基于蛋白组学分析旋毛虫原肌球蛋白过表达对小细胞肺癌NCI-H446细胞蛋白组分的影响

Proteomics-based analysis of the effect of Trichinella spiralis tropomyosin on the protein fraction of NCI-H446 small cell lung cancer cells

目的研究旋毛虫原肌球蛋白(tropomyosin,Tm)过表达对人小细胞肺癌(small cell lung cancer,SCLC)NCI-H446细胞蛋白组分的影响。方法本研究通过串联质谱标记(Tandem mass tags,TMT)技术结合生物信息学分析旋毛虫Tm过表达对NCI-H446细胞蛋白组分的影响。比较旋毛虫Tm(Tm-pcDNA3.1)过表达和转染空载体(pcDNA3.1)48 h后NCI-H446细胞的蛋白组分,利用Uniprot数据库检索差异表达蛋白(Differentially expressed protein,DEPs)并对所得DEPs进行基因本体论(GO)富集分析。从STRING数据库获得DEPs相互作用数据,利用Cytoscape 3.9.0软件构建蛋白互作网络并筛选中心节点蛋白。通过Western blot对随机选取的中心节点蛋白NDRG1进行验证。结果旋毛虫Tm转染NCI-H446细胞48 h后,筛选出DEPs 70个,其中34个上调,36个下调;GO功能富集分析显示,DEPs主要涉及转录后的调控、免疫应答的调节等生物学功能;分析蛋白互作网络获得5个下调的中心节点蛋白:细胞周期蛋白G相关激酶(GAK)、组蛋白H3-7(H3-7)、辛普勒金Symplekin(SYMPK)、N-myc下游调节因子1(NDRG1)、丝氨酸/苏氨酸蛋白激酶N2(PKN2);Westrne blot结果显示旋毛虫Tm降低NCI-H446细胞中NDRG1的表达。结论旋毛虫TM能引起NCI-H446细胞蛋白组分的显著变化;旋毛虫Tm可能通过下调NCI-H446细胞中GAK、H3-7、SYMPK、NDRG1、PKN2的表达发挥其生物学作用。

The aim of this study was to investigate the molecular function and underlying mechanisms of Trichinella spiralis tropomyosin(Tm)overexpression on NCI-H446 small cell lung cancer(SCLC)cells.The differentially expressed proteins(DEPs)in NCI-H446 overexpressing Tm were separated with tandem mass tags(TMT)for relative and absolute quantitation spectrometry,and UniProt was used to determine protein identity.Gene ontology(GO)enrichment analysis was performed,and the protein-protein interaction(PPI)network of DEPs was constructed in Cytoscape 3.9.0 software.Hub proteins were detected from the PPI network.The expression of the differentially expressed protein NDRG1 in NCI-H446 cells was detected by western blotting,whose results were consistent with the proteomics findings.A total of 70 DEPs were identified with quantitative proteomics and TMT.Among them,34 DEPs were upregulated,and 36 were downregulated.According to GO analysis,the DEPs were involved primarily in post-transcriptional regulation and the modulation of immunological responses mediated by immunoglobulins.Examination of the PPI network revealed five hub proteins:Cyclin-G-associated kinase(GAK),Histone H3-7(H3-7),Symplekin(SYMPK),N-myc downstream-regulated gene 1(NDRG1),and Serine/threonine-protein kinase N2(PKN2).In NCI-H446 cells,Trichinella spiralis Tm prevents NDRG1 from being expressed.Thus,Trichinella spiralis Tm markedly changes the transcriptome of NCI-H446 cells.The biological functions of Trichinella spiralis Tm may rely on GAK,H3-7,SYMPK,NDRG1 and PKN2 as downstream targets in NCI-H446 cells.