shRNA介导黑素瘤A375细胞Cbl-b基因沉默前后蛋白质组学分析
Proteomics analysis in A375 melanoma cells before and after short hairpin RNA-mediated Cbl-b gene silencing
目的 分析shRNA介导黑素瘤A375细胞Cbl-b基因沉默前后差异表达蛋白.方法 使用非标记定量蛋白质组学技术鉴定分别用Cbl-b shRNA慢病毒载体(Cbl-b shRNA组)和对照慢病毒载体(对照组)转染的A375细胞的差异表达蛋白.采用生物信息学方法对筛选的差异蛋白进行基因本体富集及京都基因与基因组百科全书(KEGG)通路富集分析.Western印迹验证差异蛋白(EphA2、GSK3β)表达和Cbl-b shRNA沉默后两组p-AKT表达.采用SPSS 23.0软件进行统计分析,两组间蛋白丰度比较采用两样本t检验.基因本体及KEGG富集分析结果采用Fisher精确概率法检验.结果 共鉴定3 449种蛋白,筛选出Cbl-b shRNA组和对照组差异表达蛋白74个.与对照组相比,Cbl-b shRNA组52个蛋白表达上调,22个下调.差异蛋白基因本体富集分析发现前5位显著富集生物学过程为整合素介导细胞黏附、单一生物代谢过程、整合素介导细胞黏附调节、蛋白质靶向线粒体调节、核酸代谢过程;前5位显著富集分子功能为DNA结合、2,2铁硫簇合物结合、信号受体活性、钙黏蛋白结合、细胞黏附分子结合;前5位显著富集的细胞组分为核小体、DNA包装复合体、光感器连接纤维、DNA-蛋白质复合体、胞外区部分.京都基因与基因组百科全书通路富集分析发现,前5位与黑素瘤相关的显著富集通路包括叶酸生物合成、轴突导向、细胞外基质受体相互作用、黏合连接、Wnt信号通路.Western印迹检测显示,Cbl-b shRNA组EphA2相对表达水平降低(0.369,以对照组表达水平为1计算),GSK3β表达增加(3.524),该结果与蛋白质组学检测结果一致;p-AKT相对表达水平降低(0.453).结论 Cbl-b可能通过多种生物学通路参与黑素瘤发病;EphA2/PI3K/AKT信号通路可能是Cbl-b参与黑素瘤形成的重要机制之一.
更多Objective To analyze differentially expressed proteins in A375 melanoma cells before and after short hairpin RNA (shRNA)-mediated Cbl-b gene silencing.Methods The label-free quantitative proteomics approach was performed to identify differentially expressed proteins between A375 cells transfected with lentiviral vectors containing Cbl-b shRNA (Cbl-b shRNA group) and those with control lentiviral vectors (control group).Then,the properties of differentially expressed proteins were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis.Western blot analysis was conducted to determine the expression of differential proteins (EphA2 and GSK3β) and phosphorylated protein kinase (p-AKT) after shRNA-mediated Cbl-b gene silencing.Statistical analysis was carried out by t test of two independent-samples for comparison of protein expression abundance between the two groups with SPSS 23.0 software.Additionally,the results of GO and KEGG enrichment analysis were analyzed by Fisher's exact test.Results A total of 3 449 proteins were identified and quantified,and 74 of them were differentially expressed between the Cbl-b shRNA group and control group.Compared with the control group,52 proteins were up-regulated and 22 were down-regulated in the Cbl-b shRNA group.GO enrichment analysis of differential proteins revealed that the top five significantly enriched biological processes were integrin-mediated cell adhesion,single-organism metabolic process,regulation of integrin-mediated cell adhesion,regulation of protein-targeting mitochondria and nucleic acid metabolic process.The top five significantly enriched molecular functions included DNA binding,2-iron,2-sulfur cluster binding,signaling receptor activity,cadherin binding and cell adhesion molecule binding.The top five significantly enriched cell components included nucleosome,DNA packaging complex,photoreceptor connecting cilium,DNA-protein complex and extracellular region part.KEGG enrichment analysis demonstrated that the top five significantly enriched melanoma-related signaling pathways were folate biosynthesis,axon guidance,extracellular matrix-receptor interaction,adherens junction and Wnt signaling pathways.As Western blot analysis revealed,the Cbl-b shRNA group showed lower protein expression of EphA2 (0.369),but higher protein expression of GSK3β (3.524) compared with the control group (1),which were consistent with the results of proteomics analysis.Additionally,the protein expression of p-AKT was down-regulated in Cbl-b shRNA group (0.453) compared with the control group (1).Conclusion Cbl-b may be involved in the occurrence of melanoma through a variety of biological pathways,and the EphA2/PI3K/AKT signaling pathway may be one important pathway.
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