Mstn基因干扰通过上调基因Cpt1b促进肌间脂肪的β氧化  

Mstn knockdown promotes intramuscular fatty acid metabolism byβoxidation via the up-regulation of Cpt1b

作　　者：郭亚男 杨茹艳 张芷毓 包都兰 孙莹 杨磊 李光鹏 高丽 GUO Yanan;YANG Ruyan;ZHANG Zhiyu;BAO Dulan;SUN Ying;YANG Lei;LI Guangpeng;GAO Li(Faculty of Biology Science and Technology,Baotou Teacher’s College,Baotou 014030,Inner Mongolia,China;State Key Laboratory of Reproductive Regulation&Breeding of Grassland Livestock,Inner Mongolia University,Hohhot 010070,Inner Mongolia,China)

机构地区：[1]包头师范学院生物科学与技术学院,内蒙古包头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,内蒙古呼和浩特010070

出　　处：《生物工程学报》2022年第8期3076-3089,共14页Chinese Journal of Biotechnology

基　　金：内蒙古自治区自然科学基金(2021BS08013);包头师范学院高水平研究培育项目(BSYKJ2021-ZQ03)。

摘　　要：肌生长抑制素(myostatin,Mstn)也被称为生长/分化因子-8(GDF-8)。敲减或敲除Mstn基因可促进肌肉发育、降低脂肪含量。本研究利用RNA干扰技术制备Mstn干扰小鼠,随后对其骨骼肌形态、骨骼肌甘油三酯(triglyceride,TG)含量、脂肪酸组成及含量进行了分析。结果显示,与对照组相比,Mstn干扰小鼠肌肉中Mstn的表达减少。小鼠骨骼肌肌纤维的横截面积显著增大,而TG含量显著降低,n-3/n-6和不饱和/饱和脂肪酸比值显著升高。通过实时荧光定量PCR检测脂肪酸代谢相关基因的表达,结果表明脂肪酸分解和转运相关基因表达上调,而脂肪酸合成相关基因表达下调。在这些基因中,与β氧化相关的基因Cpt1b的上调尤为明显。对骨骼肌中CPT1的酶活性进行了检测,结果与基因表达情况一致。为探讨其进一步作用机理,通过染色质免疫沉淀实验发现,Mstn基因下游转录因子SMAD3可与Cpt1b基因的启动子直接结合。上述结果表明,Mstn敲减后主要通过调控其下游转录因子SMAD3与Cpt1b基因启动子的结合,上调Cpt1b的表达,从而促进肌内脂肪酸的β氧化代谢。Myostatin(Mstn)is known as growth/differentiation factor-8(GDF-8).Knockout or knockdown of Mstn gene promotes muscle development and reduces fat content.Here we prepared Mstn knockdown mice by RNA interference,then the morphology of the skeletal muscle,the content of triglyceride(TG),the content and composition of fatty acids in the skeletal muscle were detected.The expression of Mstn reduced in muscle of Mstn knockdown mice compared to the controls.The cross sectional areas of the skeletal muscle myofibers were significantly larger while the content of TG was less than that of the controls,and the ratios of n-3/n-6 and unsat/sat in the knockdown mice increased significantly.Subsequently,we detected the expression of genes associated with fatty acid metabolism.The expression of the genes associated with lipolysis and fatty acid transportation were up-regulated,while the genes associated with fatty acid synthesis were down-regulated.Of these genes,the up-regulation of a gene associated withβoxidation,Cpt1b,was up-regulated remarkably.We further detected the enzyme activity of CPT1 in skeletal muscle and obtained the same results with gene expression.Moreover,chromatin immunoprecipitation assay was performed and we found that SMAD3,a transcription factor downstream of Mstn,directly binds to the promoter of Cpt1b gene.These results showed that knockdown of Mstn up-regulated the expression of Cpt1b through the binding of SMAD3 to the promoter of Cpt1b,then promoted theβoxidation metabolism of intramuscular fatty acids.

关 键 词：MSTN基因 RNA干扰 脂肪酸代谢 β氧化 Cpt1b 

分 类 号：Q78[生物学—分子生物学]