Elsevier

Gynecologic Oncology

Volume 132, Issue 3, March 2014, Pages 739-744
Gynecologic Oncology

The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer

https://doi.org/10.1016/j.ygyno.2014.01.034Get rights and content

Highlights

  • We demonstrated that miR-21 is overexpressed in cisplatin resistant ovarian cancer cell line.

  • The inhibition of miR-21 reversed drug resistance in vitro.

  • Overexpression of miR-21 is associated with worse patient survival.

Abstract

Background

MicroRNAs have been implicated in tumorigenesis, drug resistance, and prognosis in cancer. We investigated the role of microRNA-21 (miR-21) in regulating ovarian cancer drug resistance.

Methods

We used parental and cisplatin resistant ovarian cell lines to demonstrate the role of miR-21 in drug resistance and investigated the gene targets of miR-21. Fresh tumor specimens were used to validate our in vitro findings.

Results

Cisplatin resistant ovarian cells were four-fold more resistant compared to the parental cell line. MiR-21 was overexpressed in the resistant cell line on microRNA microarray, which was subsequently validated with qRT-PCR. Using anti-microRNA inhibitors, we demonstrated that miR-21 attenuation reversed the drug resistant phenotype in both the resistant and parental cell lines. The inhibition of miR-21 induced apoptosis based on annexin V-FITC immunostaining. Using Western blot analysis, miR-21 knockdown enhanced the expression of tumor suppressor PDCD4, and attenuated apoptosis inhibitor c-IAP2. Using 101 specimens from advanced ovarian cancer patients enrolled in The Cancer Genome Atlas, we found that women with tumors that overexpressed miR-21 were associated with a shorter progression-free survival.

Conclusion

Our data suggest that miR-21 regulates drug resistance via apoptosis and cellular survival pathways. Targeting miR-21 may have clinical utility in the treatment of resistant ovarian cancer.

Introduction

In the United States, ovarian cancer is the most lethal gynecologic malignancy associated with 15,500 deaths in 2012 [1]. Although ovarian cancer patients have a good initial response to chemotherapy, the majority will recur and succumb to their disease due to drug resistance. Novel therapies are needed to target drug resistant ovarian cancer.

MicroRNAs (miRs) are small non-coding nucleotides that post-transcriptionally regulate gene expression. These molecules may serve either as oncogenes or tumor suppressors to influence tumorigenesis and prognosis in ovarian cancer [2]. The role of microRNAs in drug resistance has received considerable attention. Investigators have shown that microRNAs may modulate response to chemotherapy, and that inhibition of these microRNAs may reverse ovarian cancer drug resistance [3], [4], [5], [6], [7], [8], [9], [10]. More specifically, prior studies have demonstrated that microRNAs regulate drug resistance via MDR1 expression, and that the attenuation of microRNAs increased sensitivity to cisplatin [11], [12].

MiR-21 has been implicated in the tumorigenesis, drug resistance, and prognosis of breast, head/neck and ovarian cancers [13], [14], [15]. In breast and head/neck cancers, the inhibition of miR-21 attenuated cell proliferation and tumor growth by augmenting apoptosis [15], [16]. Furthermore, Nam et al. showed that dysregulation of miR-21 affects ovarian cancer prognosis, while others have shown that miR-21 was associated with ovarian cancer proliferation and cell migration via the PTEN tumor suppressor pathway [17], [18]. While prior studies have identified the association of miR-21 and prognosis, few have investigated the role miR-21 regulating ovarian cancer drug resistance. In addition, most reports used cell line data without validation from fresh tumor specimens. In this current report, we explored the role of miR-21 in regulating drug resistance and identified the downstream effectors of this microRNA.

Section snippets

Cell culture

Parental A2780, cisplatin-resistant A2780-CP (Sigma-Aldrich, St. Louis, MO), or SKOV3 cells (ATCC, Manassas, VA) cells were cultured according to manufacturer's guidelines, with anti-mycotic/anti-biotic supplemented and 10% fetal bovine serum (FBS).

Transfection with anti-miR-21 inhibitor

Cells were transfected with 25 nM or 50 nM anti-miR-21 miRIDIAN Hairpin Inhibitor (Thermo Scientific Dharmacon, Lafayette, CO) using Lipofectamine 2000 transfection reagent (Invitrogen, Grand Island, NY) according to manufacture guidelines. 25 nM of

Overexpression of MiR-21

After 72 h exposure to cisplatin, the A2780-CP cells were 4-fold more resistant than parental A2780 cells based on MTT proliferation assay (IC50 15.9 vs. 4.0 μM; p = 0.02) (Fig. 1). In our DNA microarray, we identified a 2.1 fold increase in miR-21 expression in the resistant vs. parental cell lines. Other microRNAs that were differentially expressed included: miR-7d (2.1 fold), miR-10b (2.4), miR-29a (2.5), miR-98 (2.3), Let-7a (3.4), miR-10a (3.6), miR-422a (4.3), miR-27b (4.8), miR-93 (5.8), and

Discussion

Although most advanced ovarian cancer patients initially respond to chemotherapy, the majority will recur and succumb to their disease. As such, research towards defining the mechanisms and overcoming drug resistance may improve the outcomes of women with this aggressive cancer.

MicroRNAs are small non-coding nucleotides that regulate genes post-transcriptionally. Prior studies have shown that microRNAs may be implicated in cancer tumorigenesis and prognosis [13], [14]. The role of microRNAs in

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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    Financial support: This work is supported by the John Kerner Fund, the Veterans Administration Merit Review Awards (RR & D-1I01 RX000601 and BLR & D-5I01 BX000628), United States Public Health grants (R01 CA66163) and DOD grant. LYWB is a VA Senior Research Career Scientist.

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