review article role of viral mirnas and epigenetic...

Review ArticleRole of Viral miRNAs and Epigenetic Modifications inEpstein-Barr Virus-Associated Gastric Carcinogenesis

Aldo Giudice1 Giovanni DrsquoArena2 Anna Crispo1 Mario Felice Tecce3

Flavia Nocerino1 Maria Grimaldi1 Emanuela Rotondo1 Anna Maria DrsquoUrsi3

Mario Scrima3 Massimiliano Galdiero4 Gennaro Ciliberto5 Mario Capunzo6

Gianluigi Franci4 Antonio Barbieri7 Sabrina Bimonte8 and Maurizio Montella1

1Epidemiology Unit National Cancer Institute of Naples ldquoG Pascale Foundationrdquo IRCCS 80131 Naples Italy2Department of Onco-Hematology IRCCS Cancer Referral Center of Basilicata 85028 Rionero in Vulture Italy3Department of Pharmacy University of Salerno Fisciano 84084 Salerno Italy4Department of Experimental Medicine II University of Naples 81055 Naples Italy5Scientific Direction National Cancer Institute ldquoG Pascale Foundationrdquo IRCCS 80131 Naples Italy6Department of Medicine and Surgery University of Salerno Baronissi 84081 Salerno Italy7Animal Facility Unit National Cancer Institute of Naples ldquoG Pascale Foundationrdquo IRCCS 80131 Naples Italy8Division of Abdominal Surgical Oncology Hepatobiliary Unit National Cancer Institute ldquoG Pascale FoundationrdquoIRCCS 80131 Naples Italy

Correspondence should be addressed to Aldo Giudice aldogiudiceliberoit

Received 23 October 2015 Revised 12 January 2016 Accepted 14 January 2016

Academic Editor Denis Delic

Copyright copy 2016 Aldo Giudice et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

MicroRNAs are short (21ndash23 nucleotides) noncoding RNAs that typically silence posttranscriptional gene expression throughinteraction with target messenger RNAs Currently miRNAs have been identified in almost all studied multicellular eukaryotesin the plant and animal kingdoms Additionally recent studies reported that miRNAs can also be encoded by certain single-celleukaryotes and by virusesThe vastmajority of viralmiRNAs are encoded by the herpesviruses familyTheseDNAviruses includingEpstein-Barr virus encode their ownmiRNAs andormanipulate the expression of cellularmiRNAs to facilitate respective infectioncycles Modulation of the control pathways of miRNAs expression is often involved in the promotion of tumorigenesis through aspecific cascade of transduction signals Notably latent infection with Epstein-Barr virus is considered liable of causing severaltypes of malignancies including the majority of gastric carcinoma cases detected worldwide In this review we describe the role ofthe Epstein-Barr virus in gastric carcinogenesis summarizing the functions of the Epstein-Barr virus-encoded viral proteins andrelated epigenetic alterations as well as the roles of Epstein-Barr virus-encoded and virally modulated cellular miRNAs

1 Introduction

TheEpstein-Barr virus (EBV) was the first discovered humantumor-causing virus considered as the etiologic agent ofBurkittrsquos lymphoma (BL) an unusual African pediatric lym-phoma [1]

Specifically EBV is ubiquitous member of the humangamma-herpesvirus family that causes mononucleosis dur-ing acute and lytic infection and also establishes a persis-tent and latent infection in more than 90 of the human

population EBV latent infection has been demonstrated tobe involved inmultiple types of cancer that primarily developin lymphocytes and epithelial cells These include malignanttumors that develop in the immunocompromised conditionssuch as AIDS-associated lymphomas and posttransplantlymphoproliferative disease [2 3] and also several humancancers that develop in the immunocompetent patients suchas BL Hodgkinrsquos lymphoma B-cell and T-cell lymphomasepithelial nasopharyngeal carcinoma (NPC) and some formsof gastric carcinomas [4ndash6] Gastric cancer is the fourth

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 6021934 11 pageshttpdxdoiorg10115520166021934

