Regulations And Functions Of Bovine MicroRNAs On Skeletal Myoblast Development And Their Genetic Variants

The molecular regulation mechanism of skeletal muscle development can provide comprehensive genetic information on beef cattle improvement.During the embryonic stage,the myoblast differentiates into multinucleated myotubes through proliferation,differentiation and fusion.After birth,the number of the skeletal muscle fibers keep stable and mainly focus on enlarging.In addition,the damage and repair of skeletal muscle satellite cells potentially affect the beef production.The development of skeletal muscle is regulated by a variety of factors such as the myogenic regulatory factors,the sequential expression regulation of skeletal muscle-specific or non-specific skeletal muscle microRNAs,the regulation of environmental factors and nutritional level of beef cattle feeding.MicroRNAs regulate the muscle development mainly through regulating the myogenic regulatory factors and inhibiting or promoting the signal pathways that regulates the skeletal muscle development.Meanwhile,the genetic variations in the sequence of microRNAs also trigger the molecular functions to regulate the complex economic traits.This study focuses on the regulatory function of bovine micro NRAs on skeletal myoblast development and their genetic variations.The main findings are as follows:1.Screening and functional verification of the microRNA for proliferation and differentiation of primary bovine myoblastsBased on the high-throughput sequencing results,bta-mi R-365-3p was screened and verificated differentially expressed in the different development stages of skeletal muscle tissues and skeletal myoblast of Qinchuan cattle.Afterwards,the overexpression of bta-mi R-365-3p significantly reduced the expression levels of bovine skeletal myoblast proliferation related marker genes,stimulated the expression levels of skeletal muscle differentiation related marker genes and decreased the myoblast numbers in S stage and My HC positive myotubes.By contrast,bta-mi R-365-3p inhibitor showed the opposite effect.Finally,we predicted and confirmed that activin A receptor type I gene(ACVR1)could be a direct target of bta-mi R-365-3p.Consistently,knock-down of ACVR1 showed the consistent effects to skeletal myoblast with overexpression.Collectively,these data suggested that bta-mi R-365-3p repressed proliferation but promoted differentiation of bovine myoblasts through several biological mechanisms involving downregulation of ACVR1.2.Global microRNAs expression during differentiation of bovine primary myoblastsThe function of a single microRNA to skeletal myoblast differentiation can not explain the effects of the global microRNAs on the differentiation of bovine skeletal muscle.Hence,smallRNA sequence was used to identify the global microRNAs in primary bovine myoblast from Qinchuan cattle under the resveratrol treatment for differentiation for four days.The results showed that ninty-three differentially expressed(DE)microRNAs(adjusted P-value <0.05)were identified.GO enrichment analysis for the 1869 genes targeted by DE microRNAs revealed two significant GO terms(adjusted P-value < 0.05).KEGG enrichment analysis showed five significant pathways and the top three significant KEGG pathway were the insulin signaling pathway(bta04910),the ras signaling pathway(bta04014)and the MAPK(Mitogenactivated protein kinase)signaling pathway(bta04010).Our study idenfied 93 DE microRNAs induced by RSV through the global microRNA modulations,they could be regard as potentially biomarkers for primary bovine myoblast differentiation3.Integrated analysis of mRNA and microRNA co-expressed network for myoblast differentiationThe weighted correlation network analysis(WGCNA)was used to investigate the coexpressed hub messageRNAs(mRNAs)and hub microRNAs in the primary bovine myoblasts after resveratrol treatment.mRNA and microRNA analysis identified the posotive correlation of resveratrol treatments and Turquoise module(2579 mRNAs and 59 microRNAs)with the correlation coefficient higher than 0.90.After the biological enrichment analysis,2579 DE genes in the Turquoise module were significantly enriched in the actin cytoskeleton associated GO terms.The top KEGG pathway was regulation of actin cytoskeleton(bta04810).In summary,the microRNA-mRNA networks in the Turquoise module were constructed to reveal the resveratrol effect to skeletal myoblast differentiation.4.Genetic variations on bovine microRNAsThe whole-genome resequencing of 1632 cattle from 1000 Bull Genomes Project were used and identified a total of 1109,334 and 130 SNPs on the microRNA precursor,mature and seed region sequences,respectively.Population genetics effects showed that the heterozygosity value of the SNPs is below 0.3.The minimum allele frequency is mainly below0.1,and most SNPs followed Hardy-Weinberg equilibrium.Thirty-three microRNAs were selected for further analysis.These 33 microRNAs contained 37 SNPs in the seed region potentially influencing the gain or loss of target genes which enrichend in the GO terms or KEGG pathways.The selected microRNAs were mapped against bovine quantitative trait loci(QTLs)to infer their genetic variations to bovine important economic traits.