MicroRNA-101 downregulates Alzheimer’s amyloid-β precursor protein levels in human cell cultures and is differentially expressed

Abstract

The full repertoire of regulatory interactions utilized by human cells to control expression of amyloid-β precursor protein (APP) is still undefined. We investigated here the contribution of microRNA (miRNA) to this regulatory network. Several bioinformatic algorithms predicted miR-101 target sites within the APP 3′-untranslated region (3′-UTR). Using reporter assays, we confirmed that, in human cell cultures, miR-101 significantly reduced the expression of a reporter under control of APP 3′-UTR. Mutation of predicted site 1, but not site 2, eliminated this reporter response. Delivery of miR-101 directly to human HeLa cells significantly reduced APP levels and this effect was eliminated by co-transfection with a miR-101 antisense inhibitor. Delivery of a specific target protector designed to blockade the interaction between miR-101 and its functional target site within APP 3′-UTR enhanced APP levels in HeLa. Therefore, endogenous miR-101 regulates expression of APP in human cells via a specific site located within its 3′-UTR. Finally, we demonstrate that, across a series of human cell lines, highest expression of miR-101 levels was observed in model NT2 neurons.

Introduction

Amyloid-β precursor protein (APP) is a transmembrane protein that undergoes proteolytic processing by secretase enzymes to liberate soluble fragments. Sequential cleavage by β-secretase (BACE1) and the γ-secretase complex releases both the secreted beta-form of APP (sAPPβ) and the amyloid-β (Aβ) peptide, the major component of insoluble amyloid plaques found in Alzheimer’s disease (AD) [1]. Given the hypothesized centrality of Aβ to AD pathogenesis [2], understanding the processes that govern expression of its parental molecule (APP) is an essential task.

The regulation of APP expression has been extensively characterized. Cis-elements and trans-acting factors controlling APP expression have been identified in the gene promoter, and mRNA 5′-untranslated region (5′-UTR), coding sequence (CDS), and 3′-UTR [3], [4], [5], [6], [7], [8], [9], [10], [11]. However, the complete regulatory network governing expression of APP is yet to be fully elucidated.

MicroRNAs (miRNAs) are short (∼22 nucleotides), non-coding RNAs that act to inhibit protein expression by interacting with specific recognition elements in the 3′-UTR of target transcripts. These recognition elements demonstrate near perfect complementarity to the 5′ end of miRNA, termed the seed sequence, while interaction with the 3′ end of miRNA requires less stringency [12]. The miRNA guides a ribonucleoprotein complex termed RNA-induced silencing complex (RISC) to the target site in the 3′-UTR where RISC mediates either translation repression or mRNA destabilization [13]. Dysregulation of miRNAs is known to contribute to disease. Indeed, specific miRNAs have been shown to be either up or down-regulated in AD [14], [15], [16], [17]. Certain miRNAs also participate in physiological regulation of APP levels [18], [19], [20], [21], [22]. However, the regulatory roles of many predicted miRNA target sites in APP 3′-UTR and quantitation of miRNA in different cell types have yet to be studied.

With this in mind, we sought to further characterize regulation of APP expression by miRNAs. Here we report that endogenous miR-101 regulates APP levels in human cell cultures through a specific site located in the APP 3′-UTR. We suggest that modulating miR-101 may represent a novel strategy for attenuating Aβ secretion and downstream pathogenic mechanisms underlying AD.