柴胡疏肝散对SH–SY5Y细胞内miR–124表达及其相关蛋白调控作用的研究

Effect of Chaihu-Shugan-San on the Expression of miR–124 and the Regulation of Related Proteins in SH–SY5Y Cells

目的:探索中药柴胡疏肝散对人神经母细胞瘤细胞(SH–SY5Y)内微小RNA124(miR–124)的调控作用以及相关蛋白表达的影响,进一步阐明柴胡疏肝散调控神经细胞的具体作用机制和途径。方法:培养SH–SY5Y细胞,转染pCDNA3.1–miR–124质粒,并予柴胡疏肝散干预,提取细胞蛋白并酶解,同位素标记相对和绝对定量技术(iTRAQ)标记,采用高效液相色谱串联质谱法(LC–MS/MS)鉴定蛋白,软件Proteome Discoverer1.3处理数据,并进一步进行生物信息学分析,Western blot验证差异表达蛋白。结果:SH–SY5Y细胞经转染miR–124质粒后miR–124表达显著上升,而转染miR–124质粒细胞在柴胡疏肝散处理后,miR–124表达显著下降。表明对于SH–SY5Y细胞柴胡疏肝散可以下调miR–124表达。蛋白质组分析共鉴定出肽段15487个,蛋白3707个。免疫印迹检测差异蛋白NudE神经发育蛋白1(NDE1),在miR–124过量表达时NDE1蛋白表达量显著上升,而在柴胡疏肝散与miR–124共同作用时该蛋白表达量显著下调,说明柴胡疏肝散可通过下调miR–124表达,进而恢复NDE1的正常表达。结论:本研究表明柴胡疏肝散可能经miR–124调控下游相关蛋白表达以及信号通路,从而影响神经细胞的发育、增殖和分化等功能。

Objective To explore the effects of the traditional Chinese medicine Chaihu-Shugan-San(CSS)on the regulation of miR–124 and the expression of related proteins in SH–SY5Y cells.To further clarify the specific mechanism and pathways of CSS in regulating nerve cells.Methods SH–SY5Y cells were cultured,transfected with pCDNA3.1–miR–124 plasmid,and intervention of CSS.Cell proteins were extracted and enzymatically digested,labeled with isobaric tags for relative and absolute quantification(iTRAQ),identified by LC–MS/MS,processed by software Proteome Discoverer1.3 Data,and further analyzed using bioinformatics tools.Western blot were performed to verify the differentially expressed proteins.Results The expression of miR–124 in SH–SY5Y cells was significantly increased after the miR–124 plasmid was transfected,and the exogenously expressed miR–124 was significantly decreased in the cells after the treatment of CSS.It shows that CSS can down-regulate the expression of miR–124 in SH–SY5Y cells.Proteomics research were identified a total of 15,487 peptides and 3707 proteins.Several proteins with the most significant differences in expression were verified by Western blot.The expression of NDE1 protein increased significantly when miR–124 was overexpressed,and the protein expression was significantly down-regulated and was restored the normal level when the CSS and miR–124 acted together.Conclusion This study shows that CSS may regulate downstream related protein expression and signaling cascades via miR–124,thereby affecting the development,proliferation and differentiation of nerve cells.