A Novel Circular RNA circITSN2 Targets the miR-218-5p/LMO7 Axis to Promote Chicken Embryonic Myoblast Proliferation and Differentiation.

Xiaoxu Shen,Wei Liu,Jing Zhao,Yuanhang Wei,Guishuang You,Yongtong Tian,Menggen Ma,Qing Zhu,Mingxin Du,Huadong Yin,Shuyue Tang,Yao Zhang,Zhenyu Su,Jian He

doi:10.3389/fcell.2021.748844

Xiaoxu Shen, Wei Liu + Show 12 more

Open Access

https://doi.org/10.3389/fcell.2021.748844

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Abstract

Circular RNA (circRNA) is a class of endogenous non-coding RNAs without 5′ and 3′ ends; an increasing number of studies show that circRNA is involved in skeletal muscle development. From our previous sequencing data, the circRNAome in breast muscle of two chicken lines with a distinct rate of muscle development, which included a fast muscle growing broiler (FMGB) and a slow muscle growing layer (SMGL), we found a novel differentially expressed circRNA generated by intersectin 2 (ITSN2) gene (named circITSN2). We verified that circITSN2 is a skeletal muscle-enriched circRNA that promotes chicken primary myoblast (CPM) proliferation and differentiation. Further molecular mechanism analysis of circITSN2 in chicken myogenesis was performed, and we found circITSN2 directly targeting miR-218-5p. Besides, miR-218-5p inhibits CPM proliferation and differentiation, which is contrary to circITSN2. Commonly, circRNAs act as a miRNA sponge to alleviate the inhibition of miRNAs on mRNAs. Thus, we also identified that a downstream gene LIM domain 7 (LMO7) was inhibited by miR-218-5p, while circITSN2 could block the inhibitory effect of miR-218-5p by targeting it. Functional analysis revealed that LMO7 also accelerates CPM proliferation and differentiation, which was similar to circITSN2 but contrary to miR-218-5p. Taken together, these results suggested that circITSN2 promotes chicken embryonic skeletal muscle development via relieving the inhibition of miR-218-5p on LMO7. Our findings revealed a novel circITSN2/miR-218-5p/LMO7 axis in chicken embryonic skeletal muscle development, which expands our understanding of the complex muscle development regulatory network.

Highlights

With the development of animal breeding technology in the past century, extensive meat products from domestic animals prevent hunger and malnutrition
RNase R resistance test showed that circular intersectin 2 (ITSN2) RNA is more resistant to the digestion of RNase R than linear β-actin mRNA (p < 0.01; Figure 1D); the content of circITSN2 in the random primer N9 reverse transcription system was much higher than that in oligo d(T) primer reverse transcription system (p < 0.01; Figure 1E), which indicated that circITSN2 is a stable circular RNA without 3 poly (A) tail. Quantitative Real-Time PCR (qRT-PCR) analysis was used to determine the expression of circITSN2 in fast muscle growing broiler (FMGB) and slow muscle growing layer (SMGL); results showed that circITSN2 was significantly upregulated in FMGB (p < 0.05; Figure 1F)
The results of CCK-8 assay showed that knockdown of circITSN2 significantly reduced the proliferation vitality of chicken primary myoblast (CPM) (p < 0.01; Figure 2A), while the proliferation vitality was induced by circITSN2 overexpression (p < 0.05; Figure 2B)

Summary

Introduction

With the development of animal breeding technology in the past century, extensive meat products from domestic animals prevent hunger and malnutrition. A well-known example is the utilization of the Myostatin gene, which has a negative role on skeletal muscle development, and the gene mutation-caused loss of function of the Myostatin gene leads to more meat production (Aiello et al, 2018). Understanding the molecular regulation mechanism of animal skeletal muscle development can provide the necessary theoretical basis of molecular breeding. The growth and hypertrophy of animal skeletal muscle fibers are mainly attributed to the proliferation and differentiation of skeletal muscle progenitor cells (Dumont et al, 2015; Chal and Pourquié, 2017), which is regulated by many coding genes and non-coding RNAs (Zhao et al, 2019). Muscle-specific miR-133, miR-1, and miR-206 regulate muscle stem cell proliferation and differentiation via targeting different genes in many organisms (Horak et al, 2016). Expressed miR-7 inhibits chicken primary myoblast (CPM) differentiation and proliferation by targeting KLF4 (Zhang et al, 2020), and miR7 showed the same inhibitory role in mouse C2C12 myogenesis by targeting TCF12 (Gao et al, 2021)

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