基于label-free定量蛋白质组学方法筛选沉默CHAF1B基因后心肌细胞差异表达蛋白及调控网络分析  

Analysis of differentially expressed proteins and regulatory networks in cardiomyocytes after silencing CHAF1B based on label-free protein mass spectrometry

作　　者：康彦红 顾爱琴 张莹 黄帅 Kang Yanhong;Gu Aiqin;Zhang Ying;Huang Shuai(Department of Scientific Research,Xuanwu Hospital,Capital Medical University,Beijing 100053,China;Biomedical Innovation Center,Beijing Shijitan Hospital,Capital Medical University,Beijing Key Laboratory for Therapeutic Cancer Vaccines,Beijing 100038,China)

机构地区：[1]首都医科大学宣武医院科研处,北京100053 [2]首都医科大学附属北京世纪坛医院生物医学创新中心肿瘤治疗性疫苗北京市重点实验室,北京100038

出　　处：《首都医科大学学报》2024年第2期312-321,共10页Journal of Capital Medical University

基　　金：国家自然科学基金项目(82204418);首都医科大学附属北京世纪坛医院医院科研基金项目(2022-C01)。

摘　　要：目的分析沉默染色质装配因子1亚基B(chromatin assembly factor 1 subunit B,CHAF1B)基因后心肌细胞中差异表达蛋白,预测CHAF1B基因调控网络,为寻找促进心肌细胞修复的潜在治疗靶点提供参考。方法采用转染和蛋白质印迹法筛选沉默CHAF1B基因的有效小干扰RNA(small interfering RNA,siRNA)。应用有效siRNA沉默人源心肌AC16细胞CHAF1B基因后,采用细胞活力检测方法检测细胞活力;提取总蛋白质进行定量、还原、烷基化和胰蛋白酶裂解成肽段,利用高效液相串联质谱法鉴定肽段;搜索UniProt蛋白库筛选差异表达的蛋白质进行基因本体(Gene Ontology,GO)富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集和蛋白质互作网络(protein-protein interaction networks,PPI)分析。结果siRNA有效沉默CHAF1B基因后,心肌细胞存活明显受到抑制;label-free定量蛋白质组学方法鉴定结果显示,共有69个差异表达蛋白质,其中50个表达显著上调(差异倍数≥2,P<0.05),19个表达显著下调(差异倍数≤0.5,P<0.05)。GO分析显示,差异表达蛋白质主要参与大分子复合亚基体、细胞组分生物合成和组装等生物学过程,分布在细胞质和囊泡等区域,发挥蛋白质结合等分子功能。KEGG通路富集和PPI分析显示,差异表达蛋白质参与的信号通路包括蛋白酶体、氨酰tRNA生物合成、胞吞、嘧啶代谢和氨基酸生物合成等10条信号途径;表达显著上调的蛋白质如蛋白酶体亚单位A2和B7、26 S蛋白酶体调节亚单位6B和10B参与蛋白酶体途径,丝氨酸、甘氨酸、谷氨酰胺和赖氨酸tRNA合成酶介导氨酰tRNA生物合成;表达显著下调的蛋白质包括骨架相关蛋白2/3复合体亚单位3和热休克70蛋白1样参与胞吞作用,核糖核苷-二磷酸还原酶大亚基介导嘧啶代谢等通路。实时荧光定量聚合酶链式反应结果证实,转染CHAF1B siRNA后心肌细胞中合成骨架相关蛋白2/Objective To analyze differentially expressed proteins in cardiomyocytes after chromatin assembly factor 1 subunit B(CHAF1B)gene knockdown and predict the regulatory network,so as to provide reference for finding the potential therapeutic targets which can promote myocardial cell repair.Methods Cell transfection and Western blotting methods were used to screen effective small interfering RNA(siRNA).Effective siRNA was used to knock down CHAF1B in human cardiac AC16 cells and then cell viability was detected by cell counting kit-8 method.The total protein was extracted,quantified,reduced,alkylated and then cleaved into peptides by trypsin.The peptides were identified by liquid chromatography with tandem mass spectrometry method.Differentially expressed proteins were identified by searching UniProt protein resource.Gene Ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,and protein-protein interaction networks(PPI)analysis were conducted.Results The survival of cardiomyocytes was significantly inhibited after CHAF1B gene knockdown by effective siRNA;the identification results of label-free protein quantitative mass spectrometry showed that there were 69 differentially expressed proteins,of which 50 proteins were significantly up-regulated(fold change≥2,P<0.05)and 19 were significantly down regulated(fold change≤0.5,P<0.05).GO analysis showed that these proteins mainly participated in biological processes such as macromolecular composite subunit matrix,cell component biogenesis and assembly,mainly distributed in the cytoplasm,vesicles and other regions,and played molecular functions such as protein binding.KEGG pathway enrichment and PPI analysis showed that the differentially expressed proteins participated in 10 signaling pathways such as proteasome,aminoacyl tRNA biosynthesis,endocytosis,pyrimidine metabolism,and amino acid biosynthesis,etc.The significantly up-regulated proteins such as proteasome subunit alpha type-2 and beta type-7 as well as 26S proteasome regulatory subu

关 键 词：label-free定量蛋白质谱 染色质装配因子1亚基B 基因敲低 

分 类 号：R394[医药卫生—医学遗传学]