MiR-2425-5p targets RAD9A and MYOG to regulate the proliferation and differentiation of bovine skeletal muscle-derived satellite cells

Hui Li Tong,Wei Wei Zhang,Run Ying Jiang,Yun Qin Yan

doi:10.1038/s41598-017-00470-8

Abstract

Our group previously identified miR-2425-5p, a unique bovine miRNA; however, its biological function and regulation in muscle-derived satellite cells (MDSCs) remain unclear. Herein, stem-loop RT-PCR results showed that miR-2425-5p increased during MDSCs proliferation, but decreased during differentiation. Cell proliferation was examined using EdU assays, cyclin B1 (CCNB1) and proliferating cell nuclear antigen (PCNA) western blot (WB) and flow cytometry analysis. These results showed that miR-2425-5p mimics (miR-2425-M) enhanced MDSCs proliferation, whereas, miR-2425-5p inhibitor (miR-2425-I) had opposite effect. Conversely, cell differentiation studies by desmin (DES) immunofluorescence, myotubes formation, and myosin heavy chain 3 (MYH3) WB analyses revealed that miR-2425-M and miR-2425-I blocked and promoted MDSCs differentiation, respectively. Moreover, luciferase reporter, RT-PCR, and WB assays showed that miR-2425-5p directly targeted the 3′-UTR of RAD9 homolog A (RAD9A) and myogenin (MYOG) to regulate their expression. Rescue experiment showed RAD9A inhibited the proliferation of MDSCs through miR-2425-5p. In addition, we found that miR-2425-5p expression was regulated by its host gene NCK associated protein 5-like (NCKAP5L) rather than being transcribed independently as a separate small RNA. Collectively, these data indicate that miR-2425-5p is a novel regulator of bovine MDSCs proliferation and differentiation and provides further insight into the biological functions of miRNA in this species.

Highlights

MicroRNAs are a family of endogenous, small-non-coding, functional RNAs
The results showed that when compared to non-proliferating cells (P-0 h), miR-2425-5p expression was significantly increased during muscle-derived satellite cells (MDSCs) proliferation at 24 h (P-24 h) and 48 h (P-48 h) (P < 0.01), while it decreased during the differentiation
The purpose of this study was to explore the role of miR-2425-5p in bovine MDSCs proliferation and differentiation

Summary

Introduction

MicroRNAs (miRNAs) are a family of endogenous, small-non-coding, functional RNAs. MiRNAs have emerged as key post-transcriptional modulators that bind by complementary base pairing to sequences in the 3′-UTR of target mRNAs1, resulting in the repression or degradation of the transcripts. MiRNAs have been implicated in the regulation of myogenic satellite cell proliferation and differentiation. MiR-431 promotes the differentiation and regeneration of old skeletal muscle by targeting Smad[4] in mice[4]. MiRNA-222 regulates Rbm[24] alternative splicing during differentiation of skeletal muscle cells in mice[6]. MiR-101a was a positive regulator of goat skeletal muscle satellite cells differentiation[7]. MiR-2400 promotes bovine skeletal muscle satellite cell proliferation by targeting MYOG8. We found that miR-2425-5p is expressed during the proliferation and differentiation of bovine muscle-derived satellite cells (MDSCs) proliferation and differentiation; the biological functions of miR-2425-3p and miR-2425-5p remain unknown. We found that miR-2425-5p binds the 3′-UTR of RAD9A and MYOG mRNA to downregulate their expression, resulting in enhanced proliferation and attenuated differentiation of bovine MDSCs

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