miR-3940-5p reduces amyloid β production via selectively targeting PSEN1

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 APP^swe 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-APP^swe 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-APP^swe 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.

Keywords: Alzheimer’s disease; Aβ; PSEN1; SH-SY5Y cells; miRNAs.

Figure 1

miR-3940-5p specifically targets the 3′UTR of PSEN1. (A) Schematic representation of the experimental design. The wild-type (WT) PSEN1 3′UTR was cloned downstream to the renilla luciferase gene in psiCheck 2 vector to generate the reporter constructs. The HEK293 cells were transfected with the luciferase reporter constructs and with candidate miRNA (miR-9-5p, miR-302a-3p, miR-520c-3p, miR-3940-5p, or miR-4507-5p) or negative controls (NC). After 48 h, dual luciferase assay (B) was performed. As shown in (A), the PSEN1 3′UTR with mutated miR-3940-5p target site (MUT) was constructed into psiCheck 2 vector. Then, HEK293 cells were transfected with constructs containing WT or MUT 3′UTR and miR-3940-5p or NC. After 48 h, dual luciferase assay (C) was performed (n = 3, t test, data represent mean ± SEM., **p < 0.01).

Figure 2

miR-3940-5p inhibits the transcription and translation of PSEN1 in SH-SY5Y cells. (A) Transient transfection of miR-3940-5p mimics or inhibitors to SH-SY5Y cells results in miR-3940-5p overexpression or suppression. (B) miR-3940-5p mimics significantly reduce the mRNA level of PSEN1 and miR-3940-5p inhibitor increase the mRNA level of PSEN1. (C, D) Transfection with a miR-3940-5p mimic resulted in a significant decrease in PSEN1 protein levels, while addition of a miR-3940-5p inhibitor increased PSEN1 protein levels (n = 3, t test, data represent mean ± SEM., **p < 0.01, ***p < 0.001).

Figure 3

PSEN1 is down-expressed in cells stable-overexpressing miR-3940-5p. (A) After puromycin selection, the morphology of SH-SY5Y cells overexpressing miR-3940-5p were captured under brightfield and fluorescence microscopy. (B) The expression of miR-3940-5p was detected in miR-3940-5p overexpressing cells and control cells through RT-qPCR. (C) The expression of PSEN1 in overexpressing cells was quantified using RT-qPCR. (D, E) The expression level of PSEN1 was detected in miR-3940-5p overexpressing cells through western blot. (F) The expression of TENM3, CPNE8, ATXN1, CUL7, and KCNA5 was quantified using RT-qPCR in SH-SY5Y cells overexpressing miR-3940-5p (n = 3, t test, data represent mean ± SEM., *p < 0.05,**p < 0.01, ***p < 0.001).

Figure 4

miR-3940-5p inhibits the production of Aβ in SH-SY5Y-APP^swe cells. (A) RT-qPCR result shows that the APP is overexpressed in SY5Y-APP^swe cells. (B) A comparison of the Aβ₄₀ and Aβ₄₂ concentrations in SH-SY5Y-APP^swe cells and SH-SY5Y cells. (C) miR-3940-5p and PSEN1 siRNA reduce the production of Aβ in SH-SY5Y-APP^swe cells. (D) The ratio of Aβ₄₂ to Aβ₄₀ in cells treated by miR-3940-5p mimics and PSEN1 siRNA. (E) After puromycin selection, the morphology images of SH-SY5Y-APP^swe cells overexpressing miR-3940-5p were captured under brightfield and fluorescence. After overexpression of miR-3940-5p in SH-SY5Y-APP^swe cells, the production of Aβ₄₀ and Aβ₄₂ (F), and the ratio of Aβ₄₂ to Aβ₄₀ (G) were measured in this cell line (n = 3, t test, data represent mean ± SEM., ***p < 0.001).