【作者】 王燕；

【导师】 申红星；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2023， 硕士

【摘要】 背景:柯萨奇病毒B3(Coxsackievirus B3,CVB3)属Picornaviridae病毒科,肠病毒属,是引起病毒性心肌炎、无菌性脑膜炎和胰腺炎的重要病原体。然而,目前临床上治疗CVB3感染的策略十分有限。N6-甲基腺苷(N6-methyladenosine,m~6A)是RNA分子中最普遍的化学修饰,可通过调节mRNA及其编码蛋白的表达来影响基因功能。m~6A甲基化修饰的宿主细胞转录本在调节病毒复制和传染性方面有重要作用,但其在CVB3感染期间扮演的角色尚不清楚。目的:为阐明m~6A修饰的宿主转录本对CVB3感染的重要性,本研究以CVB3感染后m~6A修饰水平显著差异的基因LONP1为研究对象,拟在He La细胞(CVB3易感细胞系)内分析病毒感染期间LONP1 m~6A修饰动态变化的分子机制及其对转录表达的调节作用;并初步探讨m~6A修饰的LONP1对CVB3复制的影响及作用机制,为CVB3感染的诊断和治疗提供新的理论依据。方法:1.利用RNA甲基化免疫共沉淀-高通量测序(Methylated RNA immunoprecipitation sequencing,MeRIP-seq)技术筛选CVB3感染后m~6A修饰水平显著差异的宿主转录本—LONP1,并用SRAMP数据库对LONP1的潜在m~6A修饰位点进行定位,随后通过RNA甲基化免疫共沉淀-实时荧光定量聚合酶链反应(Methylated RNA immunoprecipitation-quantitative real time polymerase chain reaction,MeRIP-qRT-PCR)实验验证LONP1 mRNA的m~6A修饰情况以及CVB3感染前后LONP1的m~6A水平变化;同时分析CVB3感染前后LONP1的转录表达变化。2.构建LONP1野生型(LONP1 3’UTR WT)及m~6A位点突变型(LONP1 3’UTR m~6A-Mut)双荧光素酶载体,分析CVB3感染期间LONP1 m~6A甲基化修饰位点对其转录表达的调节作用。3.通过western blot、qRT-PCR、MeRIP-qRT-PCR和双荧光素酶实验综合分析CVB3感染期间m~6A相关蛋白介导LONP1甲基化修饰及转录表达的机制。4.通过RIP-qRT-PCR、qRT-PCR和western blot方法筛选与LONP1 mRNA结合的m~6A甲基化阅读蛋白,并利用放线菌素D(5μg/m L)分析CVB3感染对LONP1 mRNA和蛋白稳定性的影响,探讨病毒感染期间m~6A修饰调控LONP1转录表达的作用机制。5.过表达/沉默LONP1,通过细胞病变观察实验、western blot、病毒噬斑实验和荧光病毒实验分析LONP1对CVB3复制增殖的调控作用。6.利用western blot实验、细胞内铁离子含量测定试剂盒、ROS含量测定试剂盒分析CVB3感染期间LONP1对相关铁死亡指标的影响,进一步探讨LONP1影响CVB3复制及宿主细胞病变的分子机制。结果:1.LONP1 3’UTR区(2946bp处)存在高可信度的m~6A修饰位点,CVB3感染导致LONP1 mRNA的m~6A修饰水平显著下降(P<0.05);LONP1的mRNA及蛋白表达水平在CVB3感染7 h时明显升高(P<0.05)。2.CVB3感染可显著上调LONP1 3’UTR WT的荧光素酶活性(P<0.05),但对于LONP1 3’UTR m~6A-Mut的荧光素酶活性无明显影响(P>0.05)。3.CVB3感染显著抑制了甲基化酶WTAP的表达(P<0.05);沉默WTAP会降低LONP1的m~6A水平,升高LONP1的蛋白和mRNA水平,且荧光素酶实验显示沉默WTAP可显著上调LONP1 3’UTR WT的荧光素酶活性(P<0.05),但对于LONP1 3’UTR m~6A-Mut的荧光素酶活性无明显影响(P>0.05);CVB3感染增加了LONP1表达,但在CVB3感染的同时过表达WTAP则可使LONP1基本恢复到未感染时的水平。4.m~6A修饰的LONP1 mRNA可结合甲基化阅读蛋白YTHDF2;CVB3的感染增强了LONP1的稳定性,减缓了其在ActD处理时的衰变速率(P<0.05)。5.LONP1过表达可减轻CVB3所致的细胞病变,并抑制CVB3 VP1表达和噬斑形成,减弱CVB3-e GFP的荧光强度(P<0.05);而LONP1沉默则会加重CVB3所致的细胞病变,促进CVB3 VP1表达和噬斑形成,同时增强CVB3-e GFP的荧光强度(P<0.05)。6.在CVB3感染期间,过表达LONP1促进了铁死亡抑制基因GPX4的表达,抑制了铁死亡促进基因ACSL4的表达,同时降低细胞内铁离子含量和ROS含量(P<0.05);沉默LONP1则抑制了GPX4的表达,促进了ACSL4的表达,同时增加了细胞内铁离子含量和ROS含量(P<0.05)。结论:WTAP介导的m~6A修饰调节LONP1的稳定性,导致其在CVB3感染期间转录表达增加,表达增加的LONP1对细胞铁死亡和CVB3复制有抑制作用,这可能是一种由宿主启动的抵御病毒感染的自我保护机制。更多还原

