Identification of miRNA in Sheep Intramuscular Fat and the Role of miR-193a-5p in Proliferation and Differentiation of 3T3-L1.

Fuhui Han,Lirong Liu,Jixian Qiu,Lisheng Zhou,Nan Liu,Lei Wang,Jianning He,Le Zhao,Haijuan Li

doi:10.3389/fgene.2021.633295

Abstract

Intramuscular fat (IMF) is one of the most critical parameters affecting meat quality and mainly affected by genetic factors. MicroRNA as an important regulatory factor, which is still a lack of research in the development of sheep IMF deposition. We used RNA sequencing (RNA-seq) and cell-level validation to explore the role of miRNA in IMF deposition. As for this purpose, longissimus thoracis et lumborum (LTL) samples of 2 month-old (Mth-2) and 12 months-old (Mth-12) Aohan fine-wool sheep (AFWS) were used to identified miRNAs expression. We found 59 differentially expressed miRNAs (DE-miRNA) between these age groups and predicted their 1,796 target genes. KEGG functional enrichment analysis revealed eight pathways involved in lipid metabolism-related processes, including fatty acid elongation and the AMPK signaling pathway. A highly expressed DE-miRNA, miR-193a-5p, was found to serve a function in 3T3-L1 preadipocyte differentiation. Luciferase assay demonstrated that miR-193a-5p directly binds to the 3′-UTR region of ACAA2. By constructing mimics and inhibitor vector transfecting into 3T3-L1 cells to explore the effect of miR-193a-5p on cell proliferation and differentiation, we demonstrated that overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte proliferation, as evidenced by decreased mRNA and protein expression of CDK4 and CyclinB. CCK-8 assay showed that miR-193a-5p significantly inhibited cell proliferation. Similarly, the overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte differentiation and adipocyte-specific molecular markers’ expression, leading to a decrease in PPARγ and C/EBPα and ACAA2. Inhibition of miR-193a-5p had the opposite effects. Our study lists the miRNAs associated with intramuscular lipid deposition in sheep and their potential targets, striving to improve sheep meat quality.

Highlights

The market demand and accepted quality of mutton are being higher with its nutritional value improvement in consumer awareness
The results of determination of intramuscular fat (IMF) content have been published in previous studies (Han et al, 2021), and the results showed that the IMF content of 12 months of age was extremely significantly higher than that of 2 months of age, so Mth-2 and Mth-12 were selected for RNA sequencing (RNA-seq) to maximize the likelihood of detecting significant differences
We obtained Longissimus thoracis et lumborum (LTL) tissue samples from Mth-2 and Mth-12 rams for RNA-seq analysis, three replicates per sample, to study differential miRNAs expression related to lipid deposition

Summary

Introduction

The market demand and accepted quality of mutton are being higher with its nutritional value improvement in consumer awareness. IMF content is closely related to the flavor, juiciness, and tenderness of the meat (Dodson et al, 2015). Studies have shown that lamb IMF content must be maintained within the range of 4–5% or higher to satisfy the consumers’ flavor requirements (Mohrhauser et al, 2015). In the ongoing breeding process, the IMF content is generally low, resulting in serious negative effects on mutton tenderness and quality. Studies have shown that intramuscular lipid deposition is affected by multiple genes and signaling pathways, exploring the mechanism of fat deposition at the molecular level is an indispensable part of meat quality regulation. In order to explore the regulatory role of non-coding RNA (lncRNA, miRNA, circRNA) in the process of IMF deposition, we systematically identified the profiles of DE genes during intramuscular lipid deposition in sheep through high-throughput sequencing(Han et al, 2021)

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