Circular RNA circMYL1 Inhibit Proliferation and Promote Differentiation of Myoblasts by Sponging miR-2400.

Ibrahim Elsaeid Elnour,Zhanerke Akhatayeva,Chuzhao Lei,Toremurat Zhansaya,Rajwali Khan,Yongzhen Hung,Hong Chen,Jie Cheng,Xianyong Lan,Xiaogang Wang

doi:10.3390/cells10010176

Ibrahim Elsaeid Elnour, Zhanerke Akhatayeva + Show 8 more

Open Access

https://doi.org/10.3390/cells10010176

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Abstract

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs (ncRNAs) involved in regulating skeletal muscle development by sponging miRNAs. In this study, we found that the circMYL1 expression was down-regulated during myoblast proliferation, while gradually up-regulated in myoblast differentiation. The potential role of circMYL1 was identified in the proliferation of bovine myoblast through mRNA and protein expression of proliferation marker genes (PCNA, CyclinD1, and CDK2), cell counting kit-8 assay, flow cytometry analysis, and 5-ethynyl 2′-deoxyuridine (EdU) assay. Analysis of the expression of differentiation marker genes (MyoD, MyoG, and MYH2) and immunofluorescence of Myosin heavy chain (MyHC) was used to assess cell differentiation. The proliferation analysis revealed that circMYL1 inhibited the proliferation of bovine primary myoblast. Furthermore, the differentiation analysis demonstrated that circMYL1 promoted the differentiation of bovine primary myoblast. The luciferase screening and RNA immunoprecipitation (RIP) assays found that circMYL1 could have interaction with miR-2400. Additionally, we demonstrated that miR-2400 promoted proliferation and inhibited differentiation of bovine primary myoblast, while circMYL1 may eliminate the effects of miR-2400, as showed by rescue experiments. Together, our results revealed that a novel circular RNA of circMYL1 could inhibit proliferation and promote differentiation of myoblast by sponging miR-2400.

Highlights

Mammals have three different types of muscle tissues, including cardiac muscle, skeletal muscle, and smooth muscle [1]
Sanger sequencing for the verification of the circMYL1 junction site (Figure 1C), which was consistent with the sequencing results
Total RNAs were treated with RNase R exonuclease, and quantitative realtime polymerase chain reaction (qRT-PCR) was performed, the results showed that the circMYL1 was more resistant to RNase R than MYL1 mRNA

Summary

Introduction

Mammals have three different types of muscle tissues, including cardiac muscle, skeletal muscle, and smooth muscle [1]. Skeletal muscle represents half of the total body mass and has a critical role for voluntary motion and support [1]. The vertebrate skeletal muscle originates from the somites during embryonic development [2]. Previous studies showed that myogenesis is a complicated and orchestrated process, regulated by several genes, signalling pathways, and network regulation [3,4]. The multiple specifics myogenic regulatory transcription factors (MRFs) are important for the regulation of skeletal muscle development. Previous studies found that MRFs are a member of the basic helix-loop-helix (bHLH) family, including MYOG, MyoD, Myf, and MRF4. MRFs that significantly expressed in skeletal muscle suggested that they have an essential role during the proliferation and differentiation of skeletal muscle [5].

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