MicroRNA-205-5p inhibits three-dimensional spheroid proliferation of ErbB2-overexpressing breast epithelial cells through direct targeting of CLCN3.

Takayoshi Takeno,Koji Sato,Takuya Hasegawa,Aya Nakajima,Toshiyuki Sakamaki,Junji Okubo,Ryo Hamataka,Yasuyuki Ueno,Hiroki Hasegawa

doi:10.7717/peerj.7799

Abstract

We previously reported that microRNA-205-5p (miR-205-5p) is significantly decreased in the ErbB2-overexpressing breast epithelial cell line MCF10A-ErbB2 compared with control cells. In this study, we identified a direct target of miR-205-5p, chloride voltage-gated channel 3 (CLCN3). CLCN3 expression was induced by ErbB2 overexpression; this induced expression was then reduced to control levels by the transfection of the miR-205-5p precursor. In RNA-binding protein immunoprecipitation with Ago1/2/3 antibody, CLCN3 was significantly enriched in 293T embryonic kidney cells with miR-205-5p mimic transfection compared with negative control mimic transfection. In luciferase reporter assays using CLCN3 3′-UTR constructs, the miR-205-5p mimic significantly decreased reporter activity of both wild-type and partial mutant constructs in MCF10A-ErbB2 cells. In contrast, no inhibitory effects of the miR-205-5p mimic were detected using the complete mutant constructs. Since miR-205-5p expression in exosomes derived from MCF10A-neo cells was substantially higher than in exosomes derived from MCF10A-ErbB2 cells, we next investigated whether an exosome-mediated miR-205-5p transfer could control CLCN3 expression. To this end, exosomal miR-205-5p derived from MCF10A-neo cells was functionally transferred to MCF10A-ErbB2 cells, which served to decrease the expression of CLCN3. To assess the roles of CLCN3 in breast cancer, we next performed three-dimensional (3D) spheroid proliferation analyses using MCF10A-ErbB2 cells treated with MCF10A-neo-derived exosomes or CLCN3 shRNA stably expressing SKBR3 and MDA-MB-453 breast cancer cells. Our results showed that both treatment with MCF10A-neo-derived exosome and CLCN3 shRNA expression suppressed 3D spheroid proliferation. Collectively, these novel findings suggest that CLCN3 may be a novel direct target of miR-205-5p and this CLCN3/miR-205-5p interaction may serve a pivotal role in regulating breast cancer cellular proliferation under physiological conditions.

Highlights

MicroRNAs are a class of small noncoding RNAs that regulate gene expression post-transcriptionally through binding to the 3 -untranslated regions (3 -UTRs) of target mRNAs
Our findings in this study demonstrated that chloride voltagegated channel 3 (CLCN3) is a potential direct target of miR-205-5p and regulates 3D spheroid proliferation in ErbB2-overexpressing breast epithelial cells and breast cancer cells
To determine whether miR-205-5p expression correlates with CLCN3 expression in breast epithelial cells, we further examined CLCN3 expression in MCF10A cells, MCF10A-neo cells, MCF10A-ErbB2 cells, negative control precursor-transfected, and miR-205-5p precursor-transfected MCF10A-ErbB2 cells by western blotting

Summary

Introduction

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression post-transcriptionally through binding to the 3 -untranslated regions (3 -UTRs) of target mRNAs. A large number of miRNAs have been identified that are differentially expressed during breast cancer progression, and several have been reported to serve as diagnostic and curative targets (Bertoli, Cava & Castiglioni, 2015; Khordadmehr et al, 2019). The suspected tumor-suppressive functions of miR-205-5p in breast cancer appear to be due to the direct targeting of several oncogenes, including ERBB3, VEGFA, PKC ε, E2F1, E2F5, ZEB1 and ZEB2 (Greene, Herschkowitz & Rosen, 2010; Gregory et al, 2008; Hashiguchi et al, 2018). HMGB3, KLF12, FGF2 and ITGA5 have recently been reported to be direct targets of miR-205-5p in breast cancer (Elgamal et al, 2013; Guan et al, 2016; Hu et al, 2016; Xiao et al, 2018)

Methods

Results

Conclusion