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.
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.
Kim
Jin-Wook
JW
Department of Internal Medicine, Transplant Research Program, University of California, Davis Medical Center, Sacramento, CA 95817, USA.
Zhang
Yan-Hong
YH
Zern
Mark A
MA
Rossi
John J
JJ
Wu
Jian
J
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands; D019175:DNA Methylation; D000077209:Decitabine; D020868:Gene Silencing; D008099:Liver / Q000166:cytology*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D009711:Nucleotides; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D000077209:Decitabine; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D001374:Azacitidine