利用Label-free技术筛选MC3T3-E1细胞中miR-381-3p介导的靶蛋白和差异蛋白生物学功能分析

目的 比较小鼠前成骨细胞（MC3T3-E1）实验组（MC3-E1-in）与对照组（MC3-E1-NC-in）的蛋白质组学差异水平，以初步探究miR-381-3p所介导的靶蛋白和相关信号通路以及差异蛋白生物学功能分析。方法 通过细胞慢病毒转染构建实验模型，分为实验组与对照组；实时荧光定量PCR验证miR-381-3p在细胞中的转染效率；收集细胞样品并采用非标记定量蛋白质组学技术进行鉴定、筛选与分析；进行组间比较得到差异蛋白（differentially expressed proteins，DEPs)表达结果，绘制火山图与表达聚类分析图以及功能富集分析差异蛋白质谱，重点分析基因本体论(geneontology，GO)富集结果、KEGG 富集结果以及string-db软件预测可能的蛋白质-蛋白质相互作用结果，并对差异显著的蛋白质进行生物信息学整合分析。结果 共鉴定到4 764种差异表达蛋白，其中527种差异显著并具有统计学意义，表达上调的有357种，有170种则是表达下调；GO富集结果分析显示差异蛋白主要参与生物过程的跨膜转运、蛋白水解、细胞内蛋白质转运，还参与了细胞骨架、内质网、线粒体的分子功能过程以及参与钙离子结合、蛋白激酶活性、RNA结合等细胞组分过程。结论 己糖激酶2、驱动蛋白样蛋白22、cAMP调节的磷酸化蛋白19和脂滴包被蛋白2等差异蛋白可能参miR-381-3p调控肌肉-骨骼系统代谢疾病的发生发展过程。

Screening of miR-381-3p-mediated target protein and biological function analysis of differential protein in MC3T3-E1 cells using Label-free technology

Objective To compare the proteomics difference between the experimental group (MC3-E1-in) and the control group (MC3-E1-NC-in) of mouse preosteoblasts (MC3T3-E1), in order to explore the mediation of miR-381-3p Guide target protein and related signal pathways, as well as the biological function analysis of differential proteins. Methods In this experiment, an experimental model was constructed by cell lentivirus transfection, and grouped into an experimental group and a control group; real-time fluorescent quantitative PCR verified the miR-381-3p in cells Transfection efficiency; collected cell samples and used non-labeled quantitative proteomics technology for identification, screening and analysis; compared the expression results of differentially expressed proteins (DEPs) between groups, drew volcano maps and expression cluster analysis maps, and Functional enrichment analysis of differential protein profiles, focusing on the analysis of Gene Ontology (GO) enrichment results, KEGG enrichment results, and string-db software to predict possible protein-protein interaction results, and perform biological information on significantly different proteins Learn integrated analysis. Results Co-identified 4 764 differentially expressed proteins, of which 527 were significantly different and statistically significant, 357 were up-regulated, and 170 were down-regulated; GO enrichment analysis showed that differential proteins are mainly involved in transmembrane biological processes Transport, proteolysis, and intracellular protein transport are also involved in the molecular functional processes of the cytoskeleton, endoplasmic reticulum, and mitochondria, as well as the cellular component processes such as calcium ion binding, protein kinase activity, and RNA binding. Conclusion Differential proteins such as hexokinase 2, kinesin-like protein 22, cAMP-regulated phosphorylated protein 19 and lipid droplet coating protein 2 may be involved in miR-381-3p regulating the occurrence and development of metabolic diseases in the musculoskeletal system.