UNASSIGNED: RIP and CLIP-qPCR were performed by HT22 in vitro to observe the mechanism of hnRNP A1 regulating the expression of key proteins in glycolysis. The RNA binding domain of hnRNP A1 protein in HT22 was inhibited by VPC-80051, and the effect of hnRNP A1 on glycolysis of HT22 was observed. Lentivirus overexpression of hnRNP A1 was used to observe the effect of overexpression of hnRNP A1 on glycolysis of Aβ25-35-injured HT22. The expression of hnRNP A1 in brain tissues of wild-type mice and triple-transgenic (APP/PS1/Tau) AD mice at different ages was studied by Western blot assay.
UNASSIGNED: The results of RIP experiment showed that hnRNP A1 and HK1 mRNA were significantly bound. The results of CLIP-qPCR showed that hnRNP A1 directly bound to the 2605-2821 region of HK1 mRNA. hnRNP A1 inhibitor can down-regulate the expression of HK1 mRNA and HK1 protein in HT22 cells. Overexpression of hnRNP A1 can significantly reduce the toxic effect of Aβ25-35 on neurons via the hnRNP A1/HK1/ pyruvate pathway. In addition, inhibition of hnRNP A1 binding to amyloid precursor protein (APP) RNA was found to increase Aβ expression, while Aβ25-35 also down-regulated hnRNP A1 expression by enhancing phosphorylation of p38 MAPK in HT22. They interact to form bidirectional regulation, further down-regulating the expression of hnRNP A1, and ultimately aggravating glycolytic dysfunction. Protein immunoblotting showed that hnRNP A1 decreased with age in mouse brain tissue, and the decrease was greater in AD mice, suggesting that the decrease of hnRNP A1 may be a predisposed factor in the pathogenesis of AD.
■通过HT22体外进行RIP和CLIP-qPCR,以观察hnRNPA1调节糖酵解关键蛋白表达的机制。通过VPC-80051抑制HT22中hnRNPA1蛋白的RNA结合域,观察hnRNPA1对HT22糖酵解的影响。用慢病毒过表达hnRNPA1观察过表达hnRNPA1对Aβ25-35损伤的HT22糖酵解的影响。采用Westernblot法研究了不同年龄野生型小鼠和三转基因(APP/PS1/Tau)AD小鼠脑组织中hnRNPA1的表达。
■RIP实验结果表明hnRNPA1和HK1mRNA显著结合。CLIP-qPCR结果显示hnRNPA1直接结合HK1mRNA的2605-2821区域。hnRNPA1抑制剂可以下调HT22细胞HK1mRNA和HK1蛋白的表达。hnRNPA1过表达可显著降低Aβ25-35通过hnRNPA1/HK1/丙酮酸通路对神经元的毒性作用。此外,抑制hnRNPA1与淀粉样前体蛋白(APP)RNA的结合被发现增加Aβ表达,而Aβ25-35也通过增强HT22中p38MAPK的磷酸化下调hnRNPA1的表达。它们相互作用形成双向调节,进一步下调hnRNPA1的表达,最终加重糖酵解功能障碍。蛋白免疫印迹显示小鼠脑组织hnRNPA1随年龄增长而降低,在AD小鼠中下降更大,提示hnRNPA1的降低可能是AD发病的一个易感因素。