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RNA Interference-Mediated Inhibition of Wild-Type Torsin A Expression Increases Apoptosis Caused by Oxidative Stress in Cultured Cells

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Abstract

To assess RNAi mediated inhibition of the expression of wt-DYT1 on H2O2-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.

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Abbreviations

DYT1:

Dystonia type 1

DA:

Dopamine

ER:

Endoplasmic reticulum

KLC1:

Light chain subunit of kinesin-I

hWT:

human wild-type Torsin A

hMT:

human mutant Torsin A

LAP1:

Lamina-associated polypeptide 1

MPTP:

1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine

NE:

Nuclear envelope

RNAi:

RNA interference

shRNA:

small double-stranded hair RNA

siRNA:

small double-stranded interfering RNA

TUNEL:

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling

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Acknowledgments

This study was supported by the National Science Fund of China (No. 30400144).

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Authors and Affiliations

  1. Department of Neurology, West China Hospital, SiChuan University, 610041, Chengdu, Sichuan, China

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

  2. Neuromolecular Lab, West China Hospital, SiChuan University, 610041, Chengdu, Sichuan, China

    Bo Xiao

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  1. Xue-Ping Chen
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Correspondence to Hui-Fang Shang.

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Chen, XP., Hu, XH., Wu, SH. et al. RNA Interference-Mediated Inhibition of Wild-Type Torsin A Expression Increases Apoptosis Caused by Oxidative Stress in Cultured Cells. Neurochem Res 35, 1214–1223 (2010). https://doi.org/10.1007/s11064-010-0177-4

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