MiR-487a resensitizes mitoxantrone (MX)-resistant breast cancer cells (MCF-7/MX) to MX by targeting breast cancer resistance protein (BCRP/ABCG2)
Introduction
Breast cancer is one of the most common malignancies in women; it seriously affects the physical and mental health of patients and can severely impact their lives [1]. Chemotherapy is the mainstream method of breast cancer treatment. Despite great progress in the treatment of breast cancer, resistance to chemotherapeutic agents remains the main obstacle to successful breast cancer therapy [2].
The human breast cancer resistance protein (BCRP/ABCG2), a member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily, plays an important role in absorption, distribution, and elimination of its substrate drugs, mediating multidrug resistance (MDR) in cancer cells [3]. The 2.4 kb BCRP mRNA from the human resistant breast cancer cell line MCF-7/AdrVP was identified using RNA fingerprinting; it encodes a 655 amino acid protein with an N-terminal ATP binding domain and a C-terminal region consisting of 6 transmembrane domains [4]. BCRP has become another important MDR protein similar to multidrug resistance associated protein (MRP) [5], P-glycoprotein (P-gp) [6] and lung resistance protein (LRP) [7]. BCRP specifically pumps out various chemotherapeutic drugs, such as mitoxantrone (MX) [8], topotecan [8], [9], 7-ethyl-10-hydroxycamptothecin (SN-38) [10] and flavopiridol [11] from cancer cells using the energy from ATP hydrolysis; this reduces intracellular drug accumulation, conferring MDR in cancer cells, including breast cancer cells. Therefore, studies on targeting BCRP expression and function have become very important for reversing MDR in breast cancer.
MicroRNAs (miRNAs) are small, endogenous RNA molecules of approximately 22 nt in length that regulate gene expression by binding to their target sites in 3′-untranslated regions (3′UTRs) of mRNAs leading to cleavage or translational repression of the targeted mRNA [12]. In recent years, more attention has been paid to the role of miRNAs in reversing drug resistance. To date, several miRNAs have been reported to regulate BCRP expression by targeting the 3′UTR of BCRP mRNA in selected cancer cells. To et al. has demonstrated that has-miR-519c can decrease endogenous BCRP mRNA and protein levels by specifically interacting with the target binding site in the BCRP 3′UTR in a parental S1 colon cancer cell line that has a longer 3′UTR transcript of BCRP mRNA than its drug-resistant counterpart [13]. Additionally, miR-520h and miR-328 have been found to reduce both mRNA and protein levels of BCRP in the pancreatic cancer cell line PANC-1 [14] and drug-sensitive MCF-7 and drug-resistant MCF-7/MX100 human breast cancer cells [15]. Thus, miRNAs play an important role in regulating BCRP expression in cancer cells.
In this study, we determined by bioinformatic analysis that miR-487a was one of the miRNAs that can bind to the 3′UTR of BCRP. However, it is still unclear whether miR-487a can regulate the expression of BCRP and reverse the drug resistance of breast cancer cells. Moreover, to date, miR-487a has not been studied in any cancer cells. In the present study, we first demonstrated that miR-487a directly bound to the 3′UTR of BCRP by a luciferase reporter assay, after confirming that the expression of miR-487a was much lower in MX resistant MCF-7/MX breast cancer cells overexpressing BCRP compared with sensitive MCF-7 cells. We further determined that ectopic miR-487a expression decreased BCRP expression in MCF-7/MX cells and enhanced their sensitivity to MX. Meanwhile, inhibition of miR-487a expression increased BCRP expression in MCF-7 cells and reduced their sensitivity to MX. Furthermore, we also demonstrated that ectopic miR-487a expression increased the in vivo response of MCF-7/MX cells to MX.
Section snippets
Cell lines and mice
The human breast cancer MCF-7 cell line was purchased from the American Type Culture Collection. The Mitoxantrone (MX)-resistant MCF-7 cells (MCF-7/MX) were kindly provided by Zhirong Zhan (Molecular Therapeutics Section, Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA). These cells were maintained in Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone,
miR-487a is expressed at a lower level in MCF-7/MX cells overexpressing BCRP
To identify the differential sensitivity of the parental MCF-7 and its derivative mitoxantrone (MX)-resistant MCF-7/MX breast cancer cell line to MX (a well-known BCRP substrate), we first determined the cytotoxicity of MX in the two different cell lines by MTT assay. As shown in Fig. 1A, MCF-7/MX cells showed resistance to MX with higher IC50 values (3.63 ± 0.34 μM) than MCF-7 cells (0.52 ± 0.05 μM). Next, to investigate whether the differential expressions of MDR proteins contributed to the
Discussion
miRNAs play an important role in diverse biological processes such as cell proliferation, development, differentiation, apoptosis and pathogenesis of many malignant tumors by targeting mRNAs for cleavage or translational repression [18], [19]. To date, many studies have focused on the modulation of drug resistance by miRNAs in cancer cells [20], [21]. Despite identification of a series of miRNAs that play critical regulatory roles in conferring resistance to commonly used therapeutic agents [22]
Conflict of Interest
The authors have declared that no competing interests exist.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81102472 and 81173092), and this study was also supported by the Liaoning S&T Projects (Grant No. 2011415052) and the Shenyang Technology Projects (Grant No. F12-148-9-00)
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These authors contributed equally to this work.