[Influence of inhibited gene expression of alpha 3 nicotinic acetylcholine receptor by RNA interference on anti-oxidation in SH-SY5Y cells].
OBJECTIVE
To investigate the neuroprotective function of alpha 3 nicotinic acetylcholine receptor (nAChR) by inhibiting the gene expression in human neuroblastoma (SH-SY5Y) cells using small interference RNA (siRNA).


METHODS
The siRNA coding oligonucleotide sequences targeting alpha 3 nAChR were designed and synthesized. The annealed product was cloned into pSilencer 3.1-H1 neo vector. The recombinant alpha 3 nAChR pSilencer 3.1-H1 neo vector was transfected into the SH-SY5Y cells. The stable clones were screened by G418 medium, and the levels of alpha 3 nAChR mRNA and protein were monitored by using real-time PCR and Western blotting, respectively. After the SH-SY5Y cells with siRNA treatment were exposed to 1 micromol/L Abeta(1-42), MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], SOD, GSH-px and the lipid peroxidation were measured by spectrophotometry.


RESULTS
Compared with the controls, the expression levels of mRNA and protein in the stable SH-SY5Y clone cells transfected with the recombinant alpha 3 nAChR pSilencer 3.1-H1 neo vector were decreased with inhibitory efficiency of 98% and 66%, respectively, the MTT reduction decreased; the product of lipid peroxidation was increased and the activities of SOD and GSH-px were decreased. Biologically, the gene expression inhibition of alpha 3 nAChR enhanced the toxicity induced by Abeta in SH-SY5Y cells.


CONCLUSIONS
The expression inhibition of alpha 3 nAChR as a result of recombinant alpha 3 nAChR siRNA can induce oxidative stress and improve the toxicity of Abeta on SH-SY5Y cells, indicating that alpha 3 nAChR may play a significant neuroprotective role in the pathogenesis of Alzheimer disease.
Tang
Zhi
Z
Department of Pathology and Molecular Biology, Guiyang Medical College, Guiyang 550004, China.
An
Yu
Y
Qi
Xiao-lan
XL
Xiao
Yan
Y
Shan
Ke-ren
KR
Guan
Zhi-zhong
ZZ
D016229:Amyloid beta-Peptides / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D009447:Neuroblastoma / Q000473:pathology*; D010084:Oxidation-Reduction / Q000187:drug effects; D010446:Peptide Fragments / Q000494:pharmacology; D034622:RNA Interference / Q000276:immunology*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D011978:Receptors, Nicotinic; C075222:amyloid beta-protein (1-42); D013482:Superoxide Dismutase