Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer

Abstract

MicroRNAs are a class of small non-coding RNAs regulating gene expression. In this study, we demonstrated that retinoic acid (RA) treatment increases the expression of miR-512-3p. Overexpression of miR-512-3p inhibited cell adhesion, migration, and invasion in non-small cell lung cancer (NSCLC) cell lines A549 and H1299. miR-512-3p inhibitor partially reversed these effects in H1299 cells stably expressing miR-512. We identified DOCK3, a RAC1-GEF (guanine nucleotide exchange factor), as a target gene of miR-512-3p. Overexpression of miR-512-3p led to the decrease of DOCK3 protein but not its mRNA. Knockdown of DOCK3 resulted in similar effects on adhesion, migration, and invasion as observed of miR-512-3p overexpression. Active RAC1 pull-down assay indicated that overexpression of miR-512-3p could decrease the activity of RAC1 with a higher efficiency than that of DOCK3 knockdown. Furthermore, expression of miR-512-3p was suppressed in most NSCLC patient tumor samples compared to its paired normal controls, suggesting that miR-512-3p might play a crucial role in lung cancer development. In conclusion, our results supported that miR-512-3p could inhibit tumor cell adhesion, migration, and invasion by regulating the RAC1 activity via DOCK3 in NSCLC A549 and H1299 cell lines.

Introduction

Lung cancer is the leading cause of cancer-related death in both men and women, and non-small cell lung cancer (NSCLC) accounts for about 80% of these cases (Komaki et al., 2013). It has been demonstrated that deregulation of miRNAs is involved in the pathogenesis of human lung cancer. miRNAs are a class of small non-coding 18- to 25-nucleotide-long RNAs that negatively regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of their target mRNAs, which causes translational repression or mRNA degradation (Valencia-Sanchez et al., 2006). miRNAs are aberrantly expressed or mutated in cancer and function as either oncogenes or tumor suppressors (Zhang et al., 2010). Let-7a was the first miRNA described to be downregulated in lung tumors. Downregulation of let-7a resulted in the high expression of RAS, indicating that let-7a suppresses the expression of RAS (Johnson et al., 2005). Moreover, downregulation of Dicer, a component of the miRNA machinery, was reported in lung cancer (Esquela-Kerscher and Slack, 2006, Karube et al., 2005).

Retinoic acid (RA) is well-known for its extensive use in the treatment of acute promyelocytic leukemia. It is a potent regulator of cell growth, differentiation, and matrix formation of various cell types and during embryogenesis. In recent years, RA was also reported to involve in cell migration inhibition (Wu et al., 2006, Axel et al., 2001, Hendrix et al., 1990, Pratt et al., 1987, Joshi et al., 2005). miRNA expression profile is often used to identify novel miRNA of interest. Garzon et al. performed miRNA microarray analysis on RA-treated NB4 cells and identified deregulated miRNAs, and the RA downregulation of Bcl2 and Ras was correlated with the activation of miR-15a/miR-16-1 (Garzon et al., 2007). However, the expression profile of microRNAs upon treatment of RA in NSCLC has not been explored.miR-512-3p is located at chromosome 19q13.42. It is a member of miR-512 cluster, which contains two copies of miR-512 (miR-512-1 and miR-512-2) and 46 duplicates of miR-519 (Lichner et al., 2011). miR-512-3p can be derived from both copies of miR-512. With a frequency of more than 5% to be edited, miR-512-1 and miR-512-2 were reported to affect expression (silencing) of many genes on a global scale (Kawahara et al., 2008). Deletion of miR-512-2 was found in medulloblastomas (Lv et al., 2012). miR-512-3p was identified as a pro-apoptotic tumor suppressor in human gastric cancer cells (Saito et al., 2009), and was reported to contribute to taxol-induced apoptosis by targeting c-FLIP(Chen et al., 2010). miR-512-3p was also found to be upregulated in three germ cell tumor cell lines (Port et al., 2011), and downregulated in NSCLC A549 cell line after resveratrol treatment (Bae et al., 2011). So far, the role of miR-512-3p in NSCLC remains poorly understood and therefore needs further investigation.

DOCK3 (dedicator of cytokinesis 3) is also called as PBP (presenilin-binding protein) and MOCA (modifier of cell adhesion). As a member of the DOCK180 family, DOCK3 is known to be involved in regulation of cytoskeletal organization and cell–cell interactions, and function as a guanine nucleotide exchange factor (GEF) (Kashiwa et al., 2000, Chen et al., 2009). DOCK3 has been shown to regulate the activity of RAC1 and N-cadherin-dependent adhesion (Lu and Ravichandran, 2005). Investigations in melanoma tumor cells indicate that DOCK3 can form a complex with the adaptor protein NEDD9, and promote mesenchymal migration by activating RAC1 (Paluch and Raz, 2013). By stimulating membrane trafficking of the WAVE complex, DOCK3 can induce axonal outgrowth (Namekata et al., 2010). DOCK3 is also reported to exist in a complex with β-catenin to repress the canonical Wnt pathway and increase cell adhesion (Caspi and Rosin-Arbesfeld, 2008).

In the present study, we aimed to uncover the miRNA that might be implicated in the development and progression of NSCLC. We sought to investigate the miRNA expression profile with a miRNA microarray platform upon RA treatment, and a small number of miRNAs including miR-512-3p were found. Upregulation of miR-512-3p after RA treatment was confirmed by quantitative RT-PCR. We demonstrated that miR-512-3p inhibited adhesion, migration, and invasion in NSCLC cell lines A549 and H1299. We also revealed that miR-512-3p targets RAC1-GEF DOCK3 and therefore inhibits the activity of RAC1, which may contribute to suppression of tumor metastasis. Finally, we showed evidence that miR-512-3p was downregulated in human NSCLC tumor samples compared to the paired normal lung tissues.