O-GlcNAc糖基转移酶(OGT)基因表达的抑制对tau蛋白磷酸化作用的影响

The Inhibitory Effect of OGT Gene Expression on The Level of tau Phosphorylation

研究HEK293T细胞中O-GlcNAc糖基转移酶(OGT)基因表达抑制对与AD密切相关的tau蛋白磷酸化与糖基化修饰的影响.以OGT基因为靶点设计三段siRNA(OGT-siRNA1～3),用脂质体的方法转染OGT-siRNA1～3进入HEK293T细胞,通过RT-PCR检测OGT-siRNA1～3对OGT mRNA的抑制.将pEGFP/OGT与OGT-siRNA1～3共转染HEK293T细胞,24h后在倒置荧光显微镜下观察各组GFP/OGT的表达,根据GFP/OGT的表达量来评价OGT-siRNA1～3对OGT基因表达的抑制率.将筛选出的具有最佳沉默效果的OGT-siRNA与质粒pCI/tau441共转染HEK293T细胞,48h后Western blot检测tau蛋白糖基化与磷酸化修饰的变化.OGT-siRNA3在100nmol/L的终浓度时对OGT基因的抑制效率最高.与Mock组相比,对OGT基因mRNA及蛋白质水平的抑制率分别可达80.0%与51.3%左右.OGT基因表达的下调使tau蛋白糖基化水平下降,而磷酸化水平增高,证实tau蛋白的糖基化负调节其磷酸化,葡萄糖摄入减少或代谢降低可能在AD的发生发展中起关键作用.

To investigate the effect of OGT expression inhibited by RNAi on the alteration oftau phosphorylation and glycosylation level in HEK293T cells. The siRNAs targeting OGTgene （OGT-siRNAI-3） were designed and chemically synthesized. The OGT-siRNAI-3 were transfected into HEK293T cells via lipofectamine2000. The efficacy of RNA interference was detected by RT-PCR. The fluorescence of GFP/OGT was counted by fluorescence microscopy after pEGFP/OGT and OGT-siRNA1-3 cotransfected to HEK293T cells for 24 h. The level of tau phosphorylation and glycosylation were detected by Western blot after Plasmid pCI/tau441 and the siRNA cotransfected HEK293T cells for 48 h. OGT-siRNA3 （100 nmol/L） could effectively downregulate the expression ofOGT gene compared with Mock group （80.0% at mRNA level and 51.3% at protein level）. The level of phosphorylation at various sites of the tau was significantly upregulated and the glycosylation was downregulated following the inhibition of OGT gene expression. There is an apparent negative correlation between the modification of phosphorylation and glycosylation of tau protein, the deficient in glucose uptaken/metabolism may be an important pathogenesis of AD.