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

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.

Key words: label-free protein quantitative mass spectrometry, CHAF1B, gene knock down