MicroRNA-146a acts as a metastasis suppressor in gastric cancer by targeting WASF2

doi:10.1016/j.canlet.2013.02.031

Cancer Letters

Volume 335, Issue 1, 10 July 2013, Pages 219-224

https://doi.org/10.1016/j.canlet.2013.02.031 Get rights and content

Abstract

Previous studies have shown that miR-146a acts as either an oncogene or a tumor suppressor in various cancers. In this study, we investigated the role of miR-146a in gastric cancer cells and its potential target genes. The results showed that miR-146a expression correlated inversely with WASF2 protein expression in gastric cancer cell lines. Overexpression of miR-146a suppressed the migration and invasion of gastric cancer cells, and also the protein level of WASF2. WASF2 was shown to be a direct target gene of miR-146a by luciferase assays. Restoration of WASF2 promoted the migration and invasion of gastric cancer cells, similar to that mediated by miR-146a inhibition. This study has identified an onco-suppressive role of miR-146a in gastric cancer cells by its reduction of WASF2 expression. The newly identified miR-146a/WASF2 axis partially reveals the molecular mechanism underlying the migration and invasion of gastric cancer cells and represents a new potential therapeutic target for gastric cancer.

Introduction

Metastases account for 90% of human cancer deaths [1], yet the exact molecular mechanisms of metastases remain unclear. Gastric cancer is the second leading cause of cancer-related death worldwide [2]. Most patients with gastric cancer are diagnosed in the advanced stages of disease and present with extensive invasion and lymphatic metastasis, and only a small percentage of patients receive effective intervention [3], [4]. Therefore, it is important to fully explore the molecular mechanisms of gastric cancer progression, which might contribute to the development of novel targeted therapies.

miRNAs are small non-coding RNAs that serve as negative regulators of gene expression [5], [6], [7], [8]. Through base-pairing with the 3′-untranslated region (3′-UTR) of their target mRNAs, miRNAs cause gene silencing either by mRNA degradation or translational suppression [9]. miRNAs are involved in a wide range of important biological processes [8]. Accumulating evidence strongly suggests that miRNAs can function as novel oncogenes or tumor suppressors, and the deregulation of specific miRNAs in diverse types of cancer is associated with tumor growth, angiogenesis, apoptosis and metastasis [9], [10]. miR-146a plays a major role in the pathogenesis of many human diseases, including cancer, autoimmune disorders, virus infection, and muscle disorders [11]. Recent studies have shown the abnormal expression and paradoxical roles of miR-146a in various human cancer tissues. It functions as an oncogene in cervical cancer and anaplastic thyroid carcinoma, but as a tumor suppressor in pancreatic cancer and breast cancer [12], [13], [14], [15], [16], [17], [18]. Tchernitsa and colleagues demonstrated an important role of miR-146a in the lymph node metastasis of gastric cancer by miRNA microarray analysis [19]. Although evidence has shown that miR-146a inhibits the migration and invasion of gastric cancer cells [20], the molecular mechanisms and the related target genes are largely unknown.

WASF2 (WASP family verprolin homologous protein 2) and ROCK1 (Rho-activated protein kinase) are predicted target genes of miR-146a in Targetscan and miRBase, respectively. WASF2 is a downstream effector molecule involved in the transmission of signals from small GTPases to the actin cytoskeleton [21]. It mediates the elongated cell movement mode. ROCK1 is a protein kinase that is a key regulator of the actin cytoskeleton and cell polarity, and it is involved in the rounded cell movement mode [22], [23]. These two modes of motility have been observed in invading tumor cells [24].

In this study, we explored the effects of miR-146a on gastric cancer cells, and whether WASF2 and ROCK1 are its functional target genes, to partially clarify the molecular mechanisms of migration and invasion in gastric cancer cells.

Section snippets

Cell culture

Human gastric cancer cell lines (MKN-45, SGC-7901, HGC-27 and MGC-803), and HEK293T cells were purchased from the Cell Resource Center, Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences. Cells were maintained at 37 °C in a humidified air atmosphere containing 5% carbon dioxide in RPMI1640 (MKN-45, SGC-7901, HGC-27 and MGC-803) or Dulbecco’s Modified Eagle’s Media (HEK293T) supplemented with 10% FBS.

miRNA extraction and TaqMan real-time PCR

MicroRNAs were isolated from the gastric cancer cell lines

miR-146a expression correlates inversely with the level of WASF2 protein in gastric cancer cell lines

The expression levels of miR-146a, WASF2 and ROCK1 in a series of gastric cancer cell lines were examined (Fig. 1). As shown in Fig. 1, WASF2 was highly expressed in cell lines with a low level of endogenous miR-146a (MKN-45 and SGC-7901), while its expression level was much lower in cell lines with a high level of endogenous miR-146a (HGC-27 and MGC-803). Statistical analysis showed a significant inverse correlation between miR-146a and WASF2 protein levels (r = −0.9429, Spearman, P = 0.0167). No

Discussion

miR-146a was initially found to be substantially downregulated in gastric cancer by microarray analysis and semi-quantitative RT-PCR [19]. Further investigations demonstrated that the downregulation of miR-146a in gastric cancer modulated cell proliferation, apoptosis, migration and invasion [20], [29]. Although evidence has shown that miR-146a downregulated EGFR and IRAK1 expression in gastric cancer cells [20], it still remains unclear how miR-146a is involved in the regulation of tumor

Conflicts of interest

All authors declare that there are no conflicts of interest.

Acknowledgments

We thank Qi Hong (Institute of Biomedical Sciences, Fudan University) and Lei Liang (Liver Cancer Institute, Zhongshan Hospital) for their expert technical assistance. This study was sponsored by the National Nature Science Foundation of China (Grant No. 81170398) and the National Key Clinical Specialties Construction Projects of China.

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¹: These authors are contributed equally to this work.

²: Address: 180 Fenglin Road, Xuhui District, Shanghai 200032, China.

³: Address: 100 Haining Road, Shanghai, 200080, China.

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