miR-495-3p对山羊骨骼肌细胞增殖分化的影响

【目的】研究miR-495-3p对山羊骨骼肌细胞增殖分化的影响及其在不同组织中的表达情况，为探究miRNA在肌肉发育中的调控机制提供理论基础。【方法】利用生物信息学方法预测miR-495-3p的靶基因；实时荧光定量PCR检测miR-495-3p及其靶基因在心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、腿肌等组织中的表达水平。构建miR-495-3p的过表达(miR-495-3p mimic、miR-495-3p mimic NC)及抑制物(miR-495-3p inhibitor、miR-495-3p inhibitor NC),在山羊骨骼肌细胞汇合度达60%～70%时进行转染，并用含2%马血清的培养基进行诱导分化，用CCK-8检测细胞增殖活力，实时荧光定量PCR检测过表达和干扰效率及增殖分化相关基因配对盒基因(paired-box 7,Pax7)、细胞蛋白周期E(Cyclin E)、肌细胞生成素(myogenin, myoG)、生肌因子5(recombinant myogenic factor 5,Myf5)的表达；构建miR-495-3p靶基因的野生型和突变型载体并转染至293T细胞，用...

Effect of miR-495-3p on Proliferation and Differentiation of Goat Skeletal Muscle Cells

【Objective】 The experiment was conducted to study the effects of miR-495-3p on the proliferation and differentiation of goat skeletal muscle cells and its expression in different tissues,in order to provide a theoretical basis for exploring the regulatory mechanism of miRNA in muscle development.【Method】 Target genes of miR-495-3p were predicted by bioinformatics methods.The expression levels of miR-495-3p and its target genes in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and leg muscle were detected by Real-time quantitative PCR.The overexpression (miR-495-3p mimic,miR-495-3p mimic NC) and inhibitor (miR-495-3p inhibitor,miR-495-3p inhibitor NC) of miR-495-3p were constructed,and when the confluence of goat skeletal muscle cells reached 60%-70%,they were transfected and induced to differentiate with the medium containing 2% horse serum,CCK-8 was used to detect cell proliferation activity,and Real-time quantitative PCR was used to detect the overexpression and interference efficiency,and the expression of proliferation and differentiation related genes paired box 7 (Pax7),Cyclin E,myogenin (MyoG) and myogenic factor 5 (Myf5).The wild-type and mutant vectors of miR-495-3p target gene were constructed and transfected into 293T cells,the double luciferase report was used to verify the binding of miR-495-3p and its target gene.【Result】 Bioinformatics prediction results showed that the target gene of miR-495-3p was alpha cardiocactin 1 (ACTC1),and there were extremely significant differences of miR-495-3p and ACTC1 in longissimus dorsi muscle at the age of 1 and 10 months goats (P<0.01).miR-495-3p and ACTC1 were expressed in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and leg muscle,and the expression was higher in longissimus dorsi muscle.The results of cell transfection showed that miR-495-3p mimic significantly promoted the expression of miR-495-3p (P<0.01),and miR-495-3p inhibitor significantly inhibited the expression of miR-495-3p (P<0.01),indicating that miR-495-3p mimic and miR-495-3p inhibitor could be used in subsequent trials.Real-time quantitative PCR results showed that overexpression of miR-495-3p promoted the expression of skeletal muscle cell differentiation related marker genes MyoG and Myf5,while inhibition of miR-495-3p decreased the expression of MyoG and Myf5 genes.Overexpression and interference of miR-495-3p had no significant effect on the expression of Pax7,Cyclin E genes and cell proliferation activity of skeletal muscle cells (P>0.05).The results of double luciferase report showed that miR-495-3p had a targeting relationship with ACTC1 gene.【Conclusion】 miR-495-3p had no significant effect on the proliferation of goat skeletal muscle cells,but could promote the differentiation of goat skeletal muscle cells by targeting ACTC1 gene.