MEG3 Promotes Differentiation of Porcine Satellite Cells by Sponging miR-423-5p to Relieve Inhibiting Effect on SRF

Mengxun Li,Changchun Li,Xiaofang Cheng,Long Li,Gaoli Shi,Chengchi Fang,Lin Chen

doi:10.3390/cells9020449

Mengxun Li, Changchun Li + Show 5 more

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https://doi.org/10.3390/cells9020449

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Journal: Cells	Publication Date: Feb 15, 2020
Citations: 19	License type: CC BY 4.0

Affiliation: Huazhong Agricultural University

Abstract

Although thousands of long noncoding RNAs (lncRNAs) have been identified in porcine growth and development, the regulation mechanisms of functional lncRNAs have not been well explored. In this study, using 5′- and 3′-rapid amplification of cDNA ends (RACE) assays, we obtained two different variants of lncRNA maternally expressed gene 3 (MEG3), namely, MEG3 v1 and MEG3 v2, that were both highly expressed in porcine skeletal muscle and in the early stage of the differentiation of porcine satellite cells. Moreover, we identified the core transcript MEG3 v2. Functional analyses showed that MEG3 overexpression could effectively arrest myoblasts in the G1 phase, inhibit DNA replication, and promote myoblast differentiation, whereas MEG3 knockdown resulted in the opposite effects. Interestingly, the expression of serum response factor (SRF), a crucial transcription factor for myogenesis process, remarkably increased and decreased in mRNA and protein levels with the respective overexpression and knockdown of MEG3. Dual luciferase reporter assay showed that MEG3 could attenuate the decrease of luciferase activity of SRF induced by miR-423-5p in a dose-dependent manner. MEG3 overexpression could relieve the inhibitory effect on SRF and myoblast differentiation induced by miR-423-5p. In addition, results of RNA immunoprecipitation analysis suggested that MEG3 could act as a ceRNA for miR-423-5p. Our findings initially established a novel connection among MEG3, miR-423-5p, and SRF in porcine satellite cell differentiation. This novel role of MEG3 may shed new light on understanding of molecular regulation of lncRNA in porcine myogenesis.

Highlights

In mammalian genomes, only 2% of transcripts are translated into proteins
Large White pigs are associated with meat-producing traits [39]. These findings indicated that maternally expressed gene 3 (MEG3) may be involved in myogenesis and contribute to skeletal muscle development in pigs
The results demonstrated that miR-423-5p overexpression significantly inhibited MEG3, serum response factor (SRF), myogenic differentiation (MyoD), MyoG, and MyHC expressions at the messenger RNA (mRNA) and protein levels (Figure 5K,L)

Summary

Introduction

Only 2% of transcripts are translated into proteins. The vast majority of transcripts are noncoding RNAs, including microRNAs (miRNAs), Piwi-interacting RNAs, circularRNAs, and long noncoding RNAs (lncRNAs) [1]. Mammalian skeletal muscle development is a complex process, which includes the following phases: somite commitment into progenitors, myoblast proliferation, migration, fusion, and final adaptation into fast-twitch and slow-twitch muscle fibers [12]. A small number of lncRNAs, such as lncRNA Dum [16], MUNC [17,18], Linc-MD1 [19], Lnc-mg [20,21], LncMyoD [22], Linc-RAM [23], Linc-YY1 [24], SYISL [25], and lncRNA Irm [26], have been characterized to mediate biological processes of myoblasts during skeletal muscle development These lncRNAs regulate skeletal muscle myogenesis and regeneration through various mechanisms, including chromosome modification, transcription activation, molecular sponge activity, competitive binding, mRNA translation, and protein stability

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