MSTN基因突变通过SMAD2/SMAD3-CDKN1C调控牛肌肉卫星细胞增殖分化  

MSTN Mutation Regulates Proliferation and Differentiation of Bovine(Bos taurus)Muscle-derived Satellite Cells Through SMAD2/SMAD3-CDKN1C

作　　者：高丽 谷明娟 杨苗苗 刘云鹏[1] 卜彩虹 程鹏 白春玲 GAO Li;GU Ming-Juan;YANG Miao-Miao;LIU Yun-Peng;BU Cai-Hong;CHENG Peng;BAI Chun-Ling(School of Biology Science and Technology,Baotou Teacher's College,Baotou 014030,China;State Key Laboratory of Reproductive Regulation&Breeding of Grassland Livestock,Inner Mongolia University,Hohhot 010070,China)

机构地区：[1]包头师范学院生物科学与技术学院,包头014030 [2]内蒙古大学省部共建草原家畜生殖调控与繁育国家重点实验室,呼和浩特010070

出　　处：《农业生物技术学报》2022年第6期1128-1139,共12页Journal of Agricultural Biotechnology

基　　金：国家科技重大专项(2016ZX08007-002);内蒙古自治区自然科学基金项目(2021BS08013)。

摘　　要：肌肉生长抑制素(myostatin,MSTN)基因是肌肉发育的负调控因子,可调控细胞增殖。为探讨该基因突变在调控肌肉卫星细胞(muscle-derived satellite cells,MDSCs)增殖分化中的作用机制,本研究以分离培养的MSTN基因突变型(MSTN mutant,MT)和野生型(wild type,WT)蒙古牛(Bos taurus)MDSCs为研究对象,对其增殖及分化过程中的细胞周期和基因表达变化进行检测。EdU增殖实验结果显示,MSTN基因突变型肌肉卫星细胞增殖指数(0.78±0.06)显著高于野生型细胞(0.57±0.04),表明MSTN基因突变的细胞增殖加快。当加入诱导液向成肌分化诱导时,MSTN基因突变细胞在诱导1 d后出现分化的肌管,而野生型细胞在诱导2 d后开始分化;利用流式细胞术进一步分析成肌诱导对细胞周期的影响,发现MSTN突变型细胞在诱导1 d后细胞周期开始停滞在G1/S期,野生型细胞在诱导2 d后细胞周期出现停滞,这些结果表明,当细胞被诱导分化时,MSTN突变细胞先于野生型细胞退出细胞周期而进入分化状态。通过q PCR和Western blot对细胞周期相关基因和蛋白的表达进行检测,结果显示,在MSTN基因突变细胞中,Cyclin A表达显著上调(19.5倍),而细胞周期蛋白依赖激酶抑制因子1C(cyclin-dependent kinase inhibitor1C,CDKN1C)的表达被显著抑制(0.9%),提示Cyclin A和CDKN1C在MSTN基因突变调控肌肉卫星细胞增殖分化的过程中可能具有重要作用。通过JASPAR数据库进行在线预测,发现MSTN信号通路中关键转录因子SMAD2/SMAD3可能与CDKN1C基因的启动子结合;进一步的Ch IP-qPCR实验结果显示,SMAD2/SMAD3可与CDKN1C启动子直接结合,并促进CDKN1C基因表达。上述结果提示,MSTN基因突变可能通过下调SMAD2/SMAD3转录因子与CDKN1C启动子的结合,从而抑制CDKN1C基因表达,进而上调Cyclin A-CDK2表达,最终促进DNA合成和细胞周期进程。本研究为MSTN基因突变促进肌肉发育发掘了新的作用机制,为制备肌肉发达的基Myostatin(MSTN)is a negative regulator of muscle development.Studies have shown that it can regulate cell proliferation.In order to explore the further mechanism of MSTN mutant in regulating the proliferation and differentiation of muscle-derived satellite cells(MDSCs),in this study,the MSTN mutant(MT)and wild type(WT)bovine(Bos taurus)MDSCs were used to detect the cell cycle and gene expression in the process of proliferation and differentiation.Results of EdU proliferation assay showed that the proliferation index of MSTN mutant cells(0.78±0.0559)was significantly higher than that of wild-type cells(0.57±0.0366),indicating that MSTN mutant promoted the proliferation of MDSCs.When myogenic differentiation was induced,the MSTN mutant cells began to differentiate and formed the myotubes after 1day of induction,while the wild-type cells began to differentiate after 2 days of induction.Flow cytometry was used to further analyze the effect of myogenic induction on the cell cycle.Results also showed that the MT cells began to stagnate in the G1/S phase after 1 day of induction,and the cell cycle of WT cells stagnated after 2 days of induction.These results indicated that when cells were induced to differentiate,MSTN mutant cells exited from the cell cycle and entered to a differentiated state before wild-type cells.The expression of cell cycle-related genes and proteins was detected by q PCR and Western blot.Results showed that in MSTN mutant cells,the expression of CyclinA was significantly up-regulated(Relative expression=19.5),while the expression of cyclin-dependent kinase inhibitor 1C(CDKN1C)was significantly inhibited(Relative expression=0.009).These results showed that CyclinA and CDKN1C may play important roles in the regulation of MSTN mutant to the proliferation and differentiation of MDSCs.Online prediction through the JASPAR database showed that the key transcription factors downstream of MSTN,SMAD2/SMAD3 may bind to the promoter of CDKN1C.ChIP-qPCR results demonstrated that the SMAD2/SMAD3 transcription

关 键 词：肌肉生长抑制素基因(MSTN)突变 肌肉卫星细胞 增殖 细胞周期蛋白依赖激酶抑制因子1C(CDKN1C) SMAD2/SMAD3 

分 类 号：S823[农业科学—畜牧学]