METTL3通过IGF2BP2依赖性方式参与m6A修饰调控HBV复制的机制  

METTL3 participates in m6A modification to regulate HBV replication through an IGF2BP2-dependent manner

作　　者：余玲 张自力 曾蓉 张节 王鹏 潘万龙[1] YU Ling;ZHANG Zili;ZENG Rong;ZHANG Jie;WANG Peng;PAN Wanlong(School of Basic Medical Sciences and Forensic Medicine,North Sichuan Medical College,Affiliated Hospital of North Sichuan Medical College,Nanchong,Sichuan Province,637100,China;Department of Geriatrics,Affiliated Hospital of North Sichuan Medical College,Nanchong,Sichuan Province,637100,China)

机构地区：[1]川北医学院基础医学与法医学院,四川南充637100 [2]川北医学院附属医院老年科,四川南充637100

出　　处：《陆军军医大学学报》2024年第5期442-449,共8页Journal of Army Medical University

基　　金：南充市市校科技战略合作专项(22SXQT0318)。

摘　　要：目的探究胰岛素样生长因子2 mRNA结合蛋白2(insulin like growth factor 2 mRNA binding protein 2,IGF2BP2)协助甲基转移酶样3(methytransferase like 3,METTL3)通过N6-甲基腺苷(N6-methyladenosine,m6A)修饰调控HBV复制的分子机制。方法以HBV稳定复制细胞系HepG2.2.15及其亲本细胞HepG2为模型,斑点杂交分析m6A修饰水平,RT-qPCR和Western blot检测METTL3、IGF2BP2表达。生物信息学及免疫共沉淀分析METTL3与m6A阅读蛋白及其相互作用。将METTL3质粒、METTL3 siRNA和(或)IGF2BP2 siRNA转染至HepG2.2.15细胞,分别记为OE-METTL3、si-METTL3、si-IGF2BP2、OE-METTL3+si-IGF2BP2、si-METTL3+si-IGF2BP2、Control组;qPCR检测HBV DNA、HBV rcDNA、HBV cccDNA拷贝数,RT-qPCR或Western blot检测HBV pgRNA、METTL3、IGF2BP2表达。结果与HepG2细胞相比,在HepG2.2.15细胞中m6A修饰富集,METTL3、IGF2BP2表达水平升高(P<0.05)。与Control组相比,在OE-METTL3组中m6A修饰富集增强,IGF2BP2表达水平升高(P<0.05),HBV复制相关指标(HBV DNA、HBV rcDNA、HBV cccDNA、HBV pgRNA)升高(P<0.01);在si-METTL3组中则相反。生物信息学分析及免疫共沉淀显示METTL3与IGF2BP2为互作蛋白。与Control组相比,在si-IGF2BP2组中m6A修饰富集减弱,METTL3表达水平降低(P<0.01),HBV复制相关指标降低(P<0.01)。在OE-METTL3+si-IGF2BP2组中,HBV复制相关指标较OE-METTL3组降低(P<0.001)。在si-METTL3+si-IGF2BP2组中,HBV复制相关指标较si-METTL3组、si-IGF2BP2组降低(P<0.05)。结论METTL3依赖IGF2BP2富集m6A通过增强HBV rcDNA转化为cccDNA,进而增强pgRNA逆转录复制病毒。Objective To investigate the molecular mechanism by which methytransferase like3(METTL3)cooperates with insulin like growth factor 2 mRNA binding protein 2(IGF2BP2)to regulate HBV replication through N6-methyladenosine(m6A)modifications.Methods HBV stably replicating cell line HepG2.2.15 and its source cells HepG2 were used as models.Spot hybridization was used to analyze m6A modification level,and RT-qPCR and Western blotting were utilized to detect the expression of METTL3 and IGF2BP2 at mRNA and protein levels.Bioinformatics analysis and co-immunoprecipitation assay were applied to analyze METTL3 and m6A reading proteins and their interactions.After METTL3 plasmid,METTL3 siRNA and/or IGF2BP2 siRNA were transfected into HepG2.2.15 cells,respectively,the cells were assigned into OE-METTL3 group,si-METTL3 group,si-IGF2BP2 group,OE-METTL3+si-IGF2BP2 group,si-METTL3+si-IGF2BP2 group,and the cells untreated were subjected as control group.Copy numbers of HBV DNA,HBV rcDNA,and HBV cccDNA were detected by qPCR,and the expression of HBV pgRNA,METTL3,and IGF2BP2 were measured with RT-qPCR or Western blotting.Results Compared with HepG2 cells,m6A modification was enriched,and the expression levels of METTL3 and IGF2BP2 were elevated in HepG2.2.15 cells(P<0.05).Compared with the control group,m6A modification enrichment was enhanced and IGF2BP2 expression level was elevated in the OE-METTL3 group(P<0.05),and HBV replication-related indexes(HBV DNA,HBV rcDNA,HBV cccDNA,and HBV pgRNA)were increased(P<0.01);and opposite phenomena were observed in the si-METTL3 group.Bioinformatics analysis and co-immunoprecipitation assay showed that METTL3 and IGF2BP2 were interacting proteins.Compared with the control group,m6A modification enrichment was attenuated and METTL3 expression level was reduced(P<0.01),and HBV replication-related indexes were decreased in the si-IGF2BP2 group(P<0.01).In the OE-METTL3+si-IGF2BP2 group,HBV replication-related indexes were reduced when compared with the OE-METTL3 group(P<0.01).In the si-METTL3+si-I

关 键 词：甲基转移酶样3 胰岛素样生长因子2 mRNA结合蛋白2 N6-甲基腺苷 乙型肝炎病毒 

分 类 号：R341[医药卫生—基础医学] R373.21R394.3