Changes in intracellular and extracellular proteins after ERGIC3 knockdown in lung cancer:Proteins interacting with ERGIC3,HORN,and FLNA

Objective:Endoplasmic reticulum-Golgi intermediate compartment 3(ERGIC3)promotes cell proliferation and metastasis in lung cancer,but its molecular mechanism is unclear.Methods:The GLC-82 cells were randomly divided into the ERGIC3i group and the negative control group.The cells were transfected with ERGIC3 siRNA or control siRNA in the groups,respectively.The ERGIC3-interacting proteins expressed in cells or extracellularly were isolated by the immunoprecipitation method and detected by isobaric tags for relative and absolute quantitation and liquid chromatography-tandem mass spectrometry.The differentially expressed proteins were determined by bioinformatic methods.Results:After ERGIC3 knockdown,88 extracellular differentially expressed proteins,41 upregulated and 47 down-regulated,were detected in the supernatant of cultured cells.Among 52 intracellular differentially expressed proteins,33 were up-regulated and 19 down-regulated.Cluster analysis showed that the extracellular differential proteins are mainly involved in Ca2+binding and transport and I-kappa B kinase/NF-kappa B signal transduction.The upregulated proteins are mainly involved in the biological process of H3-K27 and H3-K4 methylation in cells.Co-immunoprecipitation assay showed that proteins interacting with ERGIC3 were rich in cytoskeleton construction and RHO GTPases activated p21-activated kinases.The intersection of these two research methods shows that ERGIC3 interacts with HORN and filamin A(FLNA).Conclusion:Proteomic analyses reveal that ERGIC3 acts as a vesicle transmembrane protein on the distribution of various extracellular and intracellular proteins and regulates the extracellular and intracellular biological processes by specifically binding hornin(HORN)and FLNA proteins.These findings maybe provide new methods and ideas for ERGIC3 as a therapeutic target for lung cancer.

人ERGIC3基因稳定表达支气管上皮细胞株的建立与迁移能力

目的构建含ERGIC3基因真核表达载体,并转染至支气管上皮细胞株BEAS-2B,筛选并建立ERGIC3稳定表达的细胞株,为进一步研究ERGIC3在肺癌中的作用奠定基础。方法采用RT-PCR方法获取人类ERGIC3基因的ORF框的DNA序列,构建逆转录病毒载体pLXSN-ERGIC3,用PT-67细胞包装后获得病毒颗粒;用病毒颗粒感染BEAS-2B细胞,通过G418筛选获取整合了外源基因ERGIC3的单克隆化细胞;采用定量RT-PCR和Western blot对ERGIC3稳定表达的细胞株进行鉴定,并用划痕实验分析稳定转染细胞株的迁移能力和形态学变化。结果获得4个ERGIC3稳定表达的BEAS-2B细胞株,ERGIC3的表达量均远高于对照组细胞,分别为对照组细胞的45.3、48.8、41.5和28.2倍,且稳定转染细胞株的迁移能力也显著增强(P<0.05),但其形态学没有明显变化。结论成功构建了含ERGIC3基因真核表达载体pLXSNERGIC3;筛选出多个ERGIC3基因稳定转染的BEAS-2B细胞株,且迁移能力明显提高。

利用TCGA数据库研究ERGIC3在肺腺癌免疫微环境中的作用及其预后评价中的价值

目的通过数据挖掘分析ERGIC3在肺腺癌(LUAD)中的表达及其在肺腺癌微环境中的可能作用与临床意义。方法通过TIMER和HPA等网站了解肺腺癌患者和正常人中ERGIC3表达量的差异,并使用UALCAN来揭示ERGIC3与临床特征的关系,应用Kaplan-meier探讨ERGIC3的预后价值,利用STRING分析与ERGIC3表达相关联蛋白质相互作用网络关系,并通过WebGestalt对上述基因进行功能富集分析,采用TIMER算法检测评估了ERGIC3表达与肺腺癌中免疫细胞浸润水平的相关性,最后借助TargetScan、starbase和miRDB预测可能与ERGIC3结合的microRNA。结果ERGIC3在肺腺癌中表达显著增加,ERGIC3高表达的肺腺癌患者总生存期、首次进展生存期和进展后生存期均明显降低(P均<0.05)。PPI网络分析发现ERGIC3与ERGIC2、SEC13和SEC63等10个蛋白存在紧密联系。与ERGIC3相互作用的基因主要富集在Localization、Endomembrane system和Protein binding等细胞功能。ERGIC3在肺腺癌免疫微环境中,与多种肿瘤免疫细胞(CD8^(+)T细胞、CD4^(+)T细胞、巨噬细胞、嗜中性粒细胞和树突状细胞)浸润呈负相关(P均<0.05)。此外,hsa-miR-4731-5p可能通过靶向ERGIC3调控肺腺癌细胞增殖和迁移。结论ERGIC3在肺腺癌中异常高表达,免疫调节在ERGIC3高表达肺腺癌的发生发展中起到重要作用,ERGIC3可能是与肺腺癌预后相关的潜在生物标志物。