FAdV-4通过lnc87774-MDM2-p53通路诱导鸡肝细胞凋亡的分子机制  

Molecular Mechanism of FAdV-4 Inducing Apoptosis of Chicken Hepatocytes through lnc87774-MDM2-p53 Pathway

作　　者：肖雅清 董雯雯 袁小远[1] 李桂明[1] 刘明超 孟凯[1] Xiao Yaqing;Dong Wenwen;Yuan Xiaoyuan;Li Guiming;Liu Mingchao;Meng Kai(Poultry Research Institute,Shandong Academy of Agricultural Sciences,Jinan 250100,China;College of Veterinary Medicine,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]山东省农业科学院家禽研究所,山东济南250100 [2]河北农业大学动物医学院,河北保定071000

出　　处：《山东农业科学》2025年第1期142-150,173,共10页Shandong Agricultural Sciences

基　　金：山东省自然科学基金项目(ZR2021MC060);山东省农业科学院农业科技创新工程项目(CXGC2023A10,CXGC2023A22);山东省重点研发计划项目(2022LZGC014);泰山产业领军人才工程专项(tscx202306046);山东省农业良种工程课题(2022LZGC013)。

摘　　要：由禽腺病毒4型(FAdV-4)引起的禽心包积液性肝炎综合征近年来在我国频频发生,给我国家禽养殖业造成了巨大损失。长链非编码RNA(lncRNA)是病毒感染期间宿主免疫反应的关键调节因子,为揭示其在FAdV-4感染时对宿主细胞的作用及机理,本研究以鸡肝癌细胞系LMH为对象,通过芯片测序、生物信息学分析及Real-time PCR等方法筛选病毒感染过程中的关键lncRNA分子,并通过荧光定位杂交、编码-非编码基因共表达(CNC)分析、Real-time PCR、酶联免疫吸附(ELISA)、RNA pull-down和蛋白免疫共沉淀(RIP)、流式细胞分析等分子生物学手段对其进行亚细胞定位及互作蛋白的筛选、验证和功能分析。结果表明,筛选出了FAdV-4感染引起的关键lncRNA分子lnc87774,其主要定位在细胞质,其靶向结合互作蛋白为E3泛素连接酶MDM2,过表达lnc87774,MDM2和Bcl-2的相对表达量显著上升,p53和Bax的相对表达量显著下降,显著抑制细胞凋亡;沉默lnc87774,上述基因表现出相反的变化趋势且显著促进细胞凋亡;进一步沉默MDM2后,p53的表达量显著上调,但同时过表达/沉默lnc87774对FAdV-4引起的细胞凋亡没有影响,表明FAdV-4感染LMH细胞过程中lnc87774通过MDM2靶向调控p53介导的细胞凋亡进程。该研究结果将增加我们对FAdV-4与宿主细胞互作的认识,为宿主抗病毒防御机制提供理论支持。Pericardial effusion hepatitis syndrome caused by fowl adenovirus serotype 4(FAdV-4)has occurred frequently in China in recent years,causing huge losses to China’s poultry industry.The lncRNA is a key regulator of host immune response during viral infection.In order to reveal its effect and mechanism on host cells during FAdV-4 infection,the chicken liver cancer cell line LMH was used as object in this study.The key lncRNA molecules during viral infection were screened by chip sequencing,bioinformatics analysis and Real-time PCR.Subcellular localization analysis and interaction protein screening,verification and functional analysis were performed by fluorescence localization hybridization,coding-non-coding gene co-expression analysis(CNC),real-time PCR,enzyme-linked immunosorbent assay(ELISA),RNA pull-down and protein immunoprecipitation(RIP),flow cytometry and other molecular biological methods.The results showed that the key lncRNA molecule lnc87774 caused by FAdV-4 infection was screened out,which was mainly located in cytoplasm,and its target binding interaction protein was E3 ubiquitin ligase MDM2.Overexpression of lnc87774,the relative expression of MDM2 and Bcl-2 increased significantly,and that of p53 and Bax decreased significantly,which significantly inhibited cell apoptosis.Silencing lnc87774,the above genes showed the opposite change trend and significantly promoted cell apoptosis.After further silencing MDM2,the expression of p53 was significantly up-regulated,but overexpression/silencing of lnc87774 had no effect on FAdV-4-induced cell apoptosis,indicating that lnc87774 targeted p53-mediated cell apoptosis through MDM2 during FAdV-4 infection of LMH cells.The results of this study could increase our understanding of the interaction between FAdV-4 and host cells and provide theoretical support for the host antiviral defense mechanism.

关 键 词：禽腺病毒4型 长链非编码RNA 蛋白互作 细胞凋亡 先天性免疫 致病机理 

分 类 号：S858.31[农业科学—临床兽医学]