Cancer Letters

Cancer Letters

Volume 275, Issue 1, 8 March 2009, Pages 44-53
Cancer Letters

miR-34a inhibits migration and invasion by down-regulation of c-Met expression in human hepatocellular carcinoma cells

https://doi.org/10.1016/j.canlet.2008.09.035Get rights and content

Abstract

Several studies have shown that miR-34a represses the expression of many genes and induces G1 arrest, apoptosis, and senescence. In the present study, we identified the role of miR-34a in the regulation of tumor cell scattering, migration, and invasion. Down-regulation of miR-34a expression was highly significant in 19 of 25 (76%) human hepatocellular carcinoma (HCC) tissues compared with adjacent normal tissues and associated with the metastasis and invasion of tumors. Furthermore, resected normal/tumor tissues of 25 HCC patients demonstrated an inverse correlation between miR-34a and c-Met-protein. In HepG2 cells, ectopic expression of miR-34a potently inhibited tumor cell migration and invasion in a c-Met-dependent manner. miR-34a directly targeted c-Met and reduced both mRNA and protein levels of c-Met; thus, decreased c-Met-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Taken together, these results provide evidence to show the suppression role of miR-34a in tumor migration and invasion through modulation of the c-Met signaling pathway.

Introduction

Hepatocellular carcinoma (HCC) is a highly malignant tumor with very poor prognosis and distant invasion and migration [1], [2]. Although invasion and migration is the overwhelming cause of mortality in patients with solid tumors, our understanding of its molecular determinant is limited [3], [4], [5]. Studies on tumor invasion and metastasis have revealed a critical role of miRNAs in these processes, since miRNAs can post-transcriptionally regulate a variety of genes pivotal for invasion or metastasis [6], [7], [8].

miRNAs are a class of 17–25 nucleotides small non-coding RNAs. In mammals, mature miRNAs are integrated into an RNA-inducing silencing complex (RISC) and associate with 3′-untranslated regions (3′-UTR) of specific target messenger RNAs (mRNAs) to suppress translation and occasionally also induce their degradation [9], [10]. miRNAs play important regulatory roles in processes such as cell differentiation, and proliferation [11], [12]. They can function as tumor suppressors or oncogenes, depending on whether they specifically target oncogenes or tumor suppressor genes [13], [14], [15]. In this regard, oncogenic miRNAs are usually overexpressed in tumors. For instance, miR-21, miR-10b, miR-373 and miR-520c are overexpressed in tumors or tumor cell lines inducing cell migration and invasion [16], [17], [18], [19], [20]. By contrast, suppressive miRNAs, such as miR-126∗, miR-335, miR-146a, miR-29c are down-regulated leading to tumor growth, carcinogenesis, and invasion [21], [22], [23], [24].

Recently, miR-34a has been demonstrated to be a direct transcriptional target of p53 and it is commonly deleted in various types of cancers. Decreased expression of miR-34a is partly due to the inactivating mutations of p53 in tumors [25], [26]. For example, loss of miR-34a expression was observed in neuroblastoma, which may be due to the relatively common deletion of a region on chromosome 1p36, which encompasses miR-34a [27]. Moreover, the expression of miR-34a was low or undetectable in 11 of 15 pancreatic cancer cell line [28]. Nine of 25 human colon cancers (36%) also showed down-regulation of miR-34a compared with counterpart normal tissues [29]. The biological targets of miR-34a have been recently identified. miR-34a induces G1 arrest, apoptosis, and senescence by regulation of critical cell cycle motors or apoptosis inhibitors including CDK4/6, cyclin E2 (CCNE2), cyclin D1 (CCND1), E2F3, Bcl-2, and MYCN [30], [31], [32]. Interestingly, miR-34a also directly represses the expression of the oncogenic tyrosine kinase receptor c-Met [8], [26]. c-Met is well-characterized as the receptor for hepatocyte growth factor (HGF) [33]. HGF-stimulated activation of c-Met triggers phosphorylation of its signal transduction intermediate molecules such as ERK1/2, the key factors influencing the tumor invasion and migration [34], [35]. Indeed, c-Met is implicated in cell scattering, migration and invasion. These evidence suggest that miR-34a might also possess a role in invasion and metastasis. In the present study we provided the evidence that miR-34a regulated cell scattering, migration and invasion in HCC tissues and HepG2 cell line, at least in part, by targeting the metastasis-related gene c-Met. Furthermore, expression levels of miR-34a and c-Met in human HCC tissues were also determined and their possible role in human HCC development was discussed.

Section snippets

Patients and tissue samples

HCC tissues and adjacent non-tumorous liver tissue counterparts used for qRT-PCR and Western blot were collected from 25 HCC patients who underwent hepatectomy between 2007 and 2008 at PLA General Hospital (Beijing, China) after informed consent and verification by a pathologist. The hard and firm tumor tissues were trimmed free of normal tissue and snap frozen in liquid nitrogen immediately after resection according to the specimen regulation of PLA General Hospital. No patient in the current

Decrease of miR-34a expression and the association of miR-34a with metastasis/invasion in human HCC tissues

To gain insight into the biological role of miR-34a in human HCC development, we analyzed the expression of miR-34a in human HCC tissues and adjacent normal hepatic tissues by qRT-PCR. Compared with adjacent normal hepatic tissues, nineteen of 25 human HCC tissues (76%) showed that miR-34a was significantly underexpressed 22-fold to 1.5-fold (Fig. 1a), only six were increased in different degree (Fig. 1b). These results suggest that down-regulation of miR-34a may be, at least in part, involved

Discussions

Comparatively low levels of miR-34a expression were reported in some human tumors and cancer cell lines [27], [38]. Quite recently, loss of miR-34a expression was observed in neuroblastoma with heterozygous deletion of chromosome 1p36, which encompasses miR-34a [27]. Moreover, the expression of miR-34a was low or undetectable in 11 of 15 pancreatic cancer cell lines [28] and 9 of 25 human colon cancer tissues [29]. Consistent with this observation, we demonstrated that the expression of miR-34a

Conflicts of interest statement

None declared.

Acknowledgements

We are grateful to Professor Lingqiang Zhang for his proofreading of the manuscript. We thank Dr. Yulan Wang for providing HCC tissues samples and technical assistance. This project was partially supported by grants from Chinese State Key Projects for Basic Research (2002CB513103, 2006CB910407, 2007CB914601), Chinese National High-tech Program (2006AA02Z127), Chinese National Natural Science Foundation (30500299) and Beijing Natural Science Foundation (5072039).

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