失神经骨骼肌萎缩模型大鼠肌肉组织中microRNA表达谱的差异分析  

Differential expression profiles of microRNAs in muscle tissue of denervated skeletal muscle atrophy rats

作　　者：刘洪文 李皎 徐文豪 聂华 刘绍江[1] 徐杰[2] 尹立[1,3] Liu Hongwen;Li Jiao;Xu Wenhao;Nie Hua;Liu Shaojiang;Xu Jie;Yin Li(Department of Orthopedics,Panzhihua Central Hospital,Panzhihua 617000,Sichuan Province,China;Department of Orthopedics,Fujian Provincial Hospital,School of Clinical Medicine,Fujian Medical University,Fuzhou 350001,Fujian Province,China;Discipline Construction Office,Panzhihua Central Hospital,Panzhihua 617000,Sichuan Province,China)

机构地区：[1]攀枝花市中心医院骨科,四川省攀枝花市617000 [2]福建医科大学省立临床医学院,福建省立医院骨科,福建省福州市350001 [3]攀枝花市中心医院学科建设办公室,四川省攀枝花市617000

出　　处：《中国组织工程研究》2023年第5期732-737,共6页Chinese Journal of Tissue Engineering Research

基　　金：四川省中管局中医药科研专项课题(2020JC0087),项目负责人:尹立;成都中医药大学学科人才科研专项课题(YYZX20211097),项目负责人:刘洪文;福建省自然科学基金面上项目(2019J01173),项目负责人:徐杰。

摘　　要：背景:失神经骨骼肌萎缩尚缺乏较有效的治疗方法,预后差,microRNA在体内的表达谱不清楚。目的:采用高通量测序技术分析失神经支配骨骼肌萎缩大鼠microRNA表达谱及转化生长因子β1/Smad3信号通路的变化,探讨microRNA在骨骼肌萎缩中的作用及其机制。方法:12只SD大鼠随机分为2组,实验组采用坐骨神经截断的方法建立失神经肌萎缩模型,对照组仅暴露坐骨神经后缝合伤口。完整取下大鼠双侧腓肠肌计算肌湿质量比,苏木精-伊红染色观察肌萎缩和纤维化情况并测定肌纤维横截面积;应用Illumina HiSeq 2500测序技术筛查骨骼肌组织中的microRNA表达谱;结合火山图、层次聚类图、基因本体论、京都基因与基因组百科全书通路分析差异表达基因参与骨骼肌代谢的生物过程;采用实时荧光定量PCR验证候选基因在肌肉组织中的差异表达;Western blot检测转化生长因子β1、p-Smad3蛋白表达。结果与结论:①与对照组相比,实验组腓肠肌湿质量比和横截面积明显降低(P<0.001),造模结果符合预期;②两组共筛查到1249个差异表达的microRNA(P<0.05;|log2 Fold Change|>0.0),筛选出显著差异表达的microRNA共14个,其中2个表达上调,12个表达下调;③生物学功能和通路分析结果提示,miR-1247-3p、miR-132-5p、miR-21-3p、miR-363-3p、miR-451-5p等具有显著差异表达的microRNAs显著富集于细胞增殖、分化、凋亡等生物过程及转化生长因子β信号通路;④实时荧光定量PCR结果显示,miR-21-3p在实验组腓肠肌组织中呈显著低表达(P<0.001),与测序结果趋势一致;⑤Western blot结果显示,转化生长因子β1、p-Smad3蛋白表达水平较对照组明显升高(P<0.05);⑥提示microRNA在骨骼肌萎缩生理病理过程中扮演关键角色,miR-21-3p可能通过激活转化生长因子β1/Smad3信号通路的生物活性,进而在骨骼肌细胞代谢中发挥作用。BACKGROUND:There is no effective treatment for denervated skeletal muscle atrophy with poor prognosis,and moreover,the expression profiles of microRNA in vivo are unclear.OBJECTIVE:To analyze the microRNA expression profiles and the changes of transforming growth factor-β1/Smad3 signaling pathway in denervated skeletal muscle atrophy rats using high-throughput sequencing technology and to explore the role and mechanism of microRNA in skeletal muscle atrophy.METHODS:Twelve Sprague-Dawley rats were randomized into two groups:experimental group and control group.Animal models of denervated muscle atrophy were established using truncation of the sciatic nerve in the experimental group.In the control group,only the sciatic nerve was exposed followed by suturing.The bilateral gastrocnemius muscles were completely removed to calculate the muscle wet weight ratio.Atrophy and fibrosis were observed by hematoxylin-eosin staining and the cross-sectional area of the muscle was determined.Illumina HiSeq 2500 sequencing technology was used to identify the microRNA expression profiles in skeletal muscle tissue.In combination with volcano plot,hierarchical clustering map,Gene Ontology,Kyoto Encyclopedia of Genes and Genomes pathway,differentially expressed genes involved in skeletal muscle metabolism were analyzed.Real-time fluorescent quantitative PCR was used to verify the differential expression of candidate genes in muscle tissue and western blot was used to detect the expression of transforming growth factor-β1 and p-Smad3 proteins.RESULTS AND CONCLUSION:Compared with the control group,the muscle wet weight ratio and cross-sectional area were significantly lower in the experimental group(P<0.05),and the modeling results were in line with expectations.A total of 1249 differentially expressed microRNAs were identified in the two groups(P<0.05;|log2 Fold Change|>0.0).There were 14 microRNAs with significant differential expression,of which 2 were up-regulated and 12 were down-regulated.The results of biological function and

关 键 词：失神经 骨骼肌萎缩 高通量测序 microRNA 转化生长因子β1 SMAD3 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R685.4