【Abstract】 Background:Coxsackievirus B3(CVB3)belongs to the Picornaviridae family of viruses,a genus of enteroviruses,and is an important pathogen causing viral myocarditis,aseptic meningitis and pancreatitis.However,there are currently limited clinical strategies to treat CVB3 infection.N6-methyladenosine(m6A)is the most prevalent chemical modification of RNA molecules that affects gene function by regulating the expression of mRNA and its encoded proteins.m6 A modified host cell transcripts are important in regulating viral replication and infectivity,but their role in CVB3 infection is unclear.Objective:In order to elucidate the importance of m6 A modified host transcripts on CVB3 infection,this research takes LONP1,a gene with significantly different levels of m6 A modification after CVB3 infection,and analyses the molecular mechanism of dynamic changes in LONP1 m6 A modification during viral infection and the regulation of its transcriptional expression in He La cells(CVB3 susceptible cell line).Initially explores the effect of m6 A modified LONP1 on CVB3 replication and its action mechanism,which provides a new theoretical basis for the diagnosis and treatment of CVB3 infection.Methods:1.Methylated RNA immunoprecipitation sequencing(Me RIP-seq)was used to screen for host transcripts with significantly different levels of m6 A modification during CVB3 infection-LONP1,and the potential m6 A sites of LONP1 were localized by SRAMP database.Subsequently,the m6 A modification of LONP1 mRNA and the change of m6 A level of LONP1 before and after CVB3 infection were verified by methylated RNA immunoprecipitation-quantitative real time polymerase chain reaction(Me RIP-q RT-PCR)assay.The transcriptional expression of LONP1 was also analysed prior to and post CVB3 infection.2.A dual luciferase vector was constructed for LONP1 wild type(LONP1 3’ UTR WT)and m6 A site mutant(LONP1 3’ UTR m6A-Mut)to analyze the regulatory role of LONP1 m6 A methylation modification site on its transcriptional expression during CVB3 infection.3.Analysis of the mechanism of m6A-related protein-mediated LONP1 methylation modification and transcriptional expression during CVB3 infection via western blot,q RT-PCR,Me RIP-q RT-PCR and dual luciferase assays.4.Screening of m6 A methylated reading proteins bound to LONP1 mRNA by RIPq RT-PCR,q RT-PCR and western blot,and analysis of the effect of CVB3 infection on LONP1 mRNA and protein stability using actinomycin D(5 μg/m L).Exploring the mechanisms of m6 A modifications regulating LONP1 transcriptional expression during viral infection.5.Overexpression/silencing of LONP1 and analysis of the regulatory role of LONP1 on CVB3 replication and proliferation by cytopathic observation assay,western blot,viral phage spot assay and fluorescent virus assay.6.Analysis of the effect of LONP1 on relevant iron death indicators during CVB3 infection using western blot assay,intracellular iron ion content assay kit and ROS content assay kit to further explore the molecular mechanisms by which LONP1 affects CVB3 replication and host cell lesions.Results:1.CVB3 infection resulted in a significant decrease in the level of m6 A modification of LONP1 mRNA(P<0.05)and a highly plausible m6 A modification site in the LONP1 3’ UTR region(at 2946 bp).mRNA and protein expression levels of LONP1 were significantly increased at 7 h of CVB3 infection(P<0.05).2.CVB3 infection significantly increased the luciferase activity of LONP1 3’ UTR WT(P<0.05),but there was no significant effect on the luciferase activity of LONP1 3’ UTR m6A-Mut(P>0.05).3.CVB3 infection significantly inhibited the expression of the methylesterase WTAP(P<0.05).silencing WTAP decreased m6 A levels and increased protein and mRNA levels in LONP1,and luciferase assays showed that silencing WTAP significantly increased luciferase activity of LONP1 3’ UTR WT(P<0.05)but not the LONP1 3’ UTR m6A-Mut(P>0.05).CVB3 infection increased LONP1 expression,but overexpression of WTAP with CVB3 infection largely restored LONP1 expression.4.m6A-modified LONP1 mRNA bound the methylated reading protein YTHDF2;CVB3 infection enhanced the stability of LONP1 and slowed its decay rate upon ActD treatment(P<0.05).5.LONP1 overexpression attenuated CVB3-induced cytopathy and inhibited CVB3 VP1 expression and phage-spot formation,and attenuated the fluorescence intensity of CVB3-e GFP(P<0.05),whereas LONP1 silencing exacerbated CVB3-induced cytopathy and promoted CVB3 VP1 expression and phage-spot formation,while enhancing the fluorescence intensity of CVB3-e GFP(P< 0.05).6.During CVB3 infection,overexpression of LONP1 promoted the expression of the iron death suppressor gene GPX4 and inhibited the expression of the iron death promoter gene ACSL4,while decreasing intracellular iron ion content and ROS content(P<0.05);silencing LONP1 inhibited GPX4 but promoted ACSL4,while increasing intracellular iron ion content and ROS content(P<0.05).Conclusion:WTAP-mediated m6 A modification regulates the stability of LONP1,leading to its increased transcriptional expression during CVB3 infection.The increased expression of LONP1 suppresses cellular iron death and CVB3 replication,which is a possible self-protective mechanism initiated by the host against viral infection.更多还原