MiR-186 bidirectionally regulates cisplatin sensitivity of ovarian cancer cells via suppressing targets PIK3R3 and PTEN and upregulating APAF1 expression

Abstract

Ovarian cancer is a highly lethal malignancy in the female reproductive system. Platinum drugs, represented by cisplatin, are the first-line chemotherapeutic agents for treatment of various malignancies including ovarian cancer, but drug resistance leads to chemotherapy failure. MicroRNAs emerged as promising molecules in reversal of cisplatin resistance. MiR-186 was reported to be downregulated in the cisplatin-resistant ovarian cell lines and miR-186 expression increased cisplatin sensitivity. However, we found the bidirectional regulatory effects of miR-186 on cisplatin sensitivity for the first time that overexpression of miR-186 at low concentration increased the cisplatin sensitivity of ovarian cancer cells A2780/DDP, while high concentration of miR-186 decreased the cisplatin sensitivity. The survival assay in other types of cancer cell lines verified the bidirectional regulatory function of miR-186 on cisplatin sensitivity in dose and cell type dependent manners. MiR-186 suppressed the protein levels of PTEN and PIK3R3 dose-dependently, which are opposite regulatory molecules of the oncogenic AKT pathway. MiR-186 also enhanced the protein levels of apoptotic gene APAF1 dose-dependently. We proposed the final effects of PTEN and APAF1 outweighed PIK3R3 when miR-186 at low concentration so as to increase the cisplatin sensitivity of ovarian cancer cells, while the final effects of PIK3R3 outweighed PTEN and APAF1 when miR-186 at high concentration so as to decrease the cisplatin sensitivity. We concluded the outcome of regulation of these opposite functional molecules contributed to the bidirectional regulatory effects of miR-186 in ovarian cancer cisplatin sensitivity. It deserves more attentions when developing therapeutic strategies based on the bidirectional functional miRNAs.

Keywords: bidirectional regulation; cisplatin sensitivity; dose-dependent; miR-186; ovarian cancer.

Figure 1

The expression levels of miR-186 in cisplatin-resistant and the parental ovarian cancer cells. (A) (B) Cells were treated with cisplatin for 48 hours, and then the IC50 of cisplatin was evaluated by cell viability assay. (C) (D) The expression levels of miR-186 was analyzed by quantitative real-time PCR in cisplatin-resistant and the parental ovarian cancer cells. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

The bidirectional regulatory effects of miR-186 on cisplatin sensitivity of ovarian cancer cells. (A) A2780/DDP cells were transfected with different concentrations of miR-186 mimic or NC, 48 hours later, cells were collected and the levels of miR-186 were assessed by quantitative real-time PCR. (B) Transfected A2780/DDP cells with different concentrations of miR-186 mimic or NC, were treated with various concentrations of cisplatin for a further 48 hours, then the IC50 was evaluated by cell viability. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

The dose-dependent effects of miR-186 on cisplatin sensitivity in other types of cancer cells. Transfected cells (including SKBR3, HepG2, A549 and HCT116 cells) with different concentrations of miR-186 mimic or NC, were exposed to cisplatin at the concentration of 20uM, and cell viability was analyzed 48 hours later. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

Multiple targets of miR-186. (A) The predicated targets PIK3R3, PTEN and APAF1 were showed in bioinformatics database. (B) HEK 293T cells were co-transfected with miR-186mimic or NC and a luciferase recombinant with the 3'-UTR of PIK3R3, or APAF1, or PTEN, then the relative luciferase activity was detected 24 hours later. (C) The protein levels of PIK3R3, APAF1 and PTEN were measured by western blotting 48 hours after transfection with miR-186 mimic or NC in A2780/DDP cells. (D) The quantitative analysis of protein levels (normalized to GAPDH) by Image J. *P<0.05.

Figure 5

The general view of multiple targets or regulated molecules contributing to the bidirectional regulatory effects of miR-186 on cisplatin sensitivity. The final effects of PTEN and APAF1 outweighed PIK3R3 when miR-186 at low concentration so as to increase the cisplatin sensitivity of ovarian cancer cells, while the final effects of PIK3R3 outweighed PTEN and APAF1 when miR-186 at high concentration so as to decrease the cisplatin sensitivity.