siRNA干扰SIAH对细胞活性和α-synuclein泛素化降解通路的影响  

Effect of siRNA Interference SIAH on Cell Activity and α-synuclein Ubiquitin Degradation Pathway

作　　者：徐晶[1] 张馨芝[1] 张永进[1] 李秀明[1] 王敏[1] 蔡增林[1] 李小民[1] 

机构地区：[1]徐州医学院附属连云港医院,江苏连云港222002

出　　处：《中国医学创新》2015年第27期1-5,共5页Medical Innovation of China

基　　金：江苏省自然基金面上项目(BK2011402)

摘　　要：目的:研究si RNA干扰SIAH基因对神经母细胞瘤细胞SH-SY5Y细胞α-synuclein泛素化、降解通路的影响。方法:用荧光标记的si RNA-FAM转染SH-SY5Y细胞后,专业设计合成3条针对SIAH的si RNA,Western Blot检测蛋白水平SIAH表达的变化,筛选出其中最高效的1条si RNA,用CCK-8法检测该si RNASIAH干扰SH-SY5Y细胞活性;流式细胞技术检测该si RNA-SIAH干扰SH-SY5Y细胞凋亡的影响;Western Blot检测α-synuclein、LC3、E1、P53蛋白水平表达的变化;q RT-PCR检测SIAH、α-synuclein、LC3、E1 m RNA水平表达的变化;免疫共聚焦分别检测SIAH、α-synuclein、LC3共定位情况。结果:3条si RNA均成功转染SH-SY5Y细胞,流式细胞术检测si RNA转染SH-SY5Y细胞转染率89%,Western结果显示其中si RNA-2#使细胞中SIAH蛋白表达明显降低,选取该序列进行研究。CCK-8法结果显示该si RNA干扰SIAH后对SH-SY5Y细胞活增高,流式细胞技术检测结果显示该si RNA干扰SIAH可抑制SH-SY5Y细胞的凋亡,与空白对照组、阴性对照组比较,差异均有统计学意义(P<0.05)。通过Western Blot检测si RNA-2#组的α-synuclein、LC3、P53蛋白水平,与空白对照组、阴性对照组比较均明显降低,E1蛋白水平增高,差异均有统计学意义(P<0.05)。通过q RT-PCR检测si RNA-2#组的SIAH、α-synuclein、LC3-Ⅱm RNA水平与空白对照组、阴性对照组比较均明显降低,而E1的m RNA水平明显升高,差异均有统计学意义(P<0.05)。结论:应用si RNA干扰SIAH基因,通过促进泛素-蛋白酶体系统作用减少SH-SY5Y中α-synuclein聚集,SIAH基因有可能成为帕金森病治疗中的一个新靶点。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.

关 键 词：帕金森病 E3泛素连接酶 Α-SYNUCLEIN 泛素化蛋白酶体 

分 类 号：R742.5[医药卫生—神经病学与精神病学]