2 Oxidative Medicine and Cellular Longevity

Gastric carcinoma andother malignancies

DNMTsinduction

Viral mRNAdegradation or

translationinhibition

Degradation DegradationTranslation Translation

Host cell mRNAdegradation or

translation inhibition

Aberrant DNA methylation andinactivation of tumor suppressor

genes

Host cell genome

Increase persistentlatent infection in the

host cells

Reduce hostimmune

surveillance

Regulate cell signalingcell division and

apoptosis

RiscViral miRNAs

Viral proteins(LMP2A)

ViralDNA

genome

Figure 1 Graphical representation of possible mechanisms by which viral miRNAs and viral proteins might contribute to EBV-associatedgastric carcinogenesis This model indicates that EBV-encoded miRNAs (eg BART miRNAs and BHRF-1 miRNAs) target viral genes tomediate immune evasion or maintenance of latency whereas some viral proteins (eg LMP2A) promote aberrant host DNA methylationand subsequent inactivation of tumor suppressor genes via DNA methyltransferases (DNMTs) induction In addition these viral miRNAsincorporated into RISC complex can also interact directly with specific host genes involved in immune surveillance cell proliferation andapoptosis playing a crucial role in the aetiology of diverse diseases including EBVaGC

most common cancer in the world and the second leadingcause of cancer-related death Globally gastric cancer posesa significant public health burden both economically andsocially [7 8] Risk factors of gastric cancer aremultifactorialhence genes diet age and chronic inflammation need tobe evaluated in connection with infectious agents (EBVHelicobacter pylori) and environmental factors (eg alcoholand smoking) [8] Notably EBV-associated gastric carcinoma(EBVaGC) represents almost 10 of all gastric carcinomacases and expresses restricted EBV latent genes (Latency I)[4 9] In recent years it has become increasingly evident thatEBV may contribute to gastric carcinogenesis through theexpression of viral proteins and microRNAs (miRNAs) [10](Figure 1) A growing body of scientific evidence also suggeststhat in addition to genetic alterations epigenetic alterationsincluding aberrant DNA methylation of CpG islands andposttranslational modifications of histones are involved inthe development and progression of EBVaGC [10 11] Thisreview briefly summarizes remarkable advancements in ourunderstanding of the functions and mechanisms of action ofherpesviral miRNAs in gastric carcinoma in recent years Inparticular it discusses how the expression of viral proteins andepigenetic alterations contribute to EBVaGC and the rolesof EBV-encoded and virally modulated cellular miRNAs in

the respective viral life cycles and in EBV-associated gastriccarcinoma

2 Biogenesis of EBV-Encoded miRNAs

The generation of viral miRNA and selection of targets aretotally dependent on the host molecular miRNA apparatusinvolved in the maturation and silencing Most viruses utilizea strategy similar to that of the host cell to produce the viralpri-miRNAs by using the host RNA polymerase II (RNAP II)[12 13] Exceptionally certain viral miRNAs (such as thosefrom mouse 120574-herpesvirus 68 MHV68) are produced fromtRNA-like genes transcribed by host RNA polymerase III(RNAP III) and processed Drosha independently by hosttRNase Z [14] Aminority of viruses from the herpesvirus andretrovirus families also utilize noncanonical pathways otherthan ldquotRNA-likerdquo to generate pre-miRNAmolecules [15ndash22]For instance herpesvirus saimiri (HVS) an oncogenic 120574-herpesvirus that infects NewWorldmonkeys expresses smallnuclear RNAs (snRNAs) of the Sm-class called HSURs whichare processed by the integrator complex to produce viralpre-miRNAs Subsequently both the MHV68 and HVS pre-miRNAs are processed by Dicer to generate miRNAs [23]Additionally some retroviruses such as foamy viruses (FVs)

