Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways

Abstract

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of nuclear factor kappaB (NF-kappaB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative reverse transcription-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-kappaB binding sites in the miR-146a promoter and identified a role for an Oct-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a-expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon-responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.

FIG. 1.

EBV LMP1 alters expression profiles of cellular miRNA. Microarray analysis of miRNA expression was performed on RNA isolated from control (Mutu pEHyg) and LMP1-expressing (Mutu pEHyg-LMP1) EBV-negative Mutu Cl.3 cells. Five separate RNA pairs were hybridized to miRNA arrays, and the data were combined for analysis. A. Western blot analysis of LMP1 expression in the EBV-positive latency type III cell lines JY, X50-7, and Jijoye, the type I latency cell line Akata, and in control or LMP1-transduced EBV-negative Mutu cells. B. Cluster analysis of miRNAs significantly altered by LMP1 at a P level of <0.01. C. MicroRNAs significantly (P < 0.01) regulated by LMP1, showing mean signal intensities and log-transformed ratios.

FIG. 2.

qRT-PCR analysis of miR-146a expression. A. Mature miR-146a. Two separate preparations of RNA were isolated from EBV-negative Mutu-pEHyg and Mutu-pEHyg-LMP1 cells and analyzed by real-time PCR to assess levels of mature miR-146a. Data were normalized to U6 small nuclear RNA expression. B. Primary miR-146a. Total RNA isolated from EBV-negative Mutu-pEHyg and Mutu-pEHyg-LMP1 cells was reverse transcribed and used in a real-time PCR assay to detect the primary miR-146a transcript. Data were normalized to GAPDH expression.

FIG. 3.

LMP1 induces miR-146a in the epithelial cell line A549. Total RNA isolated from A549 lung epithelial cells transduced with pEHyg or pEHyg-FLAG-LMP1 retroviruses was polyadenylated, reverse transcribed, and used in a qPCR analysis of mature miR-146a expression. Data were normalized to U6 small nuclear RNA expression.

FIG. 4.

Mature and primary miR-146a transcript analysis in a panel of cell lines. A. Total RNA isolated from EBV-positive, latency type III cell lines JY, IB4, X50-7 (lymphoblastoid cell lines), and Jijoye (Burkitt's lymphoma) and EBV-positive latency I Burkitt's lymphoma cell lines Akata, Rael, and Mutu was polyadenylated and reverse transcribed for qPCR analysis of mature miR-146a expression. The Western blot shows LMP1 expression in EBV latency type III cell lines but not in latency type I cell lines. B. Total RNA isolated from the indicated cell lines was reverse transcribed and subjected to qPCR to assess the levels of primary miR-146a transcripts. C. Total RNA extracted from the EBV-negative Burkitt's lymphoma cell line BL-30 and BL-30 cells infected with EBV B95-8 virus were reverse transcribed and assayed for pri-miR-146a expression by qPCR. Mature miR-146a expression was normalized to U6 small nuclear RNA expression, and pri-miR-146a expression was normalized to GAPDH expression.

FIG. 5.

miR-146a promoter analysis. A. Alignment of major homologies between human and mouse miR-146a promoter regions. Homologies were identified through a BLAST search using the human miR-146a promoter as described in Materials and Methods. Percentage scores for the indicated transcription factors were generated by TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html). B. Schematic representation of the human miR-146a promoter, with putative transcription factor binding sites. C. EBV-negative Mutu cells were cotransfected with pSG5 or pSG5-LMP1 plus wild-type or mutant miR-146a promoter-reporter vectors and harvested 48 h later. Luciferase expression is represented relative to luciferase activity of the wild-type promoter in the presence of LMP1. Error bars show the standard errors of the means. Increases for each reporter are indicated. Deletion of the dual NF-κB sites abrogated the ability of LMP1 to induce the miR-146a promoter.

FIG. 6.

Down-regulated genes with 7-mer or better miR-146a seed sequences. Transcript sequences were obtained from the website www.ensembl.org.

FIG. 7.

Differential expression levels of IRAK1 and STAT1 in miR-146a versus control cells. Potential seed sequences are shown. Transcript sequences were obtained from the website www.ensembl.org.