Department of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea.
Biochem Biophys Res Commun. 2013 Jun 28;436(2):152-5. doi: 10.1016/j.bbrc.2013.04.108. Epub 2013 May 29.
Small interfering RNAs not only modulate gene expression at a post-transcriptional level, but also induce transcriptional gene silencing by RNA interference-mediated heterochromatin formation and RNA-directed DNA methylation (RdDM). However, although established in plants, there have been controversies whether RdDM operates in mammals. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral RNA transcription, and transcriptional activity of HBV cccDNA is regulated by methylation in patients with chronic HBV infection. In this study, we stably expressed short hairpin RNA (shRNA) against HBV in human hepatoma cells to determine whether shRNA induces methylation of HBV cccDNA. HepAD38 cells which permit replication of HBV under control of tetracycline-responsive promoter were transduced with lentiviral vectors which encode sh-1580, a shRNA against the hepatitis B viral protein HBx. Bisulfite sequencing PCR analysis revealed that sh-1580 induced CpG methylations at a higher rate compared to control (31.3% vs. 12.8%, p<0.05). The sh-1580-induced CpG methylation was localized near the target sequence of sh-1580 in more than a half of the clones. Methylation-induced transcriptional suppression was confirmed by in vitro transcription assay. These results confirm the feasibility of RdDM of HBV cccDNA in human cells. Lentiviral vector-mediated transfer of shRNA may be used as a tool for novel transcriptional modulation by epigenetic modification of HBV cccDNA.
小干扰 RNA 不仅在转录后水平调节基因表达，还通过 RNA 干扰介导的异染色质形成和 RNA 指导的 DNA 甲基化（RdDM）诱导转录基因沉默。然而，尽管在植物中得到了证实，但 RdDM 是否在哺乳动物中起作用仍存在争议。乙型肝炎病毒（HBV）共价闭合环状 DNA（cccDNA）作为病毒 RNA 转录的模板，HBV cccDNA 的转录活性受慢性 HBV 感染患者中甲基化的调节。在这项研究中，我们在人肝癌细胞中稳定表达针对 HBV 的短发夹 RNA（shRNA），以确定 shRNA 是否诱导 HBV cccDNA 的甲基化。允许 HBV 在四环素反应性启动子控制下复制的 HepAD38 细胞被转导了编码针对乙型肝炎病毒蛋白 HBx 的 shRNA（sh-1580）的慢病毒载体。亚硫酸氢盐测序 PCR 分析显示，与对照相比，sh-1580 诱导 CpG 甲基化的速度更快（31.3%比 12.8%，p<0.05）。sh-1580 诱导的 CpG 甲基化在超过一半的克隆中位于 sh-1580 的靶序列附近。体外转录实验证实了甲基化诱导的转录抑制。这些结果证实了人类细胞中 HBV cccDNA 的 RdDM 的可行性。慢病毒载体介导的 shRNA 转移可作为通过 HBV cccDNA 的表观遗传修饰进行新型转录调节的工具。