Oxidative Medicine and Cellular Longevity 3

and bovine leukemia virus (BLV) are able to express pri-miRNAs via RNAP III [18 19 21] While some retroviral pri-miRNAs are processed by Drosha in the frame of RNAP IIItranscripts some others may be directly processed by Dicerbypassing Drosha processing Production of retroviral miR-NAswithout having recourse to Drosha-mediated cleavage ofthe RNA genome intermediate has been found to representan important biogenesis strategy that may eventually takean active part in reducing overall viral fitness [18 19 21]In brief viral miRNA biogenesis initiates in the nucleuswhere after transcription by host RNA polymerase II themicroprocessor complex which contains the host RNase IIIendonuclease Drosha and its interaction partner DGCR8(also known as Pasha in Drosophila) [24] cleaves pri-miRNAhairpin structure to pre-miRNAs The vast majority of pri-miRNAs contain approximately 80 nucleotide hairpin sec-ondary structures that can be intronic or exonic Approxi-mately 60 pre-miRNA nucleotides are liberated and rapidlyexported from the nucleus to the cytoplasm by exportin-5Ran GTPase pathway Once in the cytoplasm they arefurther processed by a second host RNase III endonucleaseDicer into a short double-stranded (ds) RNA or RNAduplexThe strand guide of these mature miRNAs of approximately22 nucleotides is then incorporated into a protein complexknown as the RNA-induced silencing complex (RISC) whilethe other strand called the passenger is rapidly degraded [1325] RISC complex can incorporate both strands of miRNAsgenerated in EBV-infected cells The major component ofRISC is the Argonaute (Ago) protein In mammalian cellsthere are four Ago proteins which are all incorporatedinto RISC however only Ago2 shows endonuclease activityBinding of Ago-loaded miRNAs (miRISC) to a mRNA bear-ing extensive sequence complementary to miRNA generallyresults in mRNA cleavage and degradation whereas bindingtomRNAs bearing partial complementarity tomiRNA resultsmainly in translational arrest [12 13 26] There is no proofthat any animal virus encodes additional miRNA-processingfactors or RISC components Therefore viral miRNAs bio-genesis largely relies on host-derived enzymes or proteinsInterestingly EBV miRNAs can be subdivided into twogroups Bam HI fragment H rightward open reading frame1 microRNAs (BHRF1 miRNAs) and Bam HI-A rightwardtranscripts microRNAs (BART miRNAs) based on theirlocations [27 28] Specifically BHRF1 miRNAs are locatedwithin introns of the BHRF1 gene and generated from thelong EBNA transcripts whereas BART miRNAs are insteadlocated in introns included within the BART transcripts[27 28] Nevertheless how EBV miRNA expression is finelyregulated remains largely unknown Various studies suggestthat BART transcriptsmiRNAs are transcribed fromP(1) andP(2) promoters in both B-cells and epithelial cells [29] TheBART promoter region contains putative binding sites fordiverse transcription factors Among transcription factorsCEBP120573 was reported to positively modulate the expressionof BART [30] Notably expression of the EBV miRNAs wasdetected in several human cancer cell lines in fact expressionof the EBV miRNAs was observed in EBVaGC [31] andperipheral T-cell lymphoma [32] besides being shown in B-cell lines and nasopharyngeal carcinoma EBV-infected cells

[27 28 33] Most plant miRNAs as well as some rare viralmiRNAs bind to their targets with perfect complementarityeven though this is generally uncommon in higher orderanimals Once bound to the target miRNAs behave similarto siRNAs inducing specific and irreversible endonucleolyticcleavage event in the target transcripts [34]The questionwhythis mode of miRNA-mediated action is so rarely used byanimal host miRNAs is puzzling [26]

3 Mechanism of EBV Infection Viral ProteinsExpression Profile during Latency andEBV-Associated Gastric Carcinogenesis

Mounting scientific evidence describes herpesviruses hav-ing two distinct life cycles lytic replication and latencyNotably EBV may infect host gastric epithelial cells throughdirect and indirect mechanisms In the direct infectionviral glycoproteins attach to cellular receptors and driveviral proteins conformational changes that promote fusionof the viral envelope with the epithelial cell membrane [35]In the indirect infection instead EBV initially infects Blymphocytes Subsequently EBV-infected B-cells throughdirect cell-to-cell contact infect epithelial cells via the cappedadhesion molecules [36] Following primary infection EBVafter an early replication phase establishes persistent latentinfection in the host cells During latency viral genomes existas extrachromosomal episomes in the nucleus and beinglargely silenced by host-driven methylation of CpG islandmotifs are only able to express a small subset of genesincluding latent proteins with oncogenic potential and viralmiRNAs [36]

