miR-145-5p靶向IGF 1R介导AKT通路抑制猪骨骼肌卫星细胞增殖和分化

miR-145-5p Inhibits the Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells by Targeting IGF 1R-Mediated AKT Pathway

旨在探讨miR-145-5p对猪骨骼肌卫星细胞增殖和分化的影响。本研究采用qRT-PCR检测miR-145-5p的组织表达谱和发育性表达规律;选用1 d晋汾白猪趾长伸肌进行骨骼肌卫星细胞的分离与培养,分别转染miR-145-5p模拟物(mimics)及其对照组(mimics NC),miR-145-5p抑制剂(inhibitor)及其对照组(inhibitor NC),每个处理3个重复,利用qRT-PCR、EdU和CCK-8方法检测增殖相关基因表达量、EdU阳性细胞数和细胞增殖活性;待猪骨骼肌卫星细胞分化后利用qRT-PCR、Western blot和免疫荧光染色方法探究分化相关基因mRNA和蛋白水平及肌管形成情况;采用双荧光素酶试验、qRT-PCR和Western blot探究miR-145-5p对下游IGF 1R和AKT通路的影响。结果显示,miR-145-5p基因在肝和肺中表达量最高,心和皮下脂肪中次之(P<0.05);随着日龄的增加,miR-145-5p在猪背最长肌中的表达量持续升高(P<0.05)。在猪骨骼肌卫星细胞体外培养过程中,转染miR-145-5p mimics极显著下调Ki 67和CDK 1的表达(P<0.01),显著下调PCNA和CDK 4的表达(P<0.05),阳性细胞指数极显著降低(P<0.01),细胞活性显著低于对照组(P<0.05)。在分化方面,过表达miR-145-5p极显著下调MyOD、MyOG和Myf 5的表达量(P<0.01),MyOD蛋白水平显著降低(P<0.05),MyHC阳性肌管面积少于对照组;转染miR-145-5p inhibitor则出现相反的结果。过表达miR-145-5p显著降低IGF1R mRNA和蛋白的相对表达量(P<0.05),显著降低AKT蛋白的磷酸化水平;而干扰miR-145-5p能极显著上调IGF1R mRNA和蛋白的相对表达量(P<0.01),并能挽救转染si-IGF1R对p-AKT蛋白的抑制作用(P<0.01)。本研究结果表明,miR-145-5p通过负调控IGF1R mRNA和蛋白的相对表达水平,并影响AKT通路,抑制猪骨骼肌卫星细胞的增殖和分化,进而参与骨骼肌的发育过程。研究结果丰富了猪肌肉生长发育的分子网络,并为肌肉性状的分子育种提供了靶标。

This study aimed to investigate the effect of miR-145-5p on the proliferation and differtiation of porcine skeletal muscle satellite cells.The tissue expression profile and developmental expression pattern of miR-145-5p were detected by qRT-PCR.The skeletal muscle satellite cells were isolated and cultured from the extensor digitorum longus of one-day old Jinfen White pig.The miR-145-5p simulant(mimics)and its control group(mimics NC),miR-145-5p inhibitor(inhibitor)and its control group(inhibitor NC)were transfected respectively.Each treatment had three replicates.The proliferation-related genes,the number of EdU-positive cells and the proliferative activity of cells were detected by qRT-PCR,EdU and CCK-8 methods.After the differentiation of porcine skeletal muscle satellite cells,qRT-PCR,Western blot and immunofluorescence staining were used to investigate the level of mRNA and protein of differentiation related genes and the formation of myotubes.The effects of miR-145-5p on the downstream IGF 1R and AKT pathways were investigated by double luciferase test,qRT-PCR and Western blot.The results showed that the expression of miR-145-5p was the highest in the liver and lung,followed by in heart and subcutaneous fat(P<0.05).The expression of miR-145-5p in the longissimus dorsi muscle continued to increase with the increase of age(P<0.05).During the culture procession of porcine skeletal muscle satellite cells in vitro,the expressions of Ki 67 and CDK1(P<0.01),and PCNA and CDK4(P<0.05)were down-regulated significantly after transfection of miR-145-5p mimics.The positive cell index was very significantly decreased(P<0.01),and the cell viability was also significantly decreased(P<0.05).Regarding to the differentiation of porcine skeletal muscle satellite cells,the expressions of MyOD,MyOG and Myf 5 were down-regulated significantly(P<0.01)by overexpression of miR-145-5p,the level of MyOD protein was inhibited(P<0.05),and the MyHC-positive myotube area was also less than that of the control group.Transfection of miR-145-5p inhibitor resulted in the opposite result.Overexpression of miR-145-5p significantly reduced the relative expression of IGF1R mRNA and protein(P<0.05),and significantly reduced the phosphorylation level of AKT protein.Interference with miR-145-5p increased significantly the relative expression of IGF1R mRNA and protein(P<0.01),and rescued the inhibitory effect of si-IGF1R on p-AKT protein(P<0.01).In conclusion,miR-145-5p negatively regulates the relative expression of IGF1R mRNA and protein,affects AKT pathway,inhibits the proliferation and differentiation of porcine skeletal muscle satellite cells,and then participates in the development of skeletal muscle.The results of this study enrich the molecular network of pig muscle growth and development,and provide molecular targets for molecular breeding of muscle traits.