基于iTRAQ技术探讨人胃癌细胞乙酰肝素酶调控的信号通路

目的:探讨人胃癌细胞中乙酰肝素酶(HPSE)调控的差异蛋白和信号通路,为以HPSE为靶点防治胃癌提供依据。方法:利用siRNA干扰技术,在乙酰肝素酶(HPSE)基因高表达的SGC7901细胞中转入干扰HPSE的慢病毒载体(LV-HPSE-RNAi),通过嘌呤霉素筛选出稳定株,利用qPCR和Western blot分别检测HPSE mRNA和蛋白表达;利用细胞划痕实验和Transwell侵袭实验测定细胞的迁移和侵袭能力;利用同位素标记的相对和绝对定量(iTRAQ)联合二维液相色谱串联质谱(2DLC-MS/MS)技术筛查差异蛋白,并进行生物信息学分析,对差异蛋白PKCa应用Western blot进一步验证。结果:人胃癌SGC7901细胞和沉默HPSE表达的ZSGC7901细胞对比检测出98个差异蛋白,并且富集在157条信号通路上。与肿瘤发生发展关系密切的有6条:细胞外基质和受体相互作用、局灶性黏附、PI3K-Akt信号通路、癌途径、癌中microRNAs、Wnt信号通路。且上调的FAK、ITGA、PKCa等蛋白和下调的PKA、CDK6等蛋白在通路中处于重要的位置。Western blot结果证明PKCa在沉默HPSE的ZSGC7901细胞中表现为上调,差异具有统计学意义(P<0.05),与蛋白质组学筛选结果一致。结论:HPSE在人胃癌细胞中调控的蛋白,参与细胞重要生物学过程、参与重要分子功能及重要信号途径,有望可以成为防治胃癌的新靶点。

Exploring heparinase-regulated signaling pathways in human gastric cancer cells based on iTRAQ technique

Objective:To explore the differential proteins and signaling pathways regulated by heparinase (HPSE) in human gastric cancer cells,and to provide a basis for the prevention and treatment of gastric cancer with HPSE as target.Methods:Using siRNA interference technique,lentiviral vector (LV-HPSE-Rnai) interfering with HPSE was transferred into SGC7901 cells with high expression of heparinase gene (HPSE).Stable strains were screened by purinomycin.HPSE mRNA and protein expression were detected by qPCR and Western blot,respectively.The cell migration and invasion ability were determined by cell scratch assay and Transwell invasion assay.Differential proteins were screened by isotope labeled relative and absolute quantification (iTRAQ) combined with two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) and analyzed by bioinformatics.The differential protein PKCa was further verified by Western blot.Results:A total of 98 differential proteins were detected in human gastric cancer SGC7901 cells and HPSE-silenced ZSGC7901 cells,and they were enriched in 157 signaling pathways.Six of them are closely related to tumor genesis and development:ECM-receptor interaction,focal adhesion,PI3K-Akt signaling pathway,pathway in cancer,microRNAs in cancer,and Wnt signaling pathway.Up-regulated FAK，ITGA and PKCa proteins and down-regulated PKA and CDK6 proteins are in important positions in the pathway.Western blot results showed that PKCa was up-regulated in HPSE silenced ZSGC7901 cells,and the difference was statistically significant（P＜0.05），which was consistent with the iTRAQ result.Conclusion:Proteins regulated by HPSE in human gastric cancer cells are involved in important biological processes,molecular functions and signal pathways,and are expected to become new targets for the prevention and treatment of gastric cancer.