Article Figures & Data
Figures
- 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 .
Tables
- TABLE 1.
Transcripts induced in miR-146a-expressing Akata cells (P < 0.01)
Gene Retrovirus infection (fold change) Name and function Pair 1 Pair 2 GIMAP4 7.4 7.7 GTPase, IMAP family member; decreased in T and B cells during activation STON2a 2.9 2.7 Stonin 2; involved in endocytosis AF086511 2.7 3.1 AK022044 2.6 2.2 Protein tyrosine phosphatase, receptor type, G; linked to familial breast cancer; tumor suppressor (PTPRG) 2.1 2.0 THC2247947 2.6 2.7 RAG1 2.5 2.5 Recombination activating gene 1; initiates V(D)J recombination during B-cell development OSBPL1A 2.4 2.8 Oxysterol binding protein-like 1A; likely regulates lipid metabolism SNTG2 2.3 3.0 Syntrophin; interacts with dystrophin ↵ a Data were derived from array element AK094799.
- TABLE 2.
Transcripts suppressed in miR-146a-expressing Akata cells (without known response to IFN) (P < 0.01)
Gene Retrovirus infection (fold change) miR-146a seed match Name and/or function Pair 1 Pair 2 DNAPTP6 −7.2 −12.0 8-mer DNA polymerase-transactivated protein 6; transactivated by viral polymerase SAMD9L −7.5 −5.8 Two 7-mers Sterile α-motif domain-containing 9-like EPSTI1a −6.5 −6.0 9-mer Epithelial stromal interaction 1; induced in tumor cells cocultivated with fibroblasts EPSTI1 −4.0 −4.8 RGS13 −5.3 −6.8 None Regulator of G-protein signaling 13; may mediate cytokine signaling in germinal centers; suppressed by EBV LMP1 HSPA1A −4.2 −2.0 10-mer Heat shock 70-kDa protein 1A, HSP72; antiapoptotic BCL2A1 −4.0 −3.0 7-mer Bcl2-related protein A1; induced by NF-κB, EBV-LMP1, and EBNA2; antiapoptotic BG547557 −4.0 −3.5 CCR9 −3.5 −4.0 11-mer Chemokine (c-c motif) receptor 9; receptor for TECK, involved in mucosal homing of lymphocytes PPP1R1C −3.5 −2.7 None AK026784 −3.5 −2.8 7-mer FLJ10986 −3.4 −3.5 8-mer FLJ10986 −2.7 −3.2 ITGBL1a −3.1 −4.5 None Integrin β-like 1 C1ORF21 (PIG13) −3.1 −2.1 7-mer Proliferation-induced gene 13 C1ORF21 (PIG13) −2.7 −2.1 OLFML2A −2.9 −2.2 7-mer Olfactomedin-like 2A KIAA0774 −2.7 −2.2 12-mer KIAA0774 −2.7 −3.7 C16ORF81a −2.6 −2.7 None ZBTB32 −2.6 −2.6 None Zinc finger and BTB domain-containing 32; transcription factor involved in osteoblastic differentiation AK057704 −2.4 −2.0 None TMTC2 −2.3 −2.2 None Transmembrane and tetracopeptide repeat-containing 2 SYT12 −2.1 −2.0 9-mer, two 7-mers Synaptotagmin XII, or SRG1; involved in brain disorders ITGB2 −2.0 −2.0 7-mer Integrin, β2 LMO2 −2.0 −2.0 None LIM domain only 2 (rhombotin-like 1) ↵ a Data were derived from the following array elements: EPSTI1 (ENST00000313624), ITGBL1 (ENST00000376155), and C16ORF81 (ENST00000270031).
- TABLE 3.
Transcripts suppressed in miR-146a-expressing Akata cells (known to be IFN responders) (P < 0.01)
Gene Retrovirus infection (fold change) miR-146a seed match Name and/or function Pair 1 Pair 2 IFI44L −9.2 −9.7 None Interferon-induced protein 44-like, or histocompatibility 28 IFI44 −7.4 −7.8 None Interferon-induced protein 44; induced during hepatitis virus infection MX2 −4.6 −4.9 7-mer Myxovirus (influenza virus) resistance 2, or interferon-induced GTP MX2 −3.9 −3.5 binding protein MXB RSAD2 −4.4 −3.0 None Radical S-adenosyl methionine domain-containing 2, or viperin; antiviral; RSAD2 −2.5 −2.7 induced during TLR signaling IFIT3 −3.9 −3.0 Two 8-mers Interferon-induced protein with tetratricopeptide repeats 3 OASL −3.2 −2.6 7-mer 2′-5′-oligoadenylate synthetase-like; binds double-stranded RNA and DNA TRIM22 −2.9 −2.7 Two 7-mers Tripartite motif-containing 22, or STAF50; can inhibit viral replication TRIM22 −2.7 −2.7 IFIT1 −3.3 −3.2 None Interferon-induced protein with tetratricopeptide repeats 1 IFIT5 −2.7 −2.8 None Interferon-induced protein with tetratricopeptide repeats 5 IFIT5 −2.8 −2.5 IFITM1 −2.4 −2.7 None Interferon-induced transmembrane protein 1, LEU13, or CD225; regulates cell growth IFITM3 −2.7 −2.3 None Interferon-induced transmembrane protein 3 IFITM3a −2.4 −2.4 IFITM3a −2.4 −2.5 IFITM4P −2.7 −2.9 None Interferon-induced transmembrane protein 4 pseudogene IRF7 −2.3 −2.5 None Interferon regulatory factor 7; transcriptional activator in response to IRF7 −2.6 −2.5 infection; binds to Qp of EBV EBNA1; involved in TLR signaling; colocalizes with and regulates EBV LMP1 NMI −2.7 −2.9 None N-myc (and STAT) interactor; transcription factor induced by interferon ISG15 −2.1 −2.1 None ISG15 ubiquitin-like modifier; targets STAT1, MAPK3, JAK1, etc; antiviral properties IFI27 −2.3 −2.3 None Alpha interferon-inducible protein 27 IFI27 −2.4 −2.6 ↵ a Data were derived from the array element ENST00000313624.
Supplemental material
Files in this Data Supplement:
- Supplemental file 3 -
Legends to Tables S1 and S2
PDF document, 23K. - Supplemental file 1
-
Table S1 (Differential expression of genes with twofold or greater expression and P values of <0.01 in control versus miR-146a-transduced cells for infection set 1.)
Excel document, 73K. - Supplemental file 2
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Table S2 (Differential expression of genes with twofold or greater expression and P values of <0.01 in control versus miR-146a-transduced cells for infection set 2.)
Excel document, 94K.
- Supplemental file 3 -
Legends to Tables S1 and S2
