Abstract 1: Role of MicroRNAs in Postranscriptional Regulation of Apolipoprotein B-100 mRNA

Abstract

Hepatic apolipoprotein B-100 (apoB) synthesis and secretion appears to be regulated largely at the posttranscriptional and posttranslational levels. MicroRNAs (miRNAs) are among posttranscriptional regulators of gene expression that bind to complementary sequences on target messenger RNA (mRNA) transcripts, usually resulting in translational repression or degradation. It is unknown whether specific miRNAs are involved in posttranscriptional regulation of apoB mRNA. We performed bioinformatic analysis, showing that two specific miRNAs with satisfactory E-values level (with levels indicating greater similarity between the input and its match) namely, miR-544 (E-value = 0.91) and miR-1202 (E-value=0.86) have potential to interact with 3’ and 5’ UTR of apoB, respectively. We hypothesized that the interaction of these specific miRNAs (miR-544 and miR-1202) with the 3’ and 5’UTR of apoB mRNA leads to apoB mRNA translational repression and/or activation. Using a human hepatoma cell line model, HepG2, the effects of overexpressed miRNAs and inhibition of endogenous miRNAs on the expression of apoB mRNA and apoB protein synthesis were investigated. We further examined the effect of these miRNAs on apoB mRNA traffic into cytoplasmic P-bodies. Transfection of HepG2 cells with miR-544 led to a significant reduction in apoB mRNA expression and protein synthesis and induced an increase in the co-localization of apoB mRNA into P-bodies. The opposite effect was observed when anti-miR-544 was employed to inhibit the endogenous miR-544. Results from luciferase reporter assays indicated that the effects of miR-544 may be mediated via interaction with the 3’UTR of apoB mRNA. In contrast to miR-544, miR-1202 overexpression induced an increase in apoB mRNA expression and protein synthesis. Similarly, the opposite effect was observed when using anti-miR-1202. Data from luciferase reporter assays showed an increased expression of the reporter gene in constructs carrying 5’UTR of apoB mRNA suggesting that miR-1202 may function via the 5’UTR. In summary, these data demonstrate that specific miRNAs are involved in the regulation of expression and translational control of apoB mRNA in hepatocytes. However, these miRNAs do not appear to mediate insulin regulation.