As a result the latency enables the virus to efficientlyevade the host immune response causing persistent infectionsover a lifetime This is a feature common to all herpesviruses[4 37] Considering the subset of viral genes expressedherpesviruses-associated tumors have been subdivided intofour types latency Ia latency Ib latency II and latency IIIEBVaGC belongs to latency type I where the viral genesEBV nuclear antigen 1 (EBNA1) latent membrane protein2A (LMP2A) Bam HI-A rightward transcripts (BARTs) andEBV-encoded small RNA (EBER12) may be expressed [1038 39] In particular the expression of latency genes fulfilsa relevant task in the initiation and neoplastic progressionof EBV-associated epithelial cancers including EBVaGC Forinstance the viral protein EBNA1 is absolutely required inmaintaining and replicating the viral episomal genome by thehost cell DNA polymerase machinery in all EBV-associatedmalignancies EBNA1 may contribute to cell survival afterDNA damage and to genetic instability in EBV-infectedepithelial cells [40]

Specifically in both NPC and EBVaGC EBNA1 canpromote the decline of the promyelocytic leukemia (PML)nuclear bodies including many regulatory proteins by bind-ing and modulating casein kinase 2 (CK2) or ubiquitin-specific protease 7 which in turn degrades p53 [41]ThereforeEBNA1 may increase the genetic instability of the EBV-infected epithelial cells and facilitate the oncogenesis mainlyby decreasing p53 levels following DNA damage Moreover


EBNA1 may also participate in tumor development by sup-pressing transforming growth factor-beta (TGF-120573) [42] andimproving the nuclear factor-kappa B (NF-120581B) signaling [43]In addition to EBNA1 half of all EBVaGCs also expressLMP2A and therefore EBV latency configurations shouldbe classified into Ia or Ib based on the presence or absenceof LMP2A [38 44] LMP2A viral protein therefore plays aparamount role in the transformation of epithelial cells bythe induction and maintenance of tumor phenotypes Theseactivities range from resistance to apoptosis to cell prolifera-tion invasion motility and angiogenesis [45] LMP2A alsoinhibits apoptosis by upregulation of the cellular survivingene through the NF-120581B pathway [46] In epithelial cellsLMP2A may activate not only the phosphatidylinositol 3-kinase (PI3K)protein kinase B (PKB) pathway and phospho-rylation of glycogen synthase kinase-3 but also the 120573-cateninsignaling pathway These molecular mechanisms induce sev-eral remarkable phenotypic changes including anchorage-independent growth and inhibition of differentiation [47ndash49] These data herein reviewed collectively suggest thatLMP2A viral protein not only has an impact on the NF-120581Bpathway which upregulates survivin gene therefore inhibit-ing apoptosis but also promotes the induction of cancer stemcells in EBV-associated epithelial cancers

4 Role of Epigenetic Alterations inEBV-Associated Gastric Carcinogenesis

The term epigenetics currently refers to the inheritablechanges in gene expression which occur without an alterationof the genome at the level of nucleotide sequences Epigeneticmechanisms are necessary to support the physiological organgrowth and development and moreover to insure normalgene expression in different tissues [50ndash52] However nowa-days gastric carcinogenesis processes can be explained notonly by genetic modifications [53ndash56] but also by epigeneticalterations such as DNA methylation histone modificationsand noncoding RNAs [51 57] These modifications are partof the ldquoEpigenetic coderdquo and are essential to regulate thenormal development and maintenance of tissue-specificgene expression in different mammalian cell types [51 52]Increasing evidence suggests that some environmental factorssuch as aging diet physical activity chronic inflammationand microbial infection can also affect gene methylation ingastric epithelia and promote the development of gastriccancer [58 59] Specifically EBV infection was reported asa cause of increased methylation and repression of tumorsuppressor genes in MKN7 a low methylation GC cellline [11] Further studies also confirmed that abnormalDNA methylation in the promoter regions of the genewhich provides inactivation of tumor suppressor and othercancer-related genes is the most well-defined epigeneticcharacteristic in EBVaGC but not in EBV nonassociatedGC (EBVnGC) [11 60ndash66] Specifically hypermethylationof tumor suppressor genes such as E-cadherin (CDH1)p14 p15 p16 p73 adenomatous polyposis coli (APC) andphosphatase and tensin homolog (PTEN) were observedin EBVaGC but not in EBVnGC [67 68] In addition

