Qi Yanmei, Wang Xu, Guo Xihan
School of Life Sciences, The Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Yunnan Normal University, Kunming, Yunnan, China.
Yeda Institute of Gene and Cell Therapy, Taizhou, Zhejiang, China.
Front Aging Neurosci. 2024 Mar 4;16:1346978. doi: 10.3389/fnagi.2024.1346978. eCollection 2024.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid beta (Aβ) in brain. Mounting evidence has revealed critical roles of microRNAs (miRNAs) in AD pathogenesis; however, the miRNAs directly targeting (), which encodes the catalytic core subunit of γ-secretase that limits the production of Aβ from amyloid precursor protein (APP), are extremely understudied. The present study aimed to identify miRNAs targeting and its effect on Aβ production. This study first predicted 5 candidate miRNAs that may target ,through websites such as TargetScan, miRDB, and miRwalk. Subsequently, the targeting specificity of the candidate miRNAs towards PS1 was validated using dual-luciferase reporter assays. To investigate the regulatory effect of miR-3940-5p on gene expression based on its targeting of PS1, miR-3940-5p mimics or inhibitors were transiently transfected into SH-SY5Y cells. Changes in transcription and translation in the tested cells were detected using RT-qPCR and Western Blot, respectively. Finally, to explore whether miR-3940-5p affects Aβ production, SH-SY5Y APP cells overexpressing the Swedish mutant type of APP were transiently transfected with miR-3940-5p mimics, and the expression level of Aβ was detected using ELISA. The results are as follows: The dual-luciferase reporter assays validated the targeting specificity of miR-3940-5p for . Overexpression of miR-3940-5p significantly reduced the mRNA and protein levels of in SH-SY5Y cells. Conversely, inhibition of miR-3940-5p led to an increase in mRNA levels. Transfection of miR-3940-5p mimics into SH-SY5Y-APP cells resulted in a significant reduction in Aβ and Aβ. Lentiviral-mediated overexpression of miR-3940-5p significantly decreased the expression of and did not significantly affect the expression of other predicted target genes. Furthermore, stable overexpression of miR-3940-5p in SH-SY5Y-APP cells mediated by lentivirus significantly reduced the expression of and the production of Aβ and Aβ. Therefore, our study demonstrates for the first time the functional importance of miR-3940-5p in antagonizing Aβ production through specific and direct targeting of .
阿尔茨海默病（AD）是一种进行性神经退行性疾病，其特征是大脑中β淀粉样蛋白（Aβ）的积累。越来越多的证据表明，微小RNA（miRNA）在AD发病机制中起关键作用；然而，直接靶向编码γ-分泌酶催化核心亚基（该酶可限制淀粉样前体蛋白（APP）产生Aβ）的（）的miRNA却极少被研究。本研究旨在鉴定靶向（）的miRNA及其对Aβ产生的影响。本研究首先通过TargetScan、miRDB和miRwalk等网站预测了5种可能靶向（）的候选miRNA。随后，使用双荧光素酶报告基因检测法验证了候选miRNA对早老素1（PS1）的靶向特异性。为了基于miR-3940-5p对PS1的靶向作用来研究其对基因表达的调控作用，将miR-3940-5p模拟物或抑制剂瞬时转染至SH-SY5Y细胞中。分别使用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法（Western Blot）检测受试细胞中（）转录和翻译的变化。最后，为了探究miR-3940-5p是否影响Aβ的产生，将过表达瑞典突变型APP的SH-SY5Y APP细胞瞬时转染miR-3940-5p模拟物，并使用酶联免疫吸附测定法（ELISA）检测Aβ的表达水平。结果如下：双荧光素酶报告基因检测法验证了miR-3940-5p对（）的靶向特异性。miR-3940-5p的过表达显著降低了SH-SY5Y细胞中（）的mRNA和蛋白质水平。相反，抑制miR-3940-5p导致（）mRNA水平升高。将miR-3940-5p模拟物转染至SH-SY5Y-APP细胞中导致Aβ和Aβ显著减少。慢病毒介导的miR-3940-5p过表达显著降低了（）的表达，且未显著影响其他预测靶基因的表达。此外，慢病毒介导的SH-SY5Y-APP细胞中miR-3940-5p的稳定过表达显著降低了（）的表达以及Aβ和Aβ的产生。因此，我们的研究首次证明了miR-3940-5p通过特异性直接靶向（）来拮抗Aβ产生的功能重要性。
