MiRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin β1 and Matrix Metalloproteinase2 (MMP2)

Heyong Wang,Xiaofeng Chen,Gentao Liu,Chunlian Wu,Yingchao Zhu,Yaoqin Yang,Peng Zhang,Huijun Zhang,Mingchuan Zhao,Liang Tang,Edward F Plow

doi:10.1371/journal.pone.0070192

Abstract

Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin β1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin β1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3′-UTRs of integrin β1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin β1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin β1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.

Highlights

Today, lung cancer is one of the most common cancers
We provide evidence that miR-29c expression in high-metastatic 95D cell lines was downregulated when compared to miR-29c expression in paired low-metastatic 95C cell lines, ectopic expression and siRNA knockdown of miR29c confirmed it directly targeted 39-UTR of integrin b1 and matrix metalloproteinase 2 (MMP2) mRNA and downregulated integrin b1 and MMP2 protein expression to suppress lung cancer cell adhesion to extracellular matrix (ECM) and metastasis in vitro and in vivo
Cell adhesion to the ECM is mediated via integrins, the principle cell surface adhesion receptors

Summary

Introduction

Lung cancer is one of the most common cancers. More than 90% of lung cancer patients die of metastasis rather than from their primary tumors, suggesting that metastasis is a key prognostic factor [1,2]. Mature miRNAs are short, single-stranded, endogenous and non-coding RNAs consisting of about 22 nucleotides, which regulate genes at the post-transcriptional level during the translation process. They can target the 39-UTR (untranslated regions) of mRNA, which functionally leads to a translational inhibition or deregulation of the target mRNA [4]. MiRNAs have a major influence on various biological processes, including cell differentiation, proliferation, apoptosis, stress resistance, fat metabolism, and development. They play a crucial role in different diseases including cancer [3]. The generation of therapeutic targets for malignant tumors may be miRNAs [11]

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Conclusion