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Abstract：
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 presenilin1 (PSEN1), 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 PSEN1 and its effect on Aβ production. This study first predicted 5 candidate miRNAs that may target PSEN1,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 PSEN1 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 APPswe 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 PSEN1. Overexpression of miR-3940-5p significantly reduced the mRNA and protein levels of PSEN1 in SH-SY5Y cells. Conversely, inhibition of miR-3940-5p led to an increase in PSEN1 mRNA levels. Transfection of miR-3940-5p mimics into SH-SY5Y-APPswe cells resulted in a significant reduction in Aβ42 and Aβ40. Lentiviral-mediated overexpression of miR-3940-5p significantly decreased the expression of PSEN1 and did not significantly affect the expression of other predicted target genes. Furthermore, stable overexpression of miR-3940-5p in SH-SY5Y-APPswe cells mediated by lentivirus significantly reduced the expression of PSEN1 and the production of Aβ42 and Aβ40. 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 PSEN1.
摘要：
阿尔茨海默病（AD）是一种进行性神经退行性疾病，其特征是淀粉样β（Aβ）在大脑中的积累。越来越多的证据揭示了microRNAs（miRNAs）在AD发病机制中的关键作用；然而，直接靶向前苯胺1（PSEN1）的miRNA，它编码γ-分泌酶的催化核心亚基，限制淀粉样前体蛋白（APP）产生Aβ，被极度低估了。本研究旨在鉴定靶向PSEN1的miRNA及其对Aβ产生的影响。这项研究首先通过TargetScan等网站预测了5种可能靶向PSEN1的候选miRNA，miRDB,还有miRwalk.随后，候选miRNA对PS1的靶向特异性使用双荧光素酶报告基因试验进行了验证.为了研究miR-3940-5p对基于其靶向PS1的基因表达的调节作用，将miR-3940-5p模拟物或抑制剂瞬时转染到SH-SY5Y细胞中。使用RT-qPCR和Western印迹检测测试细胞中PSEN1转录和翻译的变化，分别。最后,探讨miR-3940-5p是否影响Aβ的产生，用miR-3940-5p模拟物瞬时转染过表达瑞典突变型APP的SH-SY5YAPPswe细胞，ELISA法检测Aβ的表达水平。结果如下:双荧光素酶报告基因测定验证了miR-3940-5p对PSEN1的靶向特异性。miR-3940-5p的过表达显著降低了SH-SY5Y细胞中PSEN1的mRNA和蛋白水平。相反，抑制miR-3940-5p导致PSEN1mRNA水平增加。将miR-3940-5p模拟物转染到SH-SY5Y-APPswe细胞中导致Aβ42和Aβ40的显著减少。慢病毒介导的miR-3940-5p的过表达显著降低了PSEN1的表达,并且没有显著影响其他预测的靶基因的表达。此外，慢病毒介导的SH-SY5Y-APPswe细胞中miR-3940-5p的稳定过表达显着降低了PSEN1的表达以及Aβ42和Aβ40的产生。因此,我们的研究首次证明了miR-3940-5p在通过特异性和直接靶向PSEN1拮抗Aβ产生中的功能重要性.
