Short hairpin RNA causes the methylation of transforming growth factor-beta receptor II promoter and silencing of the target gene in rat hepatic stellate cells.

@article{citeulike:1346310, abstract = {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.}, address = {Department of Internal Medicine, Transplant Research Program, University of California, Davis Medical Center, Sacramento, CA 95817, USA; Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea.}, author = {Kim, Jin-Wook W. and Zhang, Yan-Hong H. and Zern, Mark A A. and Rossi, John J J. and Wu, Jian }, citeulike-article-id = {1346310}, doi = {10.1016/j.bbrc.2007.05.080}, issn = {0006-291X}, journal = {Biochem Biophys Res Commun}, keywords = {rnai, sirna}, month = {May}, posted-at = {2007-05-31 13:22:32}, priority = {2}, title = {Short hairpin RNA causes the methylation of transforming growth factor-beta receptor II promoter and silencing of the target gene in rat hepatic stellate cells.}, url = {http://dx.doi.org/10.1016/j.bbrc.2007.05.080}, year = {2007} }

TY - JOUR ID - citeulike:1346310 L3 - citeulike-article-id:1346310 TI - Short hairpin RNA causes the methylation of transforming growth factor-beta receptor II promoter and silencing of the target gene in rat hepatic stellate cells. JF - Biochem Biophys Res Commun AD - Department of Internal Medicine, Transplant Research Program, University of California, Davis Medical Center, Sacramento, CA 95817, USA; Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea. SN - 0006-291X N2 - 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. KW - rnai KW - sirna AU - Kim, Jin-Wook AU - Zhang, Yan-Hong AU - Zern, Mark A AU - Rossi, John J AU - Wu, Jian PY - 2007/05/22/ UR - http://dx.doi.org/10.1016/j.bbrc.2007.05.080 ER -