C19orf12的功能及其致病突变体细胞毒性机制研究  

The Function of C19orf12 and the Cytotoxic Mechanism of Its Pathogenic Mutants

作　　者：闫润 陈丽婷 朱彦霖 唐霁瑶 王学敏 韩齐 YAN Run;CHEN Li-ting;ZHU Yan-lin;TANG Ji-yao;WANG Xue-min;HAN Qi(Department of Anatomy and Physiology,Shanghai Jiao Tong University School of Medicine,Shanghai,200025,China;Cixi Biomedical Research Institute,Wenzhou Medical University,Wenzhou,Zhejiang,315302,China;Department of Emergency and Critical Disease,Songjiang Hospital Affiliated to Shanghai Jiaotong University School of Medicine,Shanghai,201600,China)

机构地区：[1]上海交通大学医学院解剖学与生理学系,上海200025 [2]浙江温州医科大学慈溪生物医药研究院,浙江温州315302 [3]上海交通大学医学院附属松江医院急危重症医学科,上海201600

出　　处：《现代生物医学进展》2024年第24期4619-4626,共8页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(82171391)。

摘　　要：目的:本研究旨在探讨神经退行性疾病基因C19orf12在H1299细胞系中的作用,分析其致病突变体(p.Ala63Pro)的潜在影响。方法:通过逆转录-荧光定量PCR(RT-qPCR)和Western Blot分析C19orf12在Hela、A549、U2OS、H1299等细胞系中的表达水平。构建靶向C19orf12的shRNA慢病毒表达载体并在H1299细胞系中敲低C19orf12。利用数据独立采集(DIA)蛋白质组学分析敲低C19orf12对细胞蛋白质表达谱的影响,对差异表达蛋白进行蛋白质-蛋白质相互作用(PPI)网络分析,并使用MCODE工具筛选出关键的蛋白互作子网络。最后,通过免疫共沉淀分析C19orf12突变体的亚细胞定位和相互作用蛋白变化。结果:C19orf12在H1299细胞中的表达水平相对较高,其敲低后显著抑制细胞生长。蛋白质组学分析鉴定出216个显著差异表达的蛋白质,其中132个上调,84个下调。通过基因本体(GO)和京都基因与基因组百科全书(KEGG)分析发现,这些差异蛋白主要参与帕金森病、阿尔茨海默病、亨廷顿病等神经退行性疾病相关通路,以及线粒体氧化磷酸化、脂质代谢和心脏疾病相关通路。C19orf12致病突变体(p.Ala63Pro)与多个关键蛋白的互作能力丧失。结论:C19orf12在细胞生长、线粒体氧化磷酸化、脂质代谢和心脏疾病等过程中发挥关键作用,其突变可导致蛋白质互作网络的改变。Objective:This study aims to investigate the role of the neurodegenerative disease gene C19orf12 in the H1299 cell line and to analyze the potential impact of its pathogenic mutant(p.Ala63Pro).Methods:The expression levels of C19orf12 in various cell lines,including Hela,A549,U2OS,and H1299,were analyzed using Reverse Transcription Quantitative PCR(RT-qPCR)and Western Blot.An shRNA lentiviral expression vector targeting C19orf12 was constructed,and C19orf12 was knocked down in the H1299 cell line.Data-independent acquisition(DIA)proteomics was utilized to analyze the effect of C19orf12 knockdown on the cellular protein expression profile.Differentially expressed proteins were subjected to protein-protein interaction(PPI)network analysis,and the MCODE tool was used to identify key protein interaction sub-networks.Finally,immunofluorescence and co-immunoprecipitation were performed to analyze the subcellular localization and interaction changes of the C19orf12 mutant proteins.Results:C19orf12 was expressed at relatively high levels in H1299 cells and its knockdown significantly inhibited cell growth.Proteomic analysis identified 216 significantly differentially expressed proteins,with 132 upregulated and 84 downregulated.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses revealed that these differentially expressed proteins were mainly involved in pathways related to neurodegenerative diseases such as Parkinson's disease,Alzheimer's disease,and Huntington's disease,as well as mitochondrial oxidative phosphorylation,lipid metabolism,and heart disease-related pathways.The pathogenic mutant of C19orf12(p.Ala63Pro)lost the ability to interact with multiple key proteins.Conclusion:C19orf12 plays a key role in cell growth,mitochondrial oxidative phosphorylation,lipid metabolism,and cardiac disease,and its mutation can lead to alterations in protein interaction networks.

关 键 词：神经退行性疾病 C19orf12 SHRNA 蛋白质组学 PPI 

分 类 号：R3[医药卫生—基础医学] Q78[生物学—分子生物学] Q513