Hino et al [46] also demonstrated that LMP2A inducedthe expression of phosphorylated signal transducer andactivator of transcription 3 (pSTAT3) which stimulated theupregulation of DNA methyltransferases (DNMT) DNMT1and DNMT2 in EBV-infected GC cells [69] Recent studiesby Zhao et al [60] also reported that hundreds of genesimplicated in cancer signaling pathways such as mitogen-activated protein kinase signaling wnt signaling pathwayand cell adhesion molecules were hypermethylated followingEBV infection [60] The same authors also suggested thatEBV infection induced aberrant CpG hypermethylation ofseveral genes by upregulation of DNMT3b through LMP2Ain EBV-positive AGS cells compared to EBV-negative AGScells Another important cellular alteration in EBVaGC isits resistance to programmed cell death (apoptosis) Thefrequency of apoptosis is markedly reduced in EBVaGCcompared to EBVnGC [70] Some studies hypothesize thatboth genes somatostatin receptor 1 (SSTR1) and glutathioneS-transferase P1 (GSTP1) are frequently hypermethylated inGC infected EBV tissues and modulate cell migration prolif-eration and apoptosis [67 68 71 72]However themolecularmechanism of the aberrant DNAmethylation following EBVinfection is still unclear As mentioned above one possiblemechanism is EBV induction of LMP2A overexpressionwhich promotes STAT3 phosphorylation further inducingDNMTs expression [60 69] Therefore LMP2A may play animportant role in cellular epigenetic dysregulation involvedin the development and maintenance of EBV-associatedcancer by increasing DNMTs expression

5 Role of EBV-Encoded miRNAs inGastric Carcinogenesis

The identification of two miRNAs miR-15a and miR-16alocated in a deletion region of 30 kb on chromosome 13q14identified in 50 of chronic lymphocytic leukemia (CLL)[73] was one of the first indications of possible involve-ment of miRNAs in human tumorigenesis After this firstexperimental evidence the correlation between the genomiclocations of a large number ofmiRNAs and cancer-associatedgenomic regions has been better identified [74]

From the functional and evolutive point of view miRNAsrepresent for viruses an element of fundamental regulationnot only of their genes but also of the host genes Comparedto genes coding for proteins the genes coding for miRNAsare small this characteristic is ideal for space-constrainedrestricted viral genomes Moreover their small size couldfacilitate rapid adaptation to new targets through smallchanges in the level of their nucleotide composition In fact amiRNA can have multiple targets and inhibit the expressionof different genes simultaneously These characteristics makemiRNAs ideal candidates for control of host-pathogen inter-actions [26] Increasing evidence suggests that EBV is a DNAtumor virus belonging to the human gamma-herpesvirusfamily which is capable of establishing a latent infectionmainly in human B lymphocytes and epithelial cells andis associated with several human lymphoid and epithelialcell malignancies including EBVaGC [1ndash6] Probably the


Table 1 List of main EBV-associated gastric carcinogenesis studies discussed in this review

First author andyear ofpublication

Study design Paper number inreferences section Summary of findings

Yau et al 2014 Review [10]

EBV infection contributes to gastric carcinogenesis through theexpression of viral proteins and microRNAs as well as aberrantDNA methylation and histone modification and relativetherapeutic implications

Shinozaki-Ushiku et al2015

Review [57]

In the present review latest findings on EBVaGC fromclinicopathological and molecular perspectives were discussed toprovide a better understanding of EBV involvement in gastriccarcinogenesis In addition to genetic and epigenetic changesposttranscriptional gene expression regulation by cellular andorEBV-derived microRNAs was also considered


Experimentalresearch [90]

Comprehensive profile of the expression of 44 known EBVmiRNAs from EBV-associated gastric carcinoma patients waspresented Of several highly expressed EBV miRNAsEBV-miR-BART4-5p plays a partial role in suppressingproapoptotic protein Bid leading to reduced apoptosis Thepresent work provides novel insights into the roles played by EBVmiRNAs in gastric carcinogenesis and identifies future potentialtherapeutic targets

Kim et al 2015 Experimentalresearch [91]

miR-BART20-5p contributes to the tumorigenesis initiationandor maintenance of EBVaGC by directly targeting 31015840-UTR ofBcl-2-associated death promoter (BAD) involved in cellproliferation and apoptosis Inhibition of miR-BART20-5p canexert a therapeutic effect for this neoplasia

