MICRO-RNA 221 INHIBITS ADAM10 PROTEIN EXPRESSION IN SH-SY5Y CELLS

Abstract

In Alzheimer's disease (AD) non-amyloidogenic pathway, A Disintegrin And Metalloprotease 10 (ADAM10) acts as α-secretase, avoiding amyloid β (Aβ) production. We propose to analyze the overexpression of miR-144–5p, miR-374 and miR-221 in SH-SY5Y cells and verify their ADAM10 protein expression correlation. The research project was approved by Brazil Ethics Platform (CAAE:02760312.0.0000.5504/112.543). Total blood of 21 AD subjects and 17 cognitively healthy elderly controls was collected and analyzed by RT-qPCR. Relative expression of approximately 700 miRNAs was performed and 21 miRNAs differentially expressed were validated. Among them, mir-144–5p, miR-374 and miR-221 were found to be downregulated in AD subjects compared to healthy controls. From this observation, we transiently transfected human neuroblastoma SH-SY5Y cells with mirVana™ mimics of three selected miRNAs. Cells were lysed and protein samples were analysed by western blotting using an anti-ADAM10 antibody. ADAM10 and endogen control (β-actin) protein expression were quantified and the adjusted volume was used to determine the arbitrary units (AU) of ADAM10 expression. ADAM10 protein levels are significantly decreased upon transient overexpression of miR-221 in SH-SY5Y cells, but not altered after overexpression of miR-144–5p and miR-374, indicating the specificity of miR-221 in the regulation of ADAM10 levels. These findings place the miR-221 as a potential blood-based biomarker for AD. Moreover, the inhibition of miRNA-221 can be a new potential therapeutic target for increasing ADAM10 in AD, thus avoiding Aβ production.