Cancer Letters

Cancer Letters

Volume 408, 1 November 2017, Pages 102-111
Cancer Letters

Original Article
The miR-491-3p/Sp3/ABCB1 axis attenuates multidrug resistance of hepatocellular carcinoma

https://doi.org/10.1016/j.canlet.2017.08.027Get rights and content

Highlights

  • MiR-491-3p inhibits both post-transcriptional level and transcriptional level of ABCB1.

  • The miR-491-3p/ABCB1/Sp3 regulatory loop increases the sensitivity of HCC cells to chemotherapeutics.

  • New therapeutic strategies targeted on the miR-491-3p/ABCB1/Sp3 axis may enhance the efficacy of chemotherapy against HCC.

Abstract

As one of main obstacles in the treatment and prognosis of hepatocellular carcinoma (HCC), multidrug resistance (MDR) is usually associated with the overexpression of the drug efflux pump P-glycoprotein (P-gp/ABCB1) which is responsible for reducing the intracellular concentration of chemotherapeutic agents. In current work, we discovered the novel role of miR-491-3p in ABCB1-mediated multidrug resistance in HCC and revealed the underlying mechanism in which miR-491-3p downregulated the expression of ABCB1 and its transcription factor Sp3 by directly targeting their 3′-UTR. Moreover, overexpressing ABCB1 or Sp3 reversed the sensitivity to chemotherapeutics in Hep3B cells induced by miR-491-3p, confirming miR-491-3p/Sp3/ABCB1 regulatory loop plays an important role in enhancing the drugs sensitivity of HCC. Meanwhile, the discovery of that the expression level of miR-491-3p was inversely correlated with that of ABCB1 and Sp3 in HCC cell lines and clinical samples pointed out the possibility of miR-491-3p in clinical use. In summary, our results reveal a pivotal role of miR-491-3p in the regulation of MDR in HCC, and suggest the potential application of miR-491-3p as a therapeutic strategy for modulating MDR in cancer cells.

Introduction

Hepatocellular carcinoma (HCC) is the fifth solid tumor worldwide and causes about half a million deaths every year [1], [2]. Although anti-cancer drugs against HCC have been used in clinic for decades, the efficient treatments of HCC still lack mostly due to the resistance of cancer cells to chemotherapy, also called multidrug resistance (MDR). MDR is mediated by high expression of adenosine triphosphate (ATP)-binding cassette (ABC) transporter family members that increase the efflux of chemotherapeutic agents out of cancer cells [3], [4], [5]. P-glycoprotein (P-gp), a 170-kDa transmembrane glycoprotein encoded by the ABCB1 gene on human chromosome 7p21, is one of the best studied ABC transporters in drug resistance [6], [7]. In addition, P-gp is considered to contribute to drug resistance in various types of cancer, and changes in its expression or function could contribute to MDR [6], [7], [8], [9], [10]. Thus, reducing P-gp expression might assist in overcoming MDR in cancer chemotherapy.

MicroRNAs (miRNAs) are a class of endogenous, single-stranded, small non-coding RNAs that are 18–25 nucleotides in length and function as negative regulators of gene expression [11]. MiRNAs can trigger either mRNA degradation or translational repression by binding to the 3′ untranslated region (3′-UTR) of specific mRNAs with perfect or near-perfect complementary, respectively [12], [13]. Emerging evidence shows that miRNAs play a relevant role in the initiation, development and progression of tumors [14], [15], [16]. In recent years, miRNAs have been reported to be involved in the modulation of MDR in various cancers [17], [18]. For instance, Wu et al. demonstrated that let-7g and let-7i enhanced cellular sensitivity to drugs by directly targeting ABCC10 [19]. Moreover, miR-181a was found to restore sensitivity to mitoxantone-resistant cells by targeting breast cancer resistance protein (BCRP/ABCG2) [20]. It was also reported that hyper-methylation of miR-129-5p island played important roles in the development of gastric cancer chemo-resistance by targeting ABC transporters [21]. Therefore, miRNA targeting drug transporters has been proven to be effective in cancer therapy.

In this study, we obtained a list of candidate miRNAs that potentially targeted the 3′-UTR of ABCB1 via using bioinformatics algorithms TargetScan and Microcosm, and then miR-491-3p was discovered to remarkably reduce P-gp expression in hepatoma cell lines. Moreover, miR-491-3p was significantly downregulated in HCC clinical samples. To better understand the regulatory network between miRNA and ABCB1, we also focused on the transcription factors of ABCB1, and found Sp3 might be another target gene of miR-491-3p. Thereby, our results suggested that miR-491-3p downregulated ABCB1 via a dual inhibitory pathway: (i) directly targeting the ABCB1 3′-UTR (post-transcriptional inhibition), and (ii) indirectly targeting the promoter of ABCB1 via its transcription factor, Sp3 (transcriptional inhibition), resulting in increased sensitivity of hepatoma cells to chemotherapeutic drugs.

Section snippets

Cell culture and tissue samples

Three human hepatocellular carcinoma cell lines Hep3B, BEL-7402 and SMMC-7721 were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Two immortalized human liver epithelial cells lines THLE-2 and THLE-3 were obtained from American Type Culture Collection (ATCC, USA). Hep3B cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, USA) and antibiotics (penicillin and streptomycin, 50

Identification of candidate miRNAs inhibiting P-gp expression in Hep3B cells

Given the crucial role of P-gp in MDR and the possibility of miRNA targeting drug transporters in cancer therapy, we predicted candidate miRNAs potentially binding to the 3′-UTR of human ABCB1 (P-gp) using the bioinformatics algorithms Targetscan and MicroCosm Targets. MiRNAs at the top of prediction list were selected to identify which one can efficiently affect P-gp expression in hepatocarcinoma. In the screening assay, miRNAs mimics (50 nM) were separately transfected into Hep3B cells, and

Discussion

MDR is a main hurdle for the successful treatment of HCC patients with chemotherapeutic agents. Efflux of anticancer drugs out of cells by ATP-binding cassette (ABC) transporters is the most commonly encountered mechanism of MDR [29], [30]. P-gp, encoded by ABCB1 gene, is the first ABC transporter to be characterized [7] and plays an important role in HCC chemoresistance [31]. Inhibiting P-gp expression and function can effectively reverse drug resistance in cancer cells [32], [33]. To date,

Acknowledgements

This work was supported by National Science and Technology Major Project of China (2015ZX09102005). We would like to thank Yizheng Wang for helpful comments. We are grateful to Ying Hou and Feng Shen the Eastern Hospital of Hepatobiliary Surgery for providing the hepatocarcinoma samples.

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