A Mechanistic Study Of MiR-3940-5p’s Role In Reducing Amyloid-β Generation In SH-SY5Y Cells By Targeting PSEN1

Alzheimer’s disease（AD）is a neurodegenerative disease with insidious onset,characterized by progressive abnormal neuronal aging and death.The main pathological features of AD are the formation of neurofibrillary tangles by hyperphosphorylation of the microtubule-associated protein tau in nerve cells and the deposition of extracellular Aβas amyloid plaques.Although there are still many unsolved mysteries in the pathogenesis of AD,Aβaggregation to increase the risk of AD is still one of the current mainstream hypotheses.Considering that PSEN1 is the catalytic subunit ofγ-secretase,the rate-limiting enzyme of Aβcascade production,reducing Aβproduction by regulating PSEN1 is expected to be a therapeutic target for AD.Micro RNAs（miRNAs）are a class of 19-24nt endogenous non-coding RNAs that can target one or more genes to affect transcription or translation.Exploring specific miRNAs and their targeting effects on AD-related genes during the occurrence and development of AD can provide a new idea for AD risk prevention,occurrence and development.In this study,five candidate miRNAs that may target PSEN1 were first predicted through targetscan,miRDB,miRwalk and other websites.The PSEN1 3’UTR sequence was cloned into a dual-luciferase reporter vector,and the recombinant vector and candidate miRNA mimics were co-transfected into HEK 293 cells,and the dual-luciferase reporter assay verified the targeting of the tested miRNAs to PSEN1.The results suggested that among the candidate miRNAs,only the dual-luciferase activity of the miR-3940-5p group was significantly decreased compared with the control（P=0.008）,suggesting its targeting to PSEN1.Subsequently,the miR-3940-5p seed sequence in the 3’UTR of PSEN1 was point mutated and then constructed into the reporter vector,and there was no significant change in the dual luciferase activity,suggesting that the targeting effect of mutated miR-3940-5p and PSEN1 was lost,which inversely confirmed the targeting of miR-3940-5p to PSEN1.To explore the regulatory effect of miR-3940-5p on gene expression based on targeting PSEN1,the effect of miR-3940-5p on endogenous PSEN1 expression was analyzed in human neuroblastoma SH-SY5Y cells.After transient transfection of miR-3940-5p mimics or inhibitor into SH-SY5Y cells,the transcription and translation changes of PSEN1 were detected by RT-q PCR and Western Blot,respectively.The results showed that miR-3940-5p mimics significantly down-regulated PSEN1transcription（P<0.01）and translation（P<0.001）,while miR-3940-5p inhibitor significantly up-regulated PSEN1 transcription（P<0.05）,but not translation level（P=0.051）.In order to further confirm the targeted regulation of PSEN1 expression by miR-3940-5p,the SH-SY5Y cell line stably overexpressing miR-3940-5p was constructed by lentiviral packaging,and it was found that the transcription and translation levels of PSEN1 in this cell line were significantly lower compared with the control（P<0.001）.Finally,to explore whether miR-3940-5p affects the production of Aβ,we transiently transfected miR-3940-5p mimic into SH-SY5Y APP^swe cells overexpressing human Swedish mutant APP.ELISA analysis showed that miR-3940-5p mimic significantly decreased the contents of Aβ₄₂（P<0.01）and Aβ₄₀（P<0.01）in the test cells.The SH-SY5Y APP^swecells stably overexpressing miR-3940-5p were further constructed by lentiviral packaging.ELISA results showed that stably overexpressing miR-3940-5p could significantly reduce intracellular Aβ₄₂（P<0.001）and Aβ₄₀（P<0.001）generation amount.In order to verify the existence of the miR-3940-5p-PSEN1-Aβaxis,we used RNA interference to knock down the expression of PSEN1（si-PSEN1）in SH-SY5Y APP^swe cells.The results showed that si-PSEN1 group Aβ₄₂、Aβ₄₀ levels were significantly lower than those in the control（P<0.01,P<0.001）.It is suggested that miR-3940-5p is involved in Aβproduction by downregulating PSEN1.In conclusion,this study found and confirmed for the first time that miR-3940-5p can target and inhibit the expression of PSEN1 at the transcriptional and translational levels,thereby indirectly participating in the regulation of the production of Aβ₄₂ and Aβ₄₀.This study provides a reference for the dynamic regulation of AD pathological targets and the intervention of AD risk with the help of specific miRNAs.