RNA interference-mediated inhibition of wild-type Torsin A expression increases apoptosis caused by oxidative stress in cultured cells

Xue Ping Chen, Xiao Hui Hu, Shu Hui Wu, Yang Wei Zhang, Bo Xiao, Hui Fang Shang

Research output: Contribution to journalArticle

Abstract

To assess RNAi mediated inhibition of the expression of wt-DYT1 on H 2O2-induced toxicity in NIH 3T3 cells and primary cortical neurons. To detect the function of wild-type Torsin A and the effect of SiRNA on the wt-DYT1 gene. The shRNA expression vector was constructed by ligating annealed complementary shRNA oligonucleotides into the down-stream of the human U6 promoter (PU6) of the RNAi-ready pSIREN-Shuttle vector. Then, the pSIREN-Shuttle-DYT1-shRNA cassette was ligated to Adeno-X Viral DNA to construct the recombinant adenoviral vector pAd-DYT1-shRNA. Cultured cerebral cortical neurons and NIH 3T3 cells were transfected with pAd-DYT1-shRNA and pSIREN-Shuttle-DYT1-shRNA. We evaluated NIH 3T3 cells and neurons in the presence of oxidative stress using a TUNEL assay under different conditions. The knockdown efficacy of the DYT1 was confirmed by real-time RT-PCR and Western Blot analysis. After exposure to H2O2, the quantity of NIH 3T3 cells transfected with pSIREN-Shuttle-DYT1-shRNA, which stained positively in the TUNEL assay, was significantly higher than the cells transfected with pSIREN-Shuttle-negative control-shRNA. (44.85 ± 1.81% vs. 8.98 ± 2.73%, t = 26.168). There were significantly more apoptotic neurons infected with pAd-DYT1-shRNA (45.63 ± 7.53%) than neurons infected with pAd-X-negative control-shRNA (17.33 ± 2.43%) (t = 9.816). The observed silencing of wild-type Torsin A expression by DYT1-shRNA was sequence-specific. RNAi-mediated inhibition of the expression of wild-type Torsin A increases apoptosis caused by oxidative stress. It is reasonable to consider that wild-type Torsin A has the capacity to protect cortical neurons against oxidative stress, and in the development of DYT1-delta GAG-dystonia the neuroprotective function of wild-type Torsin A may be compromised.

Original languageEnglish (US)
Pages (from-to)1214-1223
Number of pages10
JournalNeurochemical Research
Volume35
Issue number8
DOIs
StatePublished - Aug 1 2010

Keywords

  • Apoptosis
  • DYT1 gene
  • Dystonia
  • KLC1
  • SiRNA
  • Torsin A

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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