三结构域蛋白29（TRIM29）与HBV复制及聚乙二醇干扰素α-2b抗病毒作用的关联性分析

廖心; 张宝芳

doi:10.12449/JCH241111

三结构域蛋白29（TRIM29）与HBV复制及聚乙二醇干扰素α-2b抗病毒作用的关联性分析

DOI: 10.12449/JCH241111

廖心¹,
张宝芳^2, , ,

1.
贵州医科大学临床医学院，贵阳 550000
2.
贵州医科大学附属医院感染内科，贵阳 550000

基金项目:

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)

伦理学声明：本研究方案于2020年4月9日通过贵州医科大学附属医院伦理委员会审批，批号为2020208。所有研究对象入组前均签署了书面知情同意书。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：廖心负责课题设计，实验实施，收据收集和撰写论文；张宝芳负责课题设计，指导撰写文章并最后定稿。

详细信息

通信作者:
张宝芳， 312368145@qq.com （ORCID： 0000-0002-5396-540X）

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出版历程
- 收稿日期: 2024-02-19
- 录用日期: 2024-04-26
- 出版日期: 2024-11-25

Association of TRIM29 with HBV replication and the antiviral effect of pegylated interferon α-2b

Xin LIAO¹,
Baofang ZHANG^{2
, ,
,}

1.
School of Clinical Medicine，Guizhou Medical University，Guiyang 550000，China
2.
Department of Infectious Diseases，The Affiliated Hospital of Guizhou Medical University，Guiyang 550000，China

Research funding:

National Natural Science Foundation of China (82060114);

Science & Technology Fund Project of Guizhou Health Commission in 2023 (gzwkj2023-042);

The Science and Technology Fund Project of Guizhou Health Commission in 2024 (gzwkj2024-010)

More Information

Corresponding author: ZHANG Baofang， 312368145@qq.com （ORCID： 0000-0002-5396-540X）

摘要

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HepG2和HepG2.2.15细胞作为细胞模型，将TRIM29特异性过表达质粒或siRNA和对照转染至细胞；PEG-IFN-α-2b（0、10、100、1 000和10 000 U/mL）处理HepG2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HepG2.2.15细胞。ELISA检测HBsAg和HBeAg的浓度，qRT-PCR检测TRIM29和HBV RNA相对表达水平，Western Blot检测STING、p-TBK1、TBK1、pIRF3、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，HBsAg、HBeAg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HepG2细胞和Huh7细胞的PEG-IFN-α-2b浓度越高，TRIM29蛋白和mRNA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HepG2.2.15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。
- 乙型肝炎病毒 /
- 三结构域蛋白29 /
- 聚乙二醇干扰素α /
- 信号通路
Abstract: Objective To preliminarily investigate the association of TRIM29 with HBV replication and the antiviral effect of pegylated interferon α-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， 1 000， and 10 000 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.
- Hepatitis B Virus /
- Tripartite Motif-Containing Protein 29 /
- PEG-IFNα /
- Signaling Pathway

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图 1 TRIM29表达水平与HBV感染的关系

注： a，CHB组与健康对照组TRIM29 mRNA表达差异；b，HepG2和HepG2.2.15细胞TRIM29蛋白表达差异。

Figure 1. The relationship between TRIM29 and HBV infection

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图 2 过表达TRIM29对HBV复制的影响

注： a、c，Western Blot和qRT-PCR检测TRIM29转染效率；b、d，ELISA检测细胞培养基中HBsAg和HBeAg的浓度，qRT-PCR检测HBV RNA的相对表达水平。

Figure 2. Effect of TRIM29 overexpression on HBV replication and gene expression

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图 3 敲低TRIM29对HBV复制的影响

注： a、c，Western Blot和qRT-PCR检测TRIM29特异性si-RNA转染效率；b、d，ELISA检测细胞培养基中HBsAg和HBeAg的浓度，qRT-PCR检测HBV RNA表达水平。

Figure 3. Effect of TRIM29 knockdown on HBV replication and gene expression

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图 4 TRIM29对STING-TBK1-IRF3信号通路的调控作用

注： a，免疫共沉淀检测TRIM29和STING相互作用；b、c，qRT-PCR检测IFN-α mRNA表达水平；d、e，Western Blot检测STING、TBK1、IRF3、p-TBK1、p-IRF3蛋白水平。

Figure 4. Regulatory effect of TRIM29 on STING-TBK1-IRF3 signaling pathway

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图 5 PEG-IFN-α-2b对TRIM29表达的影响

注： a、b HepG2细胞；c、d，Huh7细胞。a、c，Western Blot检测TRIM29蛋白表达水平；b、d，qRT-PCR检测TRIM29 mRNA水平。

Figure 5. Effect of PEG-IFN-α-2b on TRIM29 expression

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图 6 TRIM29表达对PEG-IFN-α-2b的抗病毒作用的影响

Figure 6. Effect of TRIM29 expression on the antiviral effect of PEG-IFN-α-2b

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表 1 引物序列

Table 1. List of primers

引物	序列（5'-3'）
HBV RNA-F	TCTTGCCTTACTTTTGGAAG
HBV RNA-R	AGTTCTTCTTCTAGGGGACC
Hu-TRIM29-F	TGCGAGCTGCATCTCAAGC
Hu-TRIM29-R	GGTGCTATGATTCTTGTGCTCC
Hu-IFN-α-F	TGACCTCAAAGCCTGTGTGATG
Hu-IFN-α-R	AAGTATTTCCTCACAGCCAGCAG
Hu-GAPDH-F	CGGATTTGGTCGTATTGGG
Hu-GAPDH-R	TCTCGCTCCTGGAAGATGG

下载: 导出CSV

表 2 健康对照组和CHB组患者的一般资料比较

Table 2. Comparison of clinical characteristics between healthy and CHB patients

指标	健康对照组（n=30）	CHB组（n=34）	统计值	P值
年龄（岁）	38.2±10.4	33.8±5.0	t=0.451	0.328
男/女（例）	15/15	16/18	χ²=0.055	0.814
汉族/其他（例）	23/7	24/10	χ²=0.302	0.583
ALT（U/L）	19.4±4.8	17.4±4.9	t=1.702	0.094
AST（U/L）	17.3±3.3	18.4±3.3	t=-1.293	0.201
TBil（μmol/L）	6.2±3.7	8.6±2.7	t=-1.791	0.195
DBil（μmol/L）	3.4±0.8	4.6±1.6	t=-2.219	0.138

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表 3 一般临床资料

Table 3. Clinical characteristics of patients

指标	早期应答组（n=11）	早期无应答组（n=19）	t值	P值
女性（例）	7	12		>0.05
年龄（岁）	26.8±4.49	29.3±7.20	-2.041	0.643
HBV DNA （log₁₀ IU/mL）
0周	2.35±0.26	2.11±0.18	2.380	0.010
12周	1.33±0.10	1.84±0.23	2.598	<0.001
24周	1.09±0.16	1.56±0.27	-4.260	<0.001
ALT（U/L）
0周	23.64±3.70	24.09±4.00	-0.546	0.99
12周	59.78±6.30	36.25±5.73	8.127	<0.001
24周	33.66±6.74	45.68±4.96	-3.893	<0.001
TRIM29 mRNA
0周	1.57±0.43	1.32±0.75	2.07	0.047
12周	1.22±0.26	0.72±0.34	3.83	<0.001
24周	0.94±0.27	0.43±0.11	7.20	<0.001

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