Elsevier

Gynecologic Oncology

Volume 111, Issue 3, December 2008, Pages 478-486
Gynecologic Oncology

Role of microRNAs in drug-resistant ovarian cancer cells

https://doi.org/10.1016/j.ygyno.2008.08.017Get rights and content

Abstract

Objectives

Chemotherapy is the preferred therapeutic approach for the therapy of advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This work was aimed to identify selected microRNAs involved in the development of chemoresistance in ovarian cancer.

Methods

High-throughput analysis of the miRNA profile in a panel of paclitaxel- (A2780TAX, A2780TC1 and A2780TC3) and cisplatin-resistant (A2780CIS) cells was assessed using a microarray platform and subsequent validation with qPCR and Northern blots. Downstream target validation was performed for miR-130a and the target M-CSF.]

Results

Six miRNAs (let-7e, miR-30c, miR-125b, miR-130a and miR-335) were always diversely expressed in all the resistant cell lines. Let-7e was upregulated in A2780TAX cells, while it was downregulated in the other resistant cell lines. The opposite phenomenon was obtained for miR-125b, which was downregulated in A2780TAX and upregulated in the other cell lines. The miR-30c, miR-130a and miR-335 were downregulated in all the resistant cell lines, thereby suggesting a direct involvement in the development of chemoresistance. Finally downstream target validation was proven for the miR-130a, whose downregulation was linked to the translational activation of the M-CSF gene, a known resistance factor for ovarian cancer.

Conclusions

Our results indicate that ovarian cancer drug resistance is associated with a distinct miRNA fingerprint, and miRNA microarrays could represent a prognostic tool to monitor the chemotherapy outcome.

Introduction

Ovarian cancer is the deadliest cancer of the female reproductive system [1]. The main limitation to a successful treatment is the development of resistance to combined chemotherapy, such as platinum-based drugs, cisplatin or carboplatin, coupled with paclitaxel. Cisplatin binds to double-stranded DNA and forms DNA adducts, interfering with DNA replication and RNA transcription ultimately triggering apoptosis. Paclitaxel is a mitotic inhibitor which binds and hyper-stabilize microtubules, specifically binding to the β-tubulin subunit, thereby suppressing microtubule dynamicity [2], [3]. Many molecular mechanisms implicated in the rise of resistance in cellular models of ovarian cancers have been characterized, emphasising its biological complexity: increased DNA repair activity and defective DNA damage response [4], increased anti-apoptotic regulators activity [5], [6], growth factor receptor deregulation [7], [8], [9] and post-translational modification or altered expression of β-tubulin and other microtubule regulatory proteins [10]. In the past few years we audited the discovery of a new class of genomic regulators which are now universally recognized as central players in virtually any neoplasm development and progression, named microRNAs.

MicroRNAs (miRNAs) are a growing class of small (∼ 22 bp) endogenous non-coding RNAs which modulate gene expression mainly by base-pairing to the 3′-UTR of the target mRNA, and causing translational repression, mRNA cleavage, or destabilization [11], [12]. miRNA-mediated post-transcriptional gene regulation is emerging as a critical regulator of many cellular processes, both physiologic and pathologic [13], [14], [15]. Hundreds of miRNAs have been found in various organisms, suggesting their potential roles in all biological events [16], [17], [18].

Some miRNAs are abnormally expressed in human malignancies, including leukaemia [19], breast cancer [20], lung cancer [21] and thyroid carcinoma [22], and miRNA expression profiles can classify stage, subtype and prognosis of some cancers [23], [24], [25], [26], [27], [28], [29]. In this report we describe the miRNA signature of cisplatin-, paclitaxel- and cyclosporin A-resistant A2780 ovarian cancer cell line. We show that some miRNAs are deregulated in all the derived cell line and we demonstrate that the miR-130a, which is down-modulated in all the samples, targets M-CSF, previously described as predictor of poor outcome [30] and chemoresistance factor [31] in advanced ovarian cancer. We postulate that the miRNA signature of drug resistance could be pursued to develop new strategies for targeted therapies in chemorefractive ovarian carcinoma patients.

Section snippets

Cell culture

A2780 and A2780CIS human ovarian cancer cells were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK). A2780TAX, A2780TC1 and A2780TC3 cells were generated in our laboratory and are elsewhere described [32]. Culture media were selected according to ECACC suggestions. HeLa cells were cultured in DMEM medium (Gibco BRL) supplemented with 50 IU/ml of penicillin, 50 µg/ml streptomycin, Glutamax 1:100 and 10% heat-inactivated fetal bovine serum (FBS).

miRNA microarray

Total RNA was

Results

A microarray platform optimised for the analysis of a panel of 381 human microRNA was used to analyse and compare the pattern of microRNAs expression between parental human ovarian cancer A2780wt cell line and its counterparts made resistant to cisplatin (A2780CIS) and paclitaxel (A2780TAX, resistance P-glycoprotein-dependent), and TC1/TC3, made resistant to paclitaxel in the presence of cyclosporine as inhibitor of P-glycoprotein. Untransformed results were analysed using the Asterias suite (//asterias.bioinfo.cnio.es

Discussion

Drug resistance is a multifactorial process which is responsible for the absence of chemoresponse in primary and secondary tumors. In advanced ovarian cancer the first line of chemotherapy is represented by the combination carboplatin/cisplatin with paclitaxel. With this regimen around 20% of patients do not respond at the first cycle and are featured by progression upon treatment in the first year and by poor outcome [39], [40]. The remaining patients relapse after one year from the first

Conflict of interest statement

The authors have no conflicts of interest to declare.

Acknowledgments

We would like to thank Mrs. Cristiana Gaggini and Mrs. Federica Pignatelli for the expert technical assistance. This work was partially supported by a grant from the Italian Ministry of University and Research.

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