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

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2/3复合体亚单位3的基因ARPC3和氨酰tRNA生物合成关键基因QARS1的mRNA水平均显著降低。 结论  CHAF1B为心肌细胞存活的关键蛋白质，参与调控心肌细胞的胞吞和氨基酸生物合成等多种生物学过程，参考其调控网络可帮助寻找促进心肌细胞修复的干预环节。

Abstract

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 subunit 6B and 10B participated in the proteasome pathway; seryl-tRNA synthetase, glycine-tRNA synthetase, glutamine-tRNA synthetase and lysine-tRNA synthetase mediated aminoacyl tRNA biosynthesis. The significantly down regulated proteins, including actin-related protein 2/3 complex subunit 3 and heat shock 70 000 protein 1-like, participated in endocytosis; ribonucleoside-diphosphate reductase large subunit mediated pyrimidine metabolism. The real time quantitative polymerase chain reaction results confirmed that after transfection with CHAF1B siRNA, the mRNA levels of the gene ARPC3, which synthesized the skeletal related protein 2/3 complex subunit 3, and the key gene QARS1 for aminoacyl tRNA biosynthesis, were significantly reduced in cardiomyocytes. Conclusion  CHAF1B is a key protein for the survival of cardiomyocytes and participates in the regulation of various biological processes in cardiomyocytes. Referring to its regulatory network can help identify intervention steps that promote myocardial cell repair.

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

Key words

label-free protein quantitative mass spectrometry / CHAF1B / gene knock down