Elsevier

Journal of Hepatology

Volume 60, Issue 3, March 2014, Pages 590-598
Journal of Hepatology

Research Article
MicroRNA-101 inhibits human hepatocellular carcinoma progression through EZH2 downregulation and increased cytostatic drug sensitivity

https://doi.org/10.1016/j.jhep.2013.10.028Get rights and content

Background & Aims

Oncogene polycomb group protein enhancer of zeste homolog 2 (EZH2) has been proposed to be a target gene of putative tumor suppressor microRNA-101 (miR-101). The aim of our study was to investigate the functional role of both miR-101 and EZH2 in human hepatocellular carcinoma (HCC).

Methods

MiR-101 and EZH2 expressions were evaluated in tumor tissues of 99 HCC patients and 7 liver cancer cell lines by real-time PCR. Luciferase reporter assay was employed to validate whether EZH2 represents a target gene of miR-101. The effect of miR-101 on HCC growth as well as programmed cell death was studied in vitro and in vivo.

Results

MiR-101 expression was significantly downregulated in most of HCC tissues and all cell lines, whereas EZH2 was significantly overexpressed in most of HCC tissues and all cell lines. There was a negative correlation between expression levels of miR-101 and EZH2. Luciferase assay results confirmed EZH2 as a direct target gene of miR-101, which negatively regulates EZH2 expression in HCC. Ectopic overexpression of miR-101 dramatically repressed proliferation, invasion, colony formation as well as cell cycle progression in vitro and suppressed tumorigenicity in vivo. Furthermore, miR-101 inhibited autophagy and synergized with either doxorubicin or fluorouracil to induce apoptosis in tumor cells.

Conclusion

Tumor suppressor miR-101 represses HCC progression through directly targeting EZH2 oncogene and sensitizes liver cancer cells to chemotherapeutic treatment. Our findings provide significant insights into molecular mechanisms of hepatocarcinogenesis and may have clinical relevance for the development of novel targeted therapies for HCC.

Introduction

Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related death in the world [1]. Its incidence continues to increase worldwide and the long-term prognosis remains dismal [2]. An improved understanding of molecular pathogenesis of HCC has facilitated the development of new targeted therapies, which have shown a significant clinical impact [3].

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs of 20 to 25 nucleotides in length that post-transcriptionally regulate the expression of complementary messenger RNAs (mRNAs) in eukaryotes, influencing various biological processes like cell development, infection, immunity, and carcinogenesis [4], [5]. More than half of human miRNAs are associated with carcinogenesis, and this may provide the basis for the development of targeted therapies against cancer [6]. The putative tumor suppressor miR-101 counteracts tumor development and progression by downregulation of several oncogenes [7], [8]. For instance, miR-101 has been shown to inhibit tumorigenicity via targeting myeloid cell leukemia sequence 1 (Mcl-1) in HCC [8].

Polycomb group protein enhancer of zeste homolog 2 (EZH2) plays an important role in regulation of cell proliferation, determination of stem cell fate, and carcinogenesis [9], [10]. Overexpression of EZH2 has been reported for a variety of malignancies including HCC [11], [12], [13], [14]. Sudo et al. [15] were the first to report that EZH2 is upregulated in human HCC and overexpression of EZH2 is associated with a more aggressive biological behavior [16] and worse prognosis [17]. Moreover, detection of EZH2 protein by immunohistochemistry in liver needle biospies may represent a diagnostic tool for discrimination between dysplastic nodules and early HCC [18]. EZH2 has previously been proposed to be a target gene of miR-101 in prostate and bladder cancer [19], [20]. The precise regulatory mechanisms of EZH2 expression and its relationship with miR-101 in the process of hepatocarcinogenesis are still not fully elucidated. Therefore, we sought to investigate the functional role of both miR-101 and EZH2 in the pathogenesis of HCC.

Section snippets

Tissue specimens

Paired HCC tissues and adjacent non-tumor liver tissues were collected from patients who underwent surgery between November 2000 and January 2011 at the University Hospital Essen (Essen, Germany) and had not received local or systemic treatment. Additionally, normal liver tissues were collected from patients receiving liver resection for benign tumors (hepatic hemangioma, n = 4; focal nodular hyperplasia, n = 2). Tumor tissues and adjacent non-tumor liver tissues were confirmed histologically. All

MiR-101 expression is downregulated in HCC and associated with EZH2 overexpression

Significant downregulation of miR-101 was observed in 66.7% (62/93, cohort 1) and 83.3% (5/6, cohort 2) of tumor tissues (Fig. 1A) compared to respective adjacent non-tumor liver tissues and in all seven liver cancer cell lines (HepG2, Hep3B, HuH7, PLC/PRF5, SNU182, HepaRG, and BEL-7402) compared to human hepatocytes (Fig. 1D). On the contrary, EZH2 was significantly upregulated in 61.3% (57/93, cohort 1) and 66.7% (4/6, cohort 2) of tumor tissues (Fig. 1B) and in all seven liver cancer cell

Discussion

Emerging evidence demonstrates that miRNAs play important roles in both physiological and pathological processes, including tumor development and progression [4], [6], [8]. While it has been reported previously that genomic loss of miR-101 leads to overexpression of EZH2 oncogene [19], [20], their interrelationship and biological relevance have not yet been fully elucidated in HCC. In our study, the expression of miR-101 was downregulated and inversely correlated with EZH2 expression in the

Financial support

This work was supported in part by Grant 107-05710/IFORES Research Program (V.R.C.), by Grant 107-05470/IFORES Research Program (S.B.) and by the National Natural Science Foundation of China (NSFC), Grant no. 81172068 (C.L.).

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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  • Cited by (0)

    This work was presented in part at the 47th annual meeting of the European Association for the Study of the Liver (EASL), Barcelona, Spain, April 18–22, 2012.

    These authors contributed equally to this work.

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