Metformin-repressed miR-381-YAP-snail axis activity disrupts NSCLC growth and metastasis

Dan Jin,Jiwei Guo,Juanjuan Dai,Jing Du,Lijuan Yang,Xuelin Li,Shuang Miao,Guoming Su,Xiaohong Wang,Yan Wu,Kaikai Gong,Weiwei Chen

doi:10.1186/s13046-019-1503-6

Abstract

BackgroundRecent evidence indicates that metformin inhibits mammalian cancer growth and metastasis through the regulation of microRNAs. Metformin regulates miR-381 stability, which plays a vital role in tumor progression. Moreover, increased YAP expression and activity induce non-small cell lung cancer (NSCLC) tumor growth and metastasis. However, the molecular mechanism underpinning how metformin-induced upregulation of miR-381 directly targets YAP or its interactions with the epithelial-mesenchymal transition (EMT) marker protein Snail in NSCLC is still unknown.MethodsLevels of RNA and protein were analyzed using qPCR, western blotting and immunofluorescence staining. Cellular proliferation was detected using a CCK8 assay. Cell migration and invasion were analyzed using wound healing and transwell assays. Promoter activity and transcription were investigated using the luciferase reporter assay. Chromatin immunoprecipitation was used to detect the binding of YAP to the promoter of Snail. The interaction between miR-381 and the 3′UTR of YAP mRNA was analyzed using the MS2 expression system and co-immunoprecipitation with biotin.ResultsWe observed that miR-381 expression is negatively correlated with YAP expression and plays an opposite role to YAP in the regulation of cellular proliferation, invasion, migration, and EMT of NSCLC cells. The miR-381 function as a tumor suppressor was significantly downregulated in lung cancer tissue specimens and cell lines, which decreased the expression of its direct target YAP. In addition, metformin decreased cell growth, migration, invasion, and EMT via up-regulation of miR-381. Moreover, YAP, which functions as a co-transcription factor, enhanced NSCLC progression and metastasis by upregulation of Snail. Snail knockdown downregulated the mesenchymal marker vimentin and upregulated the epithelial marker E-cadherin in lung cancer cells. Furthermore, miR-381, YAP, and Snail constitute the miR-381-YAP-Snail signal axis, which is repressed by metformin, and enhances cancer cell invasiveness by directly regulating EMT.ConclusionsMetformin-induced repression of miR-381-YAP-Snail axis activity disrupts NSCLC growth and metastasis. Thus, we believe that the miR-381-YAP-Snail signal axis may be a suitable diagnostic marker and a potential therapeutic target for lung cancer.

Highlights

Recent evidence indicates that metformin inhibits mammalian cancer growth and metastasis through the regulation of microRNAs
The miR-381 level was lower in non-small cell lung cancer (NSCLC) cells (A549, H1299, Calu6, H520, and 95-D) compared with normal human bronchial epithelial cells (HBEC) (Fig. 1a)
Fluorescence in situ hybridization (FISH) results indicated that miR-381 was predominantly located in the cytoplasm, where the miR-381′s function would occur (Fig. 1b)

Summary

Introduction

Recent evidence indicates that metformin inhibits mammalian cancer growth and metastasis through the regulation of microRNAs. Metformin regulates miR-381 stability, which plays a vital role in tumor progression. Increased YAP expression and activity induce non-small cell lung cancer (NSCLC) tumor growth and metastasis. There are two mechanisms associated with the anticancer effects of metformin: metformin lowers circulating insulin, which can bind to the highly expressed insulin receptor in cancer cells, thereby indirectly decreasing cell proliferation [1]; and metformin directly activates AMPK and subsequently inhibits mammalian target of mTOR, leading to reduced cancer-cell proliferation [2]. Growing evidence indicates that metformin inhibits mammalian cancer growth and metastasis through regulation of microRNAs (miRNAs). Metformin disrupts the metastasis associated lung adenocarcinoma transcript 1 (MALAT1)/miR-142-3p sponge, decreasing the invasion and migration of cervical cancer cells [10]. Whether other regulatory mechanisms underpin the effects of metformin in NSCLC, such as metformin-decreased YAP activity by miRNAs regulation, is currently unclear

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