated that the expression levels of mTOR were significantly blocked by the increased miR-199a levels. When the miR-199a inhibitor was applied to decrease the miR-199a levels, it was observed that the mTOR expression levels were increased, while cisplatin-induced apoptosis was decreased in the OV2008 cells. The study concludes that miR-199a is able to reverse cisplatin resistance in human ovarian cancer cells through the inhibition of mTOR and that mTOR may be the target of miR-199a during this process.

Keywords: cisplatin resistance; mammalian target of rapamycin; microRNA-199a; ovarian cancer cells.

Figure 1

Expression levels of miR-199a and mTOR in OV2008 and C13* cells were detected by qPCR and western blotting. (A) Expression levels of miR-199a were, on average, 83.4-fold higher in the OV2008 cells compared with the C13* cells (P<0.05). (B) Noticeably more mTOR protein was expressed in the C13* cells compared with the OV2008 cells, as shown by western blotting. (C) The mTOR fold change in protein level is shown from three independent experiments. ^*P<0.05 vs. C13*; ^**P<0.05 vs. OV2008. mTOR, mammalian target of rapamycin.

Figure 2

Effect of miR-199a on sensitivity to cisplatin treatment in C13* and OV2008 cells. (A) C13* cells were transfected with miR-199a mimics and miR-mimic negative control (NC), then treated with 40 μM cisplatin for 24 h. Cell apoptosis was measured by flow cytometry. (B) OV2008 cells were transfected with miR-199a inhibitor and inhibitor NC, then treated with 40 μM cisplatin for 24 h. Cell apoptosis was measured by flow cytometry. (C) Cisplatin sensitivity was decreased in miR-199a inhibitor-treated OV2008 cells compared with those treated with NC, and the viability of the cells was evaluated by the CCK-8 assay. (D) C13* cells transfected with mimics of miR-199a exhibited increased sensitivity to cisplatin treatment; the viability of the cells was evaluated by the CCK-8 assay. ^*P<0.05 vs. NC. mTOR, mammalian target of rapamycin; CCK-8, cell counting kit-8.

Figure 3

Regulation of mTOR expression by miR-199a. (A) Expression of mTOR mRNA in OV2008 cells transfected with inhibitor of miR-199a. (B) Expression of mTOR mRNA in C13* cells transfected with mimics of miR-199a. (C) Expression of mTOR protein in OV2008 cells transfected with inhibitor of miR-199a. (D) Expression of mTOR protein in C13* cells transfected with mimics of miR-199a. ^*P<0.05 vs. NC. mTOR, mammalian target of rapamycin.

Figure 4

mTOR may be a target gene of miR-199a. (A) mTOR’s 3′-UTR mRNA includes a highly-conserved binding site for miR-199a. (B) C13* cells were co-transfected with psiCHECK-2™/mTOR 3′-UTR and miR-199a mimics or control oligonucleotides. At 24 h post-transfection, mTOR fluorescence intensity was detected. (C) Luciferase activity was not different between empty vector psiCHECK-2 and controls. ^*P<0.05 vs. blank. mTOR, mammalian target of rapamycin; UTR, untranslated region.