Application of RNAi inducible TNFRI knockdown cells to the analysis of TNFα-induced cytotoxicity

Fumiyo Saito, Hirofumi Yokota, Yoshihisa Sudo, Yoshikuni Yakabe, Haruko Takeyama, Tadashi Matsunaga

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

RNA interference (RNAi) has become a popular tool for downregulating in many species including mammalian cells. Therefore, suppression of target genes in mammalian cultured cells using RNAi may represent an ideal alternative to knockout studies for understanding the molecular mechanisms of chemical toxicity. Here, we assessed the potential of RNAi mediated gene knockdown in HeLa and HepG2 cells to cytotoxicity studies. Tumor necrosis factor receptor I (TNFRI) was chosen as a target gene because its signaling has been implicated in xenobiotic-induced toxicity. We optimized the design and performance of a vector-based RNAi experiment and then investigated viability of both HeLa and HepG2 cells exposed to TNFα. In addition, we examined gene expression profile of TNFRI knockdown HeLa cells after TNFα treatment, and then protein expression levels for several apoptosis-related genes of the cells. In both HeLa and HepG2 cells, TNFα exposure resulted in significantly reduced susceptibility of the knockdown cells to the cytotoxicity as compared with those of mock-transfected cells. Furthermore, the gene expression profiling and western blotting revealed that several genes including apoptosis and/or NF-κB pathway were downregulated in the knockdown HeLa cells. These results suggest that downregulation of the TNFRI gene in both HeLa and HepG2 cells by RNAi participates in resistance to TNFα-induced cytotoxicity. Therefore, this study raises the possibility that RNAi-based gene silencing in mammalian cells may be a valuable tool for elucidating the relationships between phenotypic changes and target gene functions in response to xenobiotic-induced cytotoxicity. Further exposure study using xenobiotics needs to be done to validate the potential utility of RNAi technology.

Original languageEnglish
Pages (from-to)1343-1353
Number of pages11
JournalToxicology in Vitro
Volume20
Issue number8
DOIs
Publication statusPublished - 2006 Dec
Externally publishedYes

    Fingerprint

Keywords

  • Apoptosis
  • Cytotoxicity
  • DNA microarray
  • Knockdown
  • RNA interference
  • TNFRI

ASJC Scopus subject areas

  • Toxicology

Cite this