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-β receptor (TGFβRII) 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 TGFβRII promoter in SBC10 cells transduced with the promoter-targeting lentiviral vector. In these cells, real-time RT-PCR showed a decrease in TGFβRII 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.
Original language | English (US) |
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Pages (from-to) | 292-297 |
Number of pages | 6 |
Journal | Biochemical and Biophysical Research Communications |
Volume | 359 |
Issue number | 2 |
DOIs | |
State | Published - Jul 27 2007 |
Keywords
- DNA methylation
- Promoter
- Short hairpin RNA
- Small interfering RNA
- Transcriptional gene silencing
- Transforming growth factor-β receptor
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology