Elsevier

European Journal of Cancer

Volume 49, Issue 18, December 2013, Pages 3924-3935
European Journal of Cancer

miR-133a represses tumour growth and metastasis in colorectal cancer by targeting LIM and SH3 protein 1 and inhibiting the MAPK pathway

https://doi.org/10.1016/j.ejca.2013.07.149Get rights and content

Abstract

In recent studies of microRNA expression, miR-133a deregulation was identified in colorectal carcinoma (CRC). However, the mechanisms underlying the pathogenesis and progression of CRC are poorly understood. We found that miR-133a expression was usually down-regulated in CRC cell lines and tissue specimens. Ectopic miR-133a expression inhibited cell proliferation and cell migration. Stable overexpression of miR-133a was sufficient to suppress tumour growth and intrahepatic and pulmonary metastasis in vivo. Additional studies showed that miR-133a can target the 3′ untranslated region (3′UTR) of LIM and SH3 protein 1 (LASP1) mRNA and suppress the expression of LASP1, which we identified in previous studies as a CRC-associated protein. In contrast to the phenotypes induced by miR-133a restoration, LASP1-induced cell proliferation and migration rescued miR-133a-mediated biological behaviours, as did LASP1 overexpression. Investigations of possible mechanisms underlying these behaviours revealed that miR-133a modulates the expression of key cellular molecules and participates in the MAPK pathway by inhibiting phosphorylation of ERK and MEK. miR-133a may play a key role in CRC genesis and metastasis, which suggests its potential role in the molecular therapy of cancer.

Introduction

Colorectal cancer (CRC) is the second leading cause of death from cancer worldwide. In China, CRC ranks fifth among cancer deaths and its incidence is continually increasing [1]. Despite advancements made in surgical techniques, radiotherapy, and chemotherapy for patients with CRC over the last several decades, the overall survival rate has not improved markedly. Metastasis plays a critical role in the poor prognosis. Little is known about the exact mechanisms underlying the metastasis of CRC. Once key factors in cancer progression are identified, new diagnostic strategies and drugs targeting these markers of progression can be developed accordingly.

MicroRNAs (miRNAs) comprise a class of diverse, small, non-coding RNAs that function as critical gene regulators. Bioinformatic analyses indicate that each miRNA regulates hundreds of target genes, underscoring their potential influence on almost every biological pathway [2], [3]. Recently, evidence has been provided showing that approximately half of all human miRNAs are located in cancer-associated genomic regions and can function as tumour suppressor genes or oncogenes, depending on their targets [4], [5], [6]. To date, several human miRNAs have been shown to be dysregulated in CRC—including miR-106a, miR-23a, miR-222, miR-17, miR-30a-5p and miR-34a [7], [8], [9], [10], [11], [12]—and, thus, may contribute to the development and progression of CRC. These findings suggest the involvement of miRNAs in CRC tumourigenesis. A recent systematic review of profiling studies and experimental validation revealed that miR-133a is down-regulated in CRC tissue compared with adjacent normal tissue. This finding was determined by miRNA microarray and quantitative real-time polymerase chain reaction (qRT-PCR) analysis and was consistent across four studies [13]. The role of miR-133a during different stages of CRC progression appears to be controversial, however, because in addition to tumour suppression, it may enhance brain metastasis [14]. Until now, no evidence of a functional role for miR-133a in CRC has been documented.

In this study, we investigated the involvement of miR-133a in CRC by examining its expression in human CRC cells and tissue samples; its effects on cell growth, cell-cycle distribution and cell migration; and its role in CRC tumourigenesis and metastasis in a murine model while searching for mechanism(s) underlying its activity in CRC, all with the intended goal of improving our understanding of CRC development and progression.

Section snippets

Cell culture and miRNA transfection

CRC cell lines HT29, HCT116, SW480 and SW620 were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and maintained as previously described [15]. Additionally, a human CRC cell subline with unique liver metastatic potential, designated SW480/M5, was established in our laboratory [16] and used in the analysis.

The cells were cultured in RPMI 1640 (Hyclone; Logan, UT, United States of America (USA)) supplemented with 10% foetal bovine serum (FBS) (Gibco-BRL, Invitrogen;

miR-133a was down-regulated in human CRC cell lines and clinical specimens

Our examination of miR-133a expression in five CRC cell lines and in 12 CRC and paired non-cancerous tissue specimens revealed a decrease in 10 of the CRC specimens and in all of the cell lines compared with paired non-cancerous colorectal tissue (Fig. 1A). The mean rate of expression was significantly lower in CRC tissue samples compared with controls (Fig. 1B; P < .001) and in all five CRC cell lines (Fig. 1C) compared with all of the non-cancerous colorectal tissue samples.

miR-133a inhibited cell proliferation and migration in CRC cells

CCK-8 analysis of

Discussion

The deregulation of miRNAs has been observed in various types of human cancer [18], [19]; however, the molecular mechanisms by which miRNAs modulate carcinogenesis and cancer progression are still unclear. Recently, Ma and colleagues carried out a systematic literature review that revealed that miR-133a levels may be decreased in CRC tissue [13]. The role of miR-133a at different stages of CRC progression seemed controversial, because miR-133a serves not only as a tumour suppressor but also as

Conflict of interest statement

None declared.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 30901792, 81272762, 81201635), Research Foundation of Guangzhou Medical College (2010C33), Key Project of Affiliated Tumor Hospital of Guangzhou Medical College (2011-yz-05), Guangdong Natural Science Funds for Distinguished Young Scholar (S20120011334), Guangdong Natural Science Foundation (S2012040006418).

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    These authors contributed equally to this work.

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