Identification of Novel Targets for miR-29a Using miRNA Proteomics

Abstract

MicroRNAs (miRNAs) are short regulatory RNA molecules that interfere with the expression of target mRNA by binding to complementary sequences. Currently, the most common method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and conservation. Such algorithms predict hundreds of targets for each miRNA, necessitating tedious experimentation to identify the few functional targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional miRNA targets. We used Stable Isotope Labeling by amino acids in cell culture (SILAC) based proteomics to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. Over-expression of miR-29a, a miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of a set of proteins. Gene Ontology based classification showed that a significant sub-set of these targets, including Voltage Dependent Anion Channel 1 and 2 (VDAC1 and VDAC2) and ATP synthetase, were mitochondrial proteins involved in apoptosis. Using reporter assays, we established that miR-29a targets the 3′ Untranslated Regions (3′ UTR) of VDAC1 and VDAC2. However, due to the limited number of proteins identified using this approach and the inability to differentiate between primary and secondary effects we conclude that miRNA-proteomics is of limited utility as a high-throughput alternative for sensitive and unbiased miRNA target identification. However, this approach was valuable for rapid assessment of the impact of the miRNAs on the cellular proteome and its biological role in apoptosis.

Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice

“…We tested the levels of chosen targets of the miRNA and did not find significant up-regulation of BACE1 or the other selected pro-apoptotic genes which have been reported to be targets of miR-29. However, we observed a highly significant up-regulation in the expression level of Vdac1, a mediator of apoptosis and a novel target of miR-29 identified in an miRNA-proteomics screen that we carried out previously (Bargaje et al 2012). In vitro, down-regulation of Vdac1 leads to partial rescue from neuronal death of cells transfected with LNA against miR-29.…”

“…We have recently reported the identification of a novel target of miR-29a/b, voltage-dependent anion-selective channel protein 1 (Vdac1) (Supplemental Fig. 1; Bargaje et al 2012). VDAC1 is an outer mitochondrial membrane protein porin implicated in apoptosis.…”

“…VDAC1 was identified in this screen and was shown to contain a target site in its 3 ′ UTR, although this target site is not predicted by miRNA target prediction programs-PicTar, Targetscan, or miRanda. We performed luciferase assays to experimentally show direct targeting of the Vdac1 3 ′ UTR by miR-29a in vitro (Bargaje et al 2012). We have earlier shown that VDAC1 is up-regulated in a cellular model of SCA17 in which N2a cells expressed aberrant TATA-binding protein with a pathogenic expansion of a polyglutamine repeat stretch (TBP-59Q) (Ghosh et al 2007).…”

Diverse roles of miR-29 in cancer (Review)

“…Recent studies have found that VDAC1 and VDAC2 are targets of miR-29a. Overexpression of miR-29a can lead to a high level of VDAC1 and VDAC2 and increases permeability of VDAC (70). Furthermore, as the classical pathway of apoptosis, miR-29 family members (miR-29a, miR-29b and miR-29c) increase p53 levels and induce apoptosis by a p53-dependent manner.…”

Heroin Use Promotes HCV Infection and Dysregulates HCV-Related Circulating microRNAs

“…Experimental evidence indicates that putative targets of miR-29 family include an anti-apoptotic protein, the myeloid leukemia cell differentiation protein (MCL1), an anti-inflammatory and anti-cancer gene (Mott et al 2007), Zinc finger protein 36 homolog (ZFP36) (Gebeshuber et al 2009), and another cancer related gene DNA methyltransferases DNMT3A and DNMT3B (Fabbri et al 2007). Moreover, Voltage-dependent anion channel genes VDAC1 and VDAC2 were also identified as targets of miR-29a in HEK293T cells (Bargaje et al 2012). Due to the crucial function of miRNA-29 family in both basic cellular processes and HCV related liver pathogenesis, we also observed the correlation of miRNA-29 levels and HCV infection.…”