中国医学创新  2015, Vol. 12 Issue (27): 1-5    DOI: 10.3969/j.issn.1674-4985.2015.27.001
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Effect of siRNA Interference SIAH on Cell Activity and α-synuclein Ubiquitin Degradation Pathway
Affiliated Lianyungang Hospital of Xuzhou Medical College,Lianyungang 222002,China
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Abstract  Objective:To study the effect of siRNA-SIAH on α-synuclein autophagy and UPS degradation in SH-SY5Y.Method:After transfection of siRNA-FAM cells with fluorescent labeled SH-SY5Y, 3 siRNA were professional designed for SIAH,the protein level of SIAH expression was measured by Western Blot, one of the most efficient siRNA was selected.CCK-8 assay was used to detect the activity of siRNA-SIAH in SH-SY5Y cells. Flow cytometry was used to detect the apoptosis of siRNA-SIAH in SH-SY5Y cells.Western Blot was used to detect the protein level expression ofα-synuclein,LC3,E1,and P53.qRT-PCR was used to detect the level of mRNA in SIAH,α-synuclein,LC3 and E1. Confocal microscopy was used to detect the positioning of SIAH,α-synuclein and LC3-Ⅱ.Result: 3 siRNA cells successfully transfected SH-SY5Y,the transfection efficiency was 89%. The Western results showed that the expression of SIAH was significantly decreased in siRNA-2# cells, then studied this sequence. CCK-8 assay showed that the SH-SY5Y cell activity increased after siRNA interference, and the flow cytometry results showed that siRNA could inhibit the apoptosis of SH-SY5Y cells, which was significantly different from the control group and the negative control group(P<0.05).With Western Blot method,the protein levels of α-synuclein, LC3 and P53 in siRNA-2# group were significantly lower and E1 was higher than that in control group and negative control group, the differences were statistically significant(P<0.05).With qRT-PCR method,the mRNA level of SIAH,α-synuclein and LC3-Ⅱ mRNA in siRNA-2# group were significantly lower and E1 was higher than that in control group and negative control group,the differences were statistically significant(P<0.05).Conclusion: Using siRNA to interfere with SIAH gene, SIAH gene can be used as a new target in the treatment of Parkinson’s disease by promoting the ubiquitin proteasome system to reduce SH-SY5Y in α-synuclein.
Key wordsParkinson’s disease      Seven in absentia homolog(SIAH)      α-synuclein      Ubiquitin proteasome system     
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http://www.zgyxcx.com/EN/10.3969/j.issn.1674-4985.2015.27.001     OR     http://www.zgyxcx.com/EN/Y2015/V12/I27/1
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