摘要
目的:运动神经源性聚集蛋白诱导骨骼肌表面乙酰胆碱受体(AChR)聚集簇的形成是神经肌肉接头(NMJ)的关键步骤。本研究旨在利用高通量深度测序技术,筛选调控成肌分化和AChR聚集簇形成相关的关键信号通路和潜在的重要靶基因。方法:建立小鼠成肌细胞系C2C12成肌分化和聚集蛋白诱导肌细胞表面AChR聚集簇形成的细胞模型,通过高通量深度测序技术检测C2C12成肌分化前后,以及聚集蛋白刺激分化肌管形成AChR聚集簇前后转录水平差异表达基因,并对测序结果进行Gene Ontology(GO)和KEGG pathway分析。结果:GO分析发现,成肌分化前后与聚集蛋白刺激肌管形成AChR聚集簇前后差异基因表达分子的分布、参与的生物学过程及其发挥的分子作用较为相似,均主要存在于细胞器和细胞膜,参与细胞基础代谢过程,主要发挥蛋白结合和催化反应功能。KEGG pathway分析发现,C2C12细胞成肌分化前后差异基因主要富集于聚集蛋白骨架蛋白调控信号和细胞黏附作用等,而聚集蛋白诱导肌管表面AChR聚集簇形成前后差异表达基因则主要集中于轴突导向信号、Wnt信号和肿瘤相关信号等。结论:挖掘出一批可能参与成肌分化和聚集蛋白依赖的AChR聚集簇调控的候选基因,相关分子在成肌分化和AChR聚集簇形成过程中的功能及其调控机制有待进一步深入验证。
Objective:During the development of the neuromuscular junction(NMJ),one of the critical steps is neuronal agrin induced acetylcholine receptor(AChR)clustering on the surface of skeletal muscle.The purpose of this study is to identify the crucial signaling pathways and potential candidate genes invovled in the regulation of myogenic differentiation and AChR clustering by using high throughput deep-sequencing.Methods:Firstly,mouse myoblast cell line C2C12was used to induce myogenic differentiation and agrin-induced AChR clustering.Secondly,Illumina's high throughput deep-sequencing methods was adopted to detect the differentially expressed genes before and after C2C12myogenic differentiation,and agrin-indcued AChR clustering in myotubes as well at transcriptional level.Thirdly,deep-sequencing results were analyzed using Gene Ontology(GO)and KEGG pathway enrichment methods.Results:Based on GO analysis,we found there are some similarities in biological process of differentially expressed genes between myogenic differentiation and agrin-induced AChR clustering including membrane localization,metabolic process,biological regulation in catalytic activity and molecular transducer activity.KEGG pathway analysis showed the differential genes before and after C2C12myogenic differentiation enriched in the regulation of actin cytoskeleton and focal adhesion.While major differential genes before and after agrin-induced AChR clustering enriched in pathways in cancer,axon gudiance and Wnt signaling pathway.Conclusion:We have identified a batch of candidate genes which might be involved in the regulation of myogenic differentiation and agrin-induced AChR clustering.The function and mechanisms of those candidate genes in the regulation of myogenic differentiation and AChR clustering need to be studied and verified with a series of follow-up experiments.
作者
刘凤姣
杨海红
吴燕
吴海涛
吕英涛
LIU Feng-Jiao;YANG Hai-Hong;WU Yan;WU Hai-Tao;LYU Ying-Tao(Qingdao University of Science and Technology, Qingdao 266042,China;Beijing Institute of Basic Medical Sciences, Beijing 100850, China)
出处
《生物技术通讯》
CAS
2017年第6期715-724,共10页
Letters in Biotechnology
基金
国家重点基础研究发展计划(2014CB542203)
国家自然科学基金(31371149
31522029)
北京市科学技术委员会"脑认知与脑医学"专项(Z161100000216154)
北京市科技新星项目(Z131102000413033)