信号蛋白3A对成肌细胞功能的影响及其转录调控机制

Effect of semaphorin 3A on myoblast function and its transcriptional regulation mechanism

目的探讨信号蛋白3A(Sema3A)对成肌细胞功能的影响及其转录调控机制。方法以1.0、0.1、0.01μg/ml重组Sema3A蛋白作用成肌细胞,并设置对照组,采用动态活细胞成像及划痕实验检测细胞的增殖与迁移能力,RT-qPCR检测成肌分化因子的表达。对1.0μg/ml重组Sema3A蛋白干预的成肌细胞及对照组细胞进行转录组测序,筛选差异表达基因并进行GO富集分析,对部分差异表达基因通过RT-qPCR进行转录水平验证。结果IncuCyte细胞成像计数显示,Sema3A可促进成肌细胞增殖,其中1.0μg/ml Sema3A组细胞计数较对照组升高17.36%(P<0.001)。划痕实验结果显示,Sema3A可使成肌细胞迁移能力明显增强,其中1.0μg/ml Sema3A组细胞划痕愈合率较对照组升高69.66%(P<0.001)。RT-qRCR检测结果显示,Sema3A可上调部分成肌分化基因的表达,其中1.0μg/ml Sema3A可使Myf5、MyoG基因表达分别升高37.47%±11.67%(P<0.01)、26.57%±11.31%(P<0.05)。生物信息学分析显示,Sema3A可上调成肌细胞转录组中的肌细胞与肌纤维发育、细胞与解剖结构成熟及成骨发育等相关功能通路,下调脂蛋白代谢与乳糜微粒等相关功能通路;RT-qPCR验证了富集通路中的部分差异表达基因,其中Prox1、Myh11表达分别升高22.48%±14.42%(P<0.05)、21.42%±5.81%(P<0.01),LipC、ApoC2表达分别降低23.08%±15.38%(P<0.05)、55.97%±26.51%(P<0.05)。结论外源性Sema3A蛋白可上调成肌细胞分化相关基因Myf5、MyoG的表达,且可能通过对骨骼肌发育、成熟与脂蛋白代谢等相关基因进行转录调控而促进成肌细胞的增殖、迁移。

Objective To study the effects of semaphorin 3A(Sema3A)on myoblast function and its transcriptional regulation mechanism.Methods Myoblasts were treated with 1.0μg/ml,0.1μg/ml,0.01μg/ml recombinant Sema3A protein,and the control group was set up.The proliferation and migration of myoblasts were observed by the IncuCyte system and scratch test.The expression levels of myogenic differentiation factors were detected by RT-qPCR.Transcriptome sequencing was performed for the myoblasts intervened by 1.0μg/ml recombinant Sema3A protein and those in control group.The differential expressed genes were screened out for GO enrichment analysis.Part of differential expressed genes were verified of transcription level by RT-qPCR.Results IncuCyte cell imaging count showed that Sema3A promoted proliferation of myoblasts,the cell count increased about 17.36%in 1.0μg/ml Sema3A group than in control group(P<0.001).Scratch test showed the enhanced migration ability of myoblasts by Sema3A,the cell scratch healing rate increased by 69.66%in 1.0μg/ml Sema3A group than in control group(P<0.001).RT-qRCR showed that during myoblast differentiation,1.0μg/ml Sema3A up-regulated the expression of Myf5 by 37.47%±11.67%(P<0.01),and up-regulated the expression of MyoG by 26.57%±11.31%(P<0.05).Bioinformatics analysis showed that Sema3A up-regulated the functional pathway associated with the development and maturation of muscle cells and muscle fibers,bone and chondrocyte proliferation,osteogenic differentiation,and bone mineralization;down-regulated the functional pathway associated with the lipoprotein metabolism and chylomicrons.RT-qPCR verified the partial differentially expressed genes in enrichment pathway,among which the expressions of Prox1 and Myh11 increased by 22.48%±14.42%(P<0.05)and 21.42%±5.81%(P<0.01),respectively;and of LipC and ApoC2 decreased by 23.08%±15.38%(P<0.05)and 55.97%±26.51%(P<0.05),respectively.Conclusion Exogenous Sema3A protein can up-regulate the expression of myoblast differentiation related genes Myf5 and MyoG,and may promote the proliferation and migration of myoblasts by transcriptional regulation of skeletal muscle development,maturation and lipoprotein metabolism.