Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells

Abstract

MicroRNAs are short non-coding RNA molecules able to affect stability and/or translation of mRNA, thereby regulating the expression of genes involved in many biological processes. We report here that microRNAs miR-27a and miR-451 are involved in activating the expression of P-glycoprotein, the MDR1 gene product that confers cancer cell resistance to a broad range of chemotherapeutics. We showed that expressions of miR-27a and miR-451 were up-regulated in multidrug resistant (MDR) cancer cell lines A2780DX5 and KB-V1, as compared with their parental lines A2780 and KB-3-1. Treatment of A2780DX5 cells with the antagomirs of miR-27a or miR-451 decreased the expression of P-glycoprotein and MDR1 mRNA. In contrast, the mimics of miR-27a and miR-451 increased MDR1 expression in the parental cells A2780. The sensitivity to and intracellular accumulation of cytotoxic drugs that are transported by P-glycoprotein were enhanced by the treatment with the antagomirs of miR-27a or miR-451. Our results demonstrate for the first time the roles of microRNAs in the regulation of drug resistance mediated by MDR1/P-glycoprotein, and suggest the potential for targeting miR-27a and miR-451 as a therapeutic strategy for modulating MDR in cancer cells.

Fig. 1

Effects of antagomirs and mimics of miR-27a (A) and miR-451 (B) on P-gp expression in MDR cancer cells. A2780DX5 cells were transfected with 100 nM of mimics or antagomirs of miR-27a or miR-451, or a control RNA. Forty-eight hours later, cell lysates were prepared from the transfected cells. Equal amounts (25 µg proteins) of cell lysates were separated by 8% SDS-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membrane. The membranes were immunoblotted with a monoclonal anti-P-gp antibody, C219. Detection of P-gp was performed using enzyme-linked chemiluminescence. β-Actin was used as a loading control. Protein expression was quantified using the ImageJ software. P-gp/actin ratios of the samples treated with a control RNA was arbitrarily set at 1, and the P-gp/actin ratios of the mimic or antagomir-treated samples were normalized to the control. Results represent the mean ± S.D. of triplicate determinations from one of three identical experiments.

Fig. 2

Effects of antagomirs and mimics of miR-27a and miR-451 on MDR1 mRNA expression in MDR cancer cells. A2780 or A2780DX5 cells were transfected with 100 nM of mimics or antagomirs of miR-27a ormiR-451, or a control RNA. Forty-eight hours later, total RNAs were extracted from the treated cells and quantitative real-time RT-PCR analysis of MDR1 mRNA was performed as described in Section 2. MDR1 mRNA level of the samples treated with a control RNA was arbitrarily set at 1, and the MDR1 mRNA levels of the mimic or antagomir-treated samples were normalized to the control. Results are the mean ± S.D. of triplicate determinations from one of three identical experiments.

Fig. 3

Effects of antagomir of miR-27a on sensitivity of MDR cancer cells to vinblastine and hydroxyurea. miR-27a antagomir or a control RNA-treated A2780DX5 cells were plated in 96-well plates in growth medium and incubated at 37 °C in a humidified 5% CO₂ atmosphere for 60 h in the presence of vehicle or a series of concentrations of vinblastine or hydroxyurea. At the end of incubation, the viability of cells was measured using the MTT assay. Each point represents the mean ± S.D. of quadruplicate determinations. Results are the representative of two similar experiments.

Fig. 4

Effects of antagomirs and mimics of miR-27a and miR-451 on accumulation of doxorubicin and rhodamine 123 (Rh-123) in MDR cancer cells. (A and B) Drug resistant A2780DX5 cells treated with the antagomirs ormimics of miR-27a or miR-451, and the parental, drug sensitive A2780 cells, were incubated with 25 µM of doxorubicin for 2 h. At the end of incubation, the cells were washed thrice with PBS, and then, (A) observation under a fluorescence microscope with 400× lens; (B) quantification of doxorubicin fluorescence intensity. Bars, mean ± S.E.M. **p < 0.05. (C) Cells were incubated with 2 µM of Rh-123 for 30 min at 37 °C. At the end of incubation, the cells were washed thrice with PBS to remove the free Rh-123. The fluorescence of remaining Rh-123 in the cells was measured by FACS. Results are the representative of two similar experiments.