牛胎儿骨骼肌来源成肌细胞分化相关microRNA的筛查与鉴定

摘要

This study aimed to identify differentially expressed miRNAs in myogenic differentiation of bovine fetal skeletal muscle derived myoblasts,which would provide important insight for understanding the mechanism of myoblast differentiation at bovine fetal stage.Myoblasts were isolated and cultured from skeletal muscle of 3 bovine fetal on 4 months old,and then induced myogenic differentiation in vitro.miRNAs of myoblast at the initial and terminal stage of myoblast differentiation were sequenced using high throughput sequencing technology.The sequencing data was analyzed to identify the differentially expressed miRNAs.Differentially expressed miRNAs were randomly selected to validate the sequencing accuracy using quantitative RT-PCR.The re-sults revealed 619 miRNAs in 6 libraries,and 150 miRNAs were differentially expressed.Among them,several miRNAs were related to muscle development such as bta-miR-199a-5p.The target genes of differentially expressed miRNAs were predicted and enriched to pathways related to the muscle development such as MAPK pathway.The expression of 8 randomly selected miRNAs were quantified by real-time quantitative PCR for further validation,and their results were con-sistent with those of sequencing analyses.The study identified 150 differentially expressed miRNAs in the differentiation of bovine fetal muscle derived myoblasts.Our findings will provide valuable in-sights for further exploring the regulation roles of miRNAs in the development of bovine fetal skeletal muscle.%旨在发掘牛胎儿骨骼肌来源的成肌细胞分化过程中差异表达的miRNA,为进一步研究牛胎儿期成肌细胞分化的分子机理提供理论基础.本研究体外培养3头4月龄的牛胎儿骨骼肌来源的成肌细胞,运用高通量测序技术对成肌细胞分化起始和终末两个不同时期进行miRNA测序,并对测序结果进行生物信息学分析,鉴定差异表达的miRNA.随机选取差异表达miRNA,进行荧光定量PCR验证.结果表明,本研究从6个测序文库中共鉴定出已知miRNA 619个,其中差异表达miRNA 150个.差异表达miRNA中bta-miR-199a-5p等多个miRNA已经被证实与肌肉发育相关.通过靶基因预测富集到MAPK等多个与肌肉分化相关的通路.随机选取其中的8个miRNA进行荧光定量PCR验证,定量结果与测序结果完全一致.本研究鉴定出了150个与牛胎儿骨骼肌来源的成肌细胞分化相关的差异表达miRNAs.本研究结果将为研究miRNA调控牛胎儿骨骼肌发育提供科学依据.