基于串联质谱标签的蛋白质组学联合高内涵筛选技术筛选胃癌相关基因的研究

Tandem mass tag-labeling proteomics combined with high-content screening technology reveal gastric cancer-related genes

目的:应用Tandem mass tag(TMT)技术联合高内涵筛选(HCS)寻找潜在的胃癌相关基因。方法:利用TMT技术从胃癌及癌旁组织中筛选差异表达蛋白质,应用GO和KEGG数据库进行注释和信号通路富集并结合PubMed数据库结果,筛选出胃癌中未见或较少报道的上调目的基因。通过shRNA技术敲减基因,HCS技术比较敲减对细胞增殖的影响,筛选出肿瘤增殖相关基因并通过检测mRNA含量、克隆形成和CCK8进行验证。结果:通过TMT从胃癌和癌旁组织定量到差异表达蛋白1008个(上调649个,下调359个),25个目的基因敲减后HCS结果显示SF3B4敲减抑制细胞增殖,验证发现SF3B4是潜在的胃癌细胞增殖相关基因。结论:TMT技术结合HCS为肿瘤相关基因的筛选提供新的可能性。本研究提示SF3B4是潜在的胃癌增殖相关基因,为研究胃癌发生机制提供新线索。

Objective:To screen for the gastric cancer-related genes by tandem mass tag(TMT)-labeling quantitative proteomics analysis combined with high-content screening(HCS).Methods:Proteins differentially expressed between gastric cancer tissues and para-cancer tissues were screened by TMT-based quantitative proteomics analysis.Then the differentiated expressed proteins(DEPs)were used for enrichment analysis of GO functions and the KEGG pathways.The target genes were selected by consulting the PubMed database with the results of GO and KEGG.After lentivirus-mediated shRNA technology was employed to specifically knockdown the selected target genes in the gastric cancer cell line AGS,HCS was performed to evaluate cell proliferation and screen proliferation-related genes.mRNA levels,clonogenic assay and counting kit-8(CCK8)assays were conducted to validated the genes.Results:A total of 1008 DEPs(649 up-regulated proteins and 359 down-regulated proteins)were identified in gastric cancer and para-cancer tissues by TMT proteomics.The cell proliferation experiments by HCS showed SF3B4(in 25 genes of interest)knockdown inhibited gastric cancer cell proliferation.Validations demonstrated that SF3B4 was a potential gene implicated in gastric cancer.Conclusion:TMT-labeling proteomics combined with HCS technology offers new possibilities for the screening of tumor-associated genes.This study suggests that SF3B4 was a potential gene related to gastric cancer cell proliferation,providing a new clue for the study of the pathogenesis of gastric cancer.