摘要:目的分析人 单核细胞株(THP-1)过表达人巨细胞病毒( HCMV)编码miR-US25-1-3p后蛋白质表达谱变化及价值。方法采用脂质体将 hemv-miR-US25-1-3p模拟物转染THP-1细胞构建hemv-miR-US25-1-3p过表达细胞株，RT-qPCR验证转染 效果;采用串联质谱标签标记联合液相色谱-串联质谱对hemv-miR-US25-1-3p 过表达后THP-1细胞蛋白质组学进行定量分析,生物信息学分析差异表达蛋白质生理病理功能;western blot验证重要差异蛋白表达变化。结果与对照相比， THP-1 细 胞过表达hcmv-miR-US25-1-3p后65种蛋白质呈现差异表达,其中17种上调,48种下调。GO、COG和KEGG注释及功能预测结果显示,差异表达蛋白主要参与炎症反应信号通路、代谢过程、细胞及遗传信息功能,与肿瘤、病毒性心肌炎、非酒精性脂肪 肝、原发性免疫缺陷病、类风湿关节炎、多种病原体感染、阿尔茨海默病、亨廷顿病等多种疾病相关。48种下调蛋白质中,文献调研和生物信息学分析发现，肿瘤坏死因子相关凋亡诱导配体(TRIL)和受激活调节正常T细胞表达和分泌因子(RANTES) 参与抗病毒及炎症反应,上述蛋白质下调表达经western blot 验证。结论HCMV 编码的hcmv-miR-US25-1-3p 能够影响宿主蛋白表达谱,并可能通过抑制TRAIL和RANTES表达,促进病毒潜伏、免于宿主细胞杀伤。
Abstract: Objective To analyze the changes and value of protein expression profile following overexpression of human cytomegalovirus (HCMV ) -encoded miR-US25- 1-3p in human mononuclear cell line( THP-1). Methods hemv-miR-US25- 1-3p mimics were transfected into THP-1 cells using liposomes to construct hemv -miR-US25-1-3p overexpressed cell line. The transfection effects were verified by RT-qPCR. The quantitative analysis for proteomics of THP-1 cell line after hemv-miR-US25-1-3p overexpression was performed by using tandem mass spectrometry labeling combined with liquid chromatography tandem mass spectrometry. The physiological and pathological functions of diferentially expressed proteins were analyzed by bioinformatics. The expression changes of the important differential proteins were verified by western blot. Results Compared with the controls, the hemv-miR-US25-1-3p overexpressed THP-1 cells showed differential expressions of 65 types of proteins, of which 17 were up-regulated and 48 were down-regulated. The results of annotation and functional prediction by GO, COG, and KEGG databases indicated that differentially expressed proteins were mainly involved in inflammatory response signaling pathways，metabolic processes, cellular and genetic information functions and associated with multiple diseases, e.g., cancer, viral myocarditis , nonalcoholic fatty liver disease, primary immunodeficiency disease, rheumatoid arthritis ,multiple pathogen infections, Alzheimer' s disease, Huntington's disease and others. The results of literature research and bioinformatics analysis found that tumor necrosis factor related apoptosis inducing ligand ( TRAIL) and activated regulatory normal T cell expression and secretion factors ( RANTES ) involved in antiviral and inflammatory responses among the 48 types of down-regulated proteins. The down-regulated expressions of the above proteins were verified by western blot. Conclusion HCMV encoded hemv-miR-US25-1-3p in THP-1 cells could affect the host protein expression profile, and may promote virus latency and escape from host cell killing by inhibi- ting the expression of TRAIL and RANTES.