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

Jin Wook Kim, Yan Hong Zhang, Mark A Zern, John J. Rossi, Jian Wu

Research output: Contribution to journalArticle

34 Scopus citations

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 languageEnglish (US)
Pages (from-to)292-297
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume359
Issue number2
DOIs
StatePublished - 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

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