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

作　　者：员佳乐 刘畅 黄晓宇 刘巧霞 史明月 李文霞 牛瑾 王首元 高鹏飞[1] 郭晓红[1] 李步高[1] 路畅 曹果清[1] YUN Jiale;LIU Chang;HUANG Xiaoyu;LIU Qiaoxia;SHI Mingyue;LI Wenxia;NIU Jin;WANG Shouyuan;GAO Pengfei;GUO Xiaohong;LI Bugao;LU Chang;CAO Guoqing(College of Animal Science,Shanxi Agricultural University,Taigu 030801,China;Shanxi Animal Husbandry Technology Extansion Service Center,Taiyuan 030001,China)

机构地区：[1]山西农业大学动物科学学院,太谷030801 [2]山西省畜牧技术推广服务中心,太原030001

出　　处：《畜牧兽医学报》2023年第5期1893-1904,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31872336);三晋学者支持计划专项经费资助(2016;2017);山西农业大学科技创新基金项目(2020BQ56);山西省基础研究计划项目(20210302124639)。

摘　　要：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

关 键 词：miR-145-5p IGF 1R AKT通路 增殖和分化 骨骼肌卫星细胞 

分 类 号：S828.2[农业科学—畜牧学]