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细胞周期素D1对HBV转录和复制的影响
DOI: 10.3969/j.issn.1001-5256.2023.02.010
利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。
作者贡献声明：彭思雯、关贵文负责实验，撰写修改论文；张婷负责实验和论文的撰写及修改；鲁凤民、刘佳、陈香梅负责指导撰写文章28并最后定稿。鼓思雯与关贵文对本文贡献等同，同为第一作者。
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摘要:
目的 探究细胞周期素D1(cyclin D1，基因名CCND1)对HBV复制的影响及其机制。 方法 利用GSE84044数据集，采用Spearman秩相关分析HBV相关肝纤维化患者肝组织基因表达水平与血清HBV DNA载量之间的相关性。在HBV细胞复制模型中瞬时表达cyclin D1及cyclin D1持续激活突变体(T286A)蛋白，使用时间分辨免疫荧光及实时荧光定量PCR实验分别检测细胞培养上清液中的HBsAg、HBeAg及HBV DNA水平，Western blot检测细胞内HBV core蛋白，反转录-实时荧光定量PCR法检测细胞内HBV RNA，双荧光素酶报告基因实验检测cyclin D1对HBV基本核心启动子(BCP)活性的影响。利用GSE83148数据集，分析CCND1与HBV相关调控因子表达的相关性。正态分布的计量资料两组间比较采用独立样本t检验；非正态分布的计量资料两组间比较采用Mann-Whitney U检验。 结果 在GSE84044数据中，HBV相关肝纤维化患者的肝组织中有7个细胞周期调控基因与HBV DNA载量呈显著负相关(r值均＜-0.3，P值均＜0.05)，其中包括CCND1基因(r=-0.474，P＜0.001)。外源表达cyclin D1及cyclin D1 T286A突变体能降低HBV复制细胞模型培养上清液中的HBsAg、HBeAg及HBV DNA水平，以及细胞内core蛋白和HBV RNA水平；外源表达cyclin D1显著抑制了HBV BCP的转录活性；且慢性乙型肝炎患者肝组织中CCND1表达水平与抑制HBV复制的APOBEC3G(r=0.575，＜0.001)、SMC5(r=0.341，P＜0.001)和FOXM1(r=0.333，P＜0.001)表达呈显著正相关，而与HBV进入受体NTCP(r=-0.511，P＜0.001)和HBV复制正向调控转录因子HNF1α(r=-0.430，P＜0.001)表达呈显著负相关。在HepG2细胞中过表达cyclin D1降低HNF1α及NTCP转录水平。 结论 cyclin D1抑制HBV的转录和复制，可能与其下调HNF1α及NTCP表达有关。 Abstract:Objective To investigate the effect of cyclin D1 (with CCND1 as the gene name) on HBV replication and its potential mechanism. Methods With reference to GSE84044 dataset, the Spearman's rank correlation analysis was used to investigate the correlation between the expression levels of genes in liver tissue and serum HBV DNA load in patients with HBV-related liver fibrosis. Cyclin D1 and cyclin D1 T286A mutant were transiently expressed in the HBV cell replication model, and time-resolved immunofluorescence and quantitative real-time PCR were used to measure the levels of HBsAg/HBeAg and HBV DNA in cell culture supernatant; Western blot was used to measure the level of HBV core protein in cells; reverse-transcription quantitative real-time PCR was used to measure the level of HBV RNA in cells; dual-luciferase reporter assay was used to observe the effect of cyclin D1 on the activity of HBV basic core promoter (BCP). GSE83148 dataset was used to investigate the correlation between CCND1 and HBV-related regulatory factors. The independent samples t-test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. Results The analysis of GSE84044 data showed that 7 cell cycle genes were significantly negatively correlated with HBV DNA load in liver tissue of the patients with HBV-related liver fibrosis (all r < -0.3, all P < 0.05), which included the CCND1 gene (r=-0.474, P < 0.001). Exogenous expression of cyclin D1 and cyclin D1 T286A mutant reduced the levels of HBsAg, HBeAg, and HBV DNA in culture supernatant of the HBV replication cell model, as well as the levels of HBV core protein and HBV RNA in cells. Exogenous expression of cyclin D1 significantly inhibited the transcriptional activity of HBV BCP. The expression level of CCND1 in liver tissue of chronic hepatitis B patients was significantly positively correlated with the expression of APOBEC3G (r=0.575, P < 0.001), SMC5 (r=0.341, P < 0.001), and FOXM1 (r=0.333, P < 0.001) which inhibited HBV replication, while it was significantly negatively correlated with the expression of the HBV entry receptor NTCP (r=-0.511, P < 0.001) and HNF1α as the transcription factor for positive regulation of HBV replication (r=-0.430, P < 0.001). Overexpression of cyclin D1 in HepG2 cells reduced the transcriptional levels of HNF1α and NTCP. Conclusion Cyclin D1 inhibits HBV transcription and replication possibly by downregulating the expression of HNF1α and NTCP. -
Key words:
- Hepatitis B Virus /
- Cyclin D1 /
- Virus Replication /
- Gene Transcription
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图 1 肝组织基因表达与血清HBV DNA的相关性分析
注：a，GSE84044数据集中与患者HBV DNA载量相关的肝组织宿主基因，样本按照HBV DNA载量的高低从左到右排列，蓝色代表低表达，红色代表高表达；b，765个与HBV DNA载量显著相关基因和116个细胞周期基因的交集；c，肝组织中CCND1水平与血清HBV DNA载量之间的相关性分析。
Figure 1. Correlation analysis of host gene expression in liver tissue and serum HBV DNA level
图 2 外源表达cyclin D1降低HBV的复制水平
注：a、b，外源表达cyclin D1降低HepG2和Huh-7细胞HBV core蛋白的水平；c、d，外源表达cyclin D1下调HepG2和Huh-7细胞培养上清液中的HBsAg、HBeAg与HBV DNA水平。
Figure 2. Exogenous expression of cyclin D1 reduces the replication level of HBV
图 3 外源表达cyclin D1对HBV转录水平的影响
注：a，RT-qPCR检测cyclin D1和cyclin D1(T286A)过表达对HepG2细胞内HBV RNA的影响；b，双荧光素酶报告基因实验检测过表达cyclin D1和cyclin D1(T286A)对HBV BCP启动子活性的影响。
Figure 3. Effects of exogenous expression of cyclin D1 on HBV transcript level
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