Kim Jin-Wook, Zhang Yan-Hong, Zern Mark A, Rossi John J, Wu Jian
Department of Internal Medicine, Transplant Research Program, University of California, Davis Medical Center, Sacramento, CA 95817, USA.
Biochem Biophys Res Commun. 2007 Jul 27;359(2):292-7. doi: 10.1016/j.bbrc.2007.05.080. Epub 2007 May 22.
Small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in plant and animal cells. RNA dependent DNA methylation (RdDM) accounts for TGS in plants, but it is unclear whether siRNA induces RdDM in mammalian cells. To determine whether stable expression of short hairpin siRNA (shRNA) induces DNA methylation in mammalian cells, we transduced rat hepatic stellate SBC10 cells with lentiviral vectors which encode an U6 promoter-driven shRNA expression cassette homologous to the transforming growth factor-beta receptor (TGFbetaRII) promoter region. Sequencing analysis of bisulfite-modified genomic DNA showed the methylation of cytosine residues both in CpG dinucleotides and non-CpG sites around the target region of the TGFbetaRII promoter in SBC10 cells transduced with the promoter-targeting lentiviral vector. In these cells, real-time RT-PCR showed a decrease in TGFbetaRII mRNA levels which were reversed by treatment with 5-aza-2-deoxycytidine. Our results demonstrate that recombinant lentivirus-mediated shRNA delivery resulted in the methylation of the homologous promoter area in mammalian cells, and this approach may be used as a tool for transcriptional gene silencing by epigenetic modification of mammalian cell promoters.
小分子干扰RNA（siRNA）可在植物和动物细胞中诱导转录基因沉默（TGS）。RNA依赖性DNA甲基化（RdDM）在植物中导致TGS，但尚不清楚siRNA是否在哺乳动物细胞中诱导RdDM。为了确定短发夹siRNA（shRNA）的稳定表达是否会在哺乳动物细胞中诱导DNA甲基化，我们用慢病毒载体转导大鼠肝星状细胞SBC10，该载体编码与转化生长因子-β受体（TGFbetaRII）启动子区域同源的U6启动子驱动的shRNA表达盒。亚硫酸氢盐修饰的基因组DNA的测序分析显示，在用靶向启动子的慢病毒载体转导的SBC10细胞中，TGFbetaRII启动子靶区域周围的CpG二核苷酸和非CpG位点中的胞嘧啶残基均发生了甲基化。在这些细胞中，实时RT-PCR显示TGFbetaRII mRNA水平降低，而用5-氮杂-2'-脱氧胞苷处理可使其逆转。我们的结果表明，重组慢病毒介导的shRNA传递导致哺乳动物细胞中同源启动子区域的甲基化，并且这种方法可作为通过对哺乳动物细胞启动子进行表观遗传修饰来实现转录基因沉默的工具。