Kanda et al2015


A causative relationship between BART miRNA expression andepithelial carcinogenesis in vivo was demonstrated In particular itwas shown that NDRG1 protein which is a putative target of BARTmiRNAs can be used as an epithelial differentiation marker and asuppressor of metastasis

Fu et al 2013 Review [94]

Potential mechanisms by which EBV contributes to its own latencyand the formation tumors including EBVaGC were consideredParticularly this review describes the interactions of EBV geneproducts including viral miRNAs and the Bcl-2 family membersinvolved in cell death (apoptosis) and survival pathways A betterunderstanding of this complicated network of interactions couldbe of great importance for creating novel therapeutic strategies forEBV-associated diseases

Tokunaga et al1993

Epidemiologicalresearch [104]

EBV infection contributed significantly to gastric carcinogenesis inJapan It occurred predominantly in males especially in cancers ofthe upper and middle parts of the stomach with greater cell typevariation in men suggesting that novel factors may play importantcausal roles in EBV-associated gastric carcinogenesis

induction of tumors is not the main advantage of this virusbut rather an accidental need to alter the cell cycle preventingcell death and evade the immune response [75] In EBV-associated epithelial malignancies (eg NPC and EBVaGC)the gene products encoded by EBV play a crucial role atthe beginning of the carcinogenesis process therefore onlyfew additional acquired genetic changes are required forthe neoplastic transformation [10 40 41] Specifically inEBV-associated epithelial cancers the latent genes (EBNA1EBERs andmiR-BARTs) are intensely expressed in all cancercells Unlike EBNA1 and EBERs miR-BARTs are expressed athigh levels only in EBV-infected epithelial cancers but not inEBV-transformed lymphocytes [56 76 77] pointing out their

involvement in EBV-associated epithelial cancers Interest-ingly the expression of latency genes plays an important rolein the initiation and neoplastic progression of gastric cancerby inducing strong antiapoptotic signals For instance EBER1can confer an apoptotic-resistant phenotype by increasing theexpression of insulin growth factor-1 (IGF-1) an autocrinegrowth factor which potentiates cell proliferation in EBVaGC[78] During EBV infection of epithelial cells these EBV-encoded viral regulatory RNAs may also modulate the hostinnate immune responses [79] A recent study by Banerjee etal [80] also suggested that EBERs could increase IL-6 expres-sion and activate its downstream regulator STAT3 which wasresponsible for downregulation of the cell cycle inhibitors p21


and p27 in gastric carcinoma cells and associated cancer cellresistance The same authors also demonstrated that EBERscould downregulate antimetastatic molecules RhoGD1 andKAI-1 and activate the prometastatic molecules pFAKand pPAK1 which induced cell migration In additionmany EBV miRNAs also repress apoptosis by targetingthe proapoptotic proteins p53-upregulated modulator ofapoptosis (PUMA) Bcl-2 interacting mediator of cell death(BIM) and translocase of outer mitochondrial membrane 22homolog (TOMM22) respectively [81ndash83] Specifically Choyand colleagues [81] have demonstrated that EBVmiR-BART5inhibits production of the proapoptotic protein PUMA bytargeting mRNA which encodes the cellular factor PUMAPUMA is one of the sixmembers of theBH3-only group in theBcl-2 family The BH3-only proteins the essential initiatorsof apoptosis are responsible for controlling the releaseof cytochrome C from the mitochondrial intermembrane[84] In EBV-infected carcinoma cells miR-BART5 depletioninduces high levels of PUMA-mediated apoptosis suggestinga crucial role of miR-BART5 in the inhibition of apoptosisboth in EBV-infected epithelial cells and in EBV-transformedcellsThereforemiR-BART5may contribute to the survival ofinfected cells during the natural viral infection and influencethe cell survival in virus-associated cancers Another BH3-only group proapoptotic protein Bim has been reportedto be a target of both EBV miR-BART4 and miR-BART15[82 85] It is possible that this activity is linked to the observedinhibition of apoptosis by miRNAs of the BART cluster inthe human gastric carcinoma cell line AGS InterestinglyBimrsquos 31015840-UTR is not responsive to any of the individual miR-BARTs in stable transfectants indicating possible cooperationof miR-BARTs in cluster 1 for Bim expression Anotherpotential target of miR-BART-16 is TOMM22 a proteinforming part of a mitochondrial pore complex that standsas a receptor for the proapoptotic protein Bcl-2-associatedX (Bax) [83] EBV can gain benefit from the repression ofTOMM22 since siRNA-mediated knockdown of TOMM22has been shown to inhibit the association of Bax protein tomitochondria therefore preventing Bax-dependent apopto-sis [86] Other EBV miRNAs of the BART cluster variablyexpressed in EBVaGC tissue samples and cell lines are EBV-miR-BART1-3p 5-5p 7-3p 15-3p 19-3p and 22-3p [87ndash89] Recently Shinozaki-Ushiku et al [90] also reportedthat EBV-miR-BART4-5p exerts a crucial role in gastrictumorigenesis through modulation of apoptosis Specificallythese authors demonstrated that reduction of apoptosis inclinical samples from EBVaGC patients was attributableto the expression of EBV-miR-BART4-5p which plays apartial role in suppressing proapoptotic protein Bid alsotermed as the BH3 interacting domain death agonist Anotherimportant finding has been recently reported by Kim et al[91] These authors suggested that miR-BART20-5p reducedBcl-2-associated death promoter (BAD) expression in EBV-infected GC cells as opposed to EBV-negative GC cells bydirectly targeting 31015840-UTR of BAD in order to promote hostcell survival Recently Kanda et al [92] also demonstratedthat multiple EBV-encoded miRNAs contribute to EBVaGCby targeting N-myc downstream regulated gene 1 (NDRG1)This oncosuppressor protein was highly expressed in primary

