CAJ | 학술논문

目的：研究siRNA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siRNA-FAM转染SH-SY5Y细胞后，专业设计合成3条针对SIAH的siRNA， 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siRNA，用CCK-8法检测该siRNA-SIAH干扰SH-SY5Y细胞活性；流式细胞技术检测该siRNA-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；qRT-PCR检测SIAH、α-synuclein、LC3、E1 mRNA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siRNA均成功转染SH-SY5Y细胞，流式细胞术检测siRNA转染SH-SY5Y细胞转染率89%，Western结果显示其中siRNA-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siRNA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siRNA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siRNA-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qRT-PCR检测siRNA-2#组的SIAH、α-synuclein、LC3-Ⅱ mRNA水平与空白对照组、阴性对照组比较均明显降低，而E1的mRNA水平明显升高，差异均有统计学意义（P<0.05）。结论：应用siRNA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。
목적：연구siRNA간우SIAH기인대신경모세포류세포SH-SY5Y세포α-synuclein범소화、강해통로적영향。방법：용형광표기적siRNA-FAM전염SH-SY5Y세포후，전업설계합성3조침대SIAH적siRNA， Western Blot검측단백수평SIAH표체적변화，사선출기중최고효적1조siRNA，용CCK-8법검측해siRNA-SIAH간우SH-SY5Y세포활성；류식세포기술검측해siRNA-SIAH간우SH-SY5Y세포조망적영향；Western Blot검측α-synuclein、LC3、E1、P53단백수평표체적변화；qRT-PCR검측SIAH、α-synuclein、LC3、E1 mRNA수평표체적변화；면역공취초분별검측SIAH、α-synuclein、LC3공정위정황。결과：3조siRNA균성공전염SH-SY5Y세포，류식세포술검측siRNA전염SH-SY5Y세포전염솔89%，Western결과현시기중siRNA-2#사세포중SIAH단백표체명현강저，선취해서렬진행연구。CCK-8법결과현시해siRNA간우SIAH후대SH-SY5Y세포활증고，류식세포기술검측결과현시해siRNA간우SIAH가억제SH-SY5Y세포적조망，여공백대조조、음성대조조비교，차이균유통계학의의（P<0.05）。통과Western Blot검측siRNA-2#조적α-synuclein、LC3、P53단백수평，여공백대조조、음성대조조비교균명현강저，E1단백수평증고，차이균유통계학의의（P<0.05）。통과qRT-PCR검측siRNA-2#조적SIAH、α-synuclein、LC3-Ⅱ mRNA수평여공백대조조、음성대조조비교균명현강저，이E1적mRNA수평명현승고，차이균유통계학의의（P<0.05）。결론：응용siRNA간우SIAH기인，통과촉진범소-단백매체계통작용감소SH-SY5Y중α-synuclein취집，SIAH기인유가능성위파금삼병치료중적일개신파점。
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.