摘要
目的三结构域蛋白29(TRIM29)参与多种疾病的发生和发展,与部分DNA和RNA病毒复制密切相关,本研究对TRIM29与HBV复制及聚乙二醇干扰素(PEG-IFN)α-2b抗病毒作用之间的关系展开初步讨论。方法选取2021年10月—2022年6月在贵州医科大学附属医院感染内科门诊就诊的CHB患者64例,其中未治疗患者34例(CHB组),经PEG-IFN-α-2b治疗患者30例,体检中心30例健康志愿者作为对照(健康对照组)。收集志愿者年龄、性别、ALT、AST、TBil、DBil、HBV DNA和外周血单个核细胞(PBMC)。采用Hep G2和Hep G2.2.15细胞作为细胞模型,将TRIM29特异性过表达质粒或si RNA和对照转染至细胞;PEG-IFN-α-2b(0、10、100、1000和10000 U/mL)处理Hep G2细胞和Huh7细胞;TRIM29特异性si-RNA或阴性对照联合PEG-IFN-α-2b处理Hep G2.2.15细胞。ELISA检测HBs Ag和HBe Ag的浓度,q RT-PCR检测TRIM29和HBV RNA相对表达水平,Western Blot检测STING、p-TBK1、TBK1、p IRF3、IRF3、MX1和IFIT1蛋白表达情况,免疫共沉淀检测TRIM29与STING蛋白相互作用关系。正态分布的计量资料两组间比较采用成组t检验,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。计数资料两组间比较采用χ2检验或Fisher检验。结果CHB组患者外周血TRIM29表达明显高于健康对照组(P<0.001)。在细胞实验中,HBs Ag、HBe Ag和HBV RNA的表达均随TRIM29表达上调而升高,随TRIM29表达下调而降低(P值均<0.05)。TRIM29与STING互相结合,并通过蛋白酶降解STING,与对照组相比,过表达TRIM29对TBK1和IRF3总蛋白无明显变化,STING、p-TBK1和p-IRF3蛋白表达水平均降低(P值均<0.05)。处理Hep G2细胞和Huh7细胞的PEG-IFN-α-2b浓度越高,TRIM29蛋白和m RNA的表达水平越低(P值均<0.01)。CHB患者在PEG-IFN-α-2b治疗期间,TRIM29 mRNA表达水平逐渐降低,且早期应答组和无应答组间差异均有统计学意义(P值均<0.05)。在等量PEG-IFN-α-2b处理下,与对照相比,敲低TRIM29后Hep G2.2.15细胞的MX1和IFIT1蛋白表达水平明显增高(P值均<0.05)。在PEG-IFN-α-2b治疗早期,CHB患者PBMC中TRIM29表达逐渐降低。结论TRIM29靶向并降解STING,通过抑制STING-TBK1-IRF3信号通路促进HBV复制。TRIM29干扰PEG-IFN-α-2b的抗病毒作用,CHB患者PBMC中TRIM29的表达水平可能作为预测患者对PEG-IFN-α-2b治疗应答的指标。
Objective To preliminarily investigate the association of TRIM29 with HBV replication and the antiviral effect of pegylated interferonα-2b(PEG-IFN-α-2b),since TRIM29 protein is involved in the development and progression of a variety of diseases and is closely associated with the replication of some DNA and RNA viruses.Methods A total of 64 chronic hepatitis B(CHB)patients who attended the outpatient service of Department of Infectious Diseases,The Affiliated Hospital of Guizhou Medical University,from October 2021 to June 2022 were enrolled,among whom there were 34 treatment-naïve patients and 30 patients treated with PEG-IFN-α-2b,and 30 healthy volunteers in Physical Examination Center were enrolled as controls.Related data were collected,including age,sex,alanine aminotransferase,aspartate aminotransferase,total bilirubin,direct bilirubin,HBV DNA,and peripheral blood mononuclear cells(PBMCs).HepG2 and HepG2.2.15 cells were used as cell models and were transfected with TRIM29-specific overexpressed plasmid or siRNA and control plasmid.HepG2 cells and Huh7 cells were treated with PEG-IFN-α-2b(0,10,100,1000,and 10000 U/mL),and HepG2.2.15 cells were treated with TRIM29-specific siRNA or negative control combined with PEG-IFN-α-2b.ELISA was used to measure the concentrations of HBsAg and HBeAg;qRT-PCR was used to measure the relative expression levels of TRIM29 and HBV RNA;Western blot was used to measure the protein expression levels of STING,p-TBK1,TBK1,pIRF3,IRF3,MX1,and IFIT1;co-immunoprecipitation assay was used to observe the interaction between TRIM29 and STING protein.The independent-samples t test was used for comparison of normally distributed continuous data between two groups,and a one-way analysis of variance was used for comparison between multiple groups,with the least significant difference t-test for further comparison between two groups;the chi-square test or the Fisher’s exact test was used for comparison of categorical data between two groups.Results The CHB patients had a significantly higher expression level of TRIM29 in peripheral blood than the healthy controls(P<0.001).In cell experiments,the expression levels of HBsAg,HBeAg,and HBV RNA increased with the upregulation of TRIM29 expression and decreased with downregulation of TRIM29 expression(P<0.05).TRIM29 bound to STING and degraded STING via protease,and compared with the control group,there were no significant changes in the total protein levels of TBK1 and IRF3 after overexpression of TRIM29,while there were significant reductions in the expression levels of STING,p-TBK1,and p-IRF3(P<0.05).The protein and mRNA expression levels of TRIM29 decreased with the increase in the concentration of PEG-IFN-α-2b for the treatment of HepG2 and Huh7 cells(P<0.01).During the treatment with PEG-IFN-α-2b,the CHB patients had a gradual reduction in the mRNA expression level of TRIM29,and there was a significant difference between the early response group and the non-response group(P<0.05).In the context of treatment with an equal volume of PEG-IFN-α-2b,compared with the control group,there were significant increases in the protein expression levels of Mx1 and IFIT1 in HepG2.2.15 cells after TRIM29 knockdown(P<0.05).There was a gradual reduction in the expression of TRIM29 in CHB patients during the early stage of PEG-IFN-α-2b treatment.Conclusion TRIM29 targets and degrades STING and promotes HBV replication by inhibiting the STING-TBK1-IRF3 signaling pathway.TRIM29 interferes with the antiviral effect of PEG-IFN-α-2b,and the expression level of TRIM29 in PBMCs of CHB patients may be used as an indicator for predicting the response of patients to PEG-IFN-α-2b therapy.
作者
廖心
张宝芳
LIAO Xin;ZHANG Baofang(School of Clinical Medicine,Guizhou Medical University,Guiyang 550000,China;Department of Infectious Diseases,The Affiliated Hospital of Guizhou Medical University,Guiyang 550000,China)
出处
《临床肝胆病杂志》
CAS
北大核心
2024年第11期2191-2200,共10页
Journal of Clinical Hepatology
基金
国家自然科学基金地区基金(82060114)
贵州省卫健委2023年度科技基金项目(gzwkj2023-042)
贵州省卫生健康委员会2024年科技基金项目(gzwkj2024-010)。