Elsevier

Gynecologic Oncology

Volume 119, Issue 1, October 2010, Pages 125-130
Gynecologic Oncology

MiR-27a modulates MDR1/P-glycoprotein expression by targeting HIPK2 in human ovarian cancer cells

https://doi.org/10.1016/j.ygyno.2010.06.004Get rights and content

Abstract

Objective

MicroRNAs (miRNAs) are non-coding, single-stranded small RNAs that regulate gene expression negatively, which is involved in fundamental cellular processes and the initiation, development and progression of human cancer. In this study, we investigated the role of miR-27a in the development of drug resistance in ovarian cancer cells.

Methods

Expression of miR-27a in ovarian cancer cell lines A2780 and A2780/Taxol were detected by stem-loop real-time PCR. A2780 and A2780/Taxol cells were transfected with the mimics or inhibitors of miR-27a or negative control RNA (NC) by Lipofectamine 2000. The expression levels of MDR1 mRNA, P-glycoprotein (P-gp) and Homeodomain-interacting protein kinase-2 (HIPK2) proteins were assessed by real-time PCR and western blot respectively. Drug sensitivity was analyzed by MTT assay while apoptosis and the fluorescence intensity of intracellular Rhodamine 123 (Rh-123) were measured by FACS.

Results

The expression levels of miR-27a and P-gp were up-regulated in paclitaxel-resistant ovarian cancer cell line A2780/Taxol as compared with its parental line A2780. Transfection of A2780/Taxol cells with the inhibitors of miR-27a decreased the expression of MDR1 mRNA and P-gp protein, increased HIPK2 protein expression, enhanced the sensitivity of A2780/taxol cells to paclitaxel, increased paclitaxel-induced apoptosis and the fluorescence intensity of intracellular Rh-123. Expression of MDR1 mRNA was increased while the sensitivity to paclitaxel was decreased in A2780 cells management with the mimics of miR-27a.

Conclusions

The deregulation of miR-27a may be involved in the development of drug resistance, regulating the expression of MDR1/P-gp, at least in part, by targeting HIPK2 in ovarian cancer cells.

Introduction

Ovarian cancer is the most common cause of death from gynecologic malignancy in the world, which is seriously harmful to the women's health. Due to the lack of effective biomarkers screening test and no early symptoms, more than two thirds of women with ovarian cancer have advanced (Stage III or IV) disease at the time of diagnosis [1]. Currently the standard treatment for advanced-stage ovarian cancer is primary cytoreductive surgery, followed by platinum and paclitaxel combination chemotherapy. However, most of these patients will relapse between one to two years and only 20%–30% will be alive after 5 years [2]. Resistance of cancer cells to chemotherapy is a major clinical obstacle to successful treatment and leads to poor prognosis for the patients. Although the specific mechanisms of drug resistance are not clear, several major mechanisms have been demonstrated to play crucial role in the resistance of chemotherapy, such as selective expression of beta-tubulin isotypes, increased repair of DNA damage, reduced apoptosis, altered metabolism of drugs and the overexpression of P-glycoprotein [3], [4]. Among them, P-glycoprotein, a 170-kDa transmembrane glycoprotein encoded by the MDR1 (ABCB1) gene on human chromosome 7p21, belongs to the ATP-binding cassette (ABC) transporters and involves in efflux of drugs from cancer cells. In addition, P-gp overexpression is considered to contribute to drug resistance in many tumors including ovarian cancer [5], [6].

MicroRNAs (miRNAs) are a cluster of short non-protein-coding RNAs, about 19–23 nucleotides in length [7]. In the cytoplasm, pre-miRNAs are cleaved to mature miRNAs which is incorporated into the RNA-induced silencing complex (RISC) and binds mainly to the 3′-untranslated region (3′-UTR) of the target mRNA by way of a 7- to 8-nucleotide seed sequence, leading to either translational inhibition or degradation of the target mRNA [8]. Previous reports demonstrated that miRNAs were involved in fundamental cellular processes such as cellular differentiation, proliferation, apoptosis and metabolism processes [9]. Recently, increasing studies have indicated that miRNAs are not only associated with the initiation, progression and metastasis of human cancers but also play a vital role in tumor cells response to chemotherapeutic agents by acting as oncogenes and tumor suppressors [10], [11], [12]. And miR-328, miR-15b, miR-16, miR-130a, miR-34a, miR-27a and miR-451 have been reported to correlate with the anticancer drug resistance [13], [14], [15], [16], [17], [18].

In the present study, we examined the role of miR-27a in the development of drug resistance in human ovarian cancer cells. The results showed that aberrant miR-27a expression may involve in the resistance of A2780/taxol cells to paclitaxel by negative regulation target gene.

Section snippets

Cell line and culture

Human ovarian cancer cell line A2780, purchased from China Center for Type Culture Collection (CCTCC), was cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Gibco, Melbourne, Australia), 100 IU/ml of penicillin and 100 μg/ml of streptomycin in a humidified atmosphere incubator with 5% CO2 at 37 °C.

Establishment of paclitaxel-resistant ovarian cancer cell line

The paclitaxel-resistant ovarian cancer cell line A2780/Taxol was established by stepwise increased concentrations of paclitaxel in our laboratory. First, the

Expressions of miR-27a and P-gp in A2780 and A2780/Taxol ovarian cancer cells

We detected the expression levels of miR-27a and P-gp in A2780 and A2780/Taxol cell lines using stem-loop real-time PCR and Western blot, respectively. It was shown that miR-27a had an average of 2.2-fold higher expression level in A2780/Taxol cells than in A2780 cells (P < 0.05, Fig. 1A). As shown in Fig. 1B, the expression of P-gp was very high in A2780/Taxol cell line, but it was absent in the parental A2780 cell line.

MiR-27a regulates expression of MDR1/P-gp

To determine whether miR-27a is involved in regulation the expression of

Discussion

Most recently, accumulating evidence revealed that aberrant microRNA expression is strongly implicated in the development of drug resistance. The down-regulation or up-regulation of miRNAs may affect the expression of target proteins which could be drug transporters, drug targets or cell apoptosis and cell-cycle-related components, resulting in variations of sensitivity of cells to chemotherapeutic drugs. The studies about the roles of miRNAs in the development of drug resistance have attracted

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

This work was supported by grant no. 30901585 from the National Natural Science Foundation of China.

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