Overexpression of miR-584-5p inhibits proliferation and induces apoptosis by targeting WW domain-containing E3 ubiquitin protein ligase 1 in gastric cancer

Qing Li,Liang Chen,Lu Wang,Zhipeng Xu,Hao Xu,Guangli Sun,Zheng Chen,Jianghao Xu,Bowen Li,Lei Zhang,Yiwen Xia,Song Wei,Weizhi Wang,Zheng Li,Qiang Li,Zekuan Xu,Diancai Zhang

doi:10.1186/s13046-017-0532-2

Abstract

BackgroundMicroRNAs are endogenously expressed, small non-coding RNAs that modulate gene expression by targeting specific mRNAs, resulting in translational repression or mRNA degradation. Although miR-584-5p has been reported to play a vital role in various malignancies, its role and the molecular mechanisms underlying the effects of miR-584-5p in gastric cancer (GC) remain to be clarified. In this study, we investigated the role of miR-584-5p in GC.MethodsThe expression of miR-584-5p and its specific target gene were determined in human GC specimens and cell lines by microRNA real-time polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR) and Western blot. The effects of miR-584-5p depletion or ectopic expression on GC proliferation were evaluated in vitro using CCK-8 proliferation assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, colony formation assays and cell-cycle assays and the in vivo effects were investigated using a mouse tumorigenicity model. Cell apoptosis was evaluated by in vitro flow cytometric analysis, cell viability assays and in vivo TUNEL assays. Luciferase reporter assays were employed to identify interactions between miR-584-5p and its specific target gene.ResultsA series of in vitro and in vivo gain- and loss-of-function assays revealed that miR-584-5p inhibited GC cell proliferation, while apoptosis was induced. Luciferase reporter assays and Western blot analysis revealed WWP1 to be a direct target of miR-584-5p. The effects of miR-584-5p-mimic were rescued by WWP1 overexpression. In contrast, the effects of the miR-584-5p-inhibitor were impaired by WWP1-shRNA. Furthermore, miR-584-5p expression levels correlated negatively with WWP1 protein expression in GC tissues and GC cell lines. A series of investigations indicated that miR-584-5p promoted senescence and activated the TGFβ signaling pathway by downregulation of WWP1.ConclusionTaken together, these results suggest that downregulation of miR-584-5p contributes to tumor progression by downregulation of WWP1, thus, highlighting the potential of miR-584-5p as a therapeutic target for human GC.

Highlights

MicroRNAs are endogenously expressed, small non-coding RNAs that modulate gene expression by targeting specific messenger RNAs (mRNAs), resulting in translational repression or mRNA degradation
MiR-584-5p is down-regulated in human gastric cancer tissues and cells To explore whether miR-584-5p is dysregulated in GC tissues, we collected 75 human GC tissue samples and paired adjacent normal tissues to detect the expression of miR-584-5p by miRNA RT-polymerase chain reaction (PCR)
MiR-584-5p activates the TGFβ signaling pathway by downregulation of WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) Having shown that miR-584-5p promoted proliferation and induced apoptosis of GC cells, we investigated the possibility that these effects are mediated partially by activation of the TGFβ signaling pathway following WWP1 downregulation

Summary

Introduction

MicroRNAs are endogenously expressed, small non-coding RNAs that modulate gene expression by targeting specific mRNAs, resulting in translational repression or mRNA degradation. MiR-584-5p has been reported to play a vital role in various malignancies, its role and the molecular mechanisms underlying the effects of miR-584-5p in gastric cancer (GC) remain to be clarified. MicroRNAs (miRNAs) are endogenously expressed, small, non-coding RNAs consisting of 20–24 nucleotides [4]. MiRNAs downregulate gene expression by binding to the 3′-untranslated regions (3′-UTR) of specific target messenger RNAs (mRNAs), resulting in inhibition of translation or mRNA degradation [5]. The biological functions and molecular mechanisms underlying the role of miR-584-5p in GC remain to be elucidated. We discovered that miR-584-5p was downregulated in GC and that miR-584-5p overexpression suppressed the progression of GC both in vitro and in vivo

Methods

Results

Discussion

Conclusion