epithelial cells and significantly reduced in the BART(+)virus-infected epithelial cells playing an important role incarcinogenesis and preventing the metastasis and invasion ofgastric cancer cells These data also implicate that NDRG1could be utilized as a prognostic andor diagnostic markeras well as a potential therapeutic target against gastric cancer[92 93] (Table 1) Additional studies by Fu et al [94] alsodemonstrated that EBV BHRF1 miRNAs can promote cellsurvival by interacting with several proapoptotic proteinssuch as Bcl-2 homologous antagonistkiller (Bak) Bcl-2-related ovarian death gene (Bod) Bcl-2-related ovarian killer(Bok) Bim and Bcl-2-interacting killer (Bik) in variouscell lines including gastric carcinoma cell lines The sameauthors also indicate that BARF1 (Bam HI-A fragment right-ward reading frame 1) might promote cell transformationby activating antiapoptotic protein Bcl-2 [94] In additionto these effects EBV infection may also affect host cellmiRNA expression and exert effects on immune responsesand oncogenesis For instance studies by Shinozaki et al[95] have demonstrated downregulation of the cellular miR-200 family (eg miR-200a and miR-200b) in EBV-associatedgastric carcinoma by repressing transcription of pri-miRNAsand by posttranscriptional dysregulation of the miRNAin EBVaGC compared to EBVnGC and adjacent mucosaSpecifically these authors indicate that all the latency type Igenes such as Bam HI-A rightward reading frame (BARF0)EBERs EBNA1 and LMP2A have a synergetic effect onthese processes and contribute to the downregulation of themature miR-200 family and the subsequent upregulation ofthe E-cadherin transcription repressors zinc finger E-boxbinding factor 1 (ZEB1) and ZEB2 resulting in the inhibitionof E-cadherin expression and induction of the epithelial-to-mesenchymal transition (EMT) [95] This is a pivotal stagein the process of carcinogenesis of EBV-associated gastriccarcinoma Another report recently published by Du andcolleagues [96] also revealed that miR-141 a member ofthe miR-200 family was downregulated in 80 of primarygastric cancer tissues showing its inhibitory effect on cellproliferation A recent study by Marquitz et al [97] suggeststhat by infecting EBV-negative AGS gastric carcinoma celllines with a recombinant EBV a clear hallmark of transfor-mation becomes apparent with an anchorage independentphenotype Specifically the authors showed that the cellshave levels of BART miRNAs similar to EBV-positive gastriccancer They also reported that EBNA1 overexpression inAGS gastric carcinoma cell lines significantly reduced miR-143 expression which acts as tumor suppressor in severaltypes of cancer [98ndash100] More recently Marquitz et al[101] also indicate that EBV infection induces downregula-tion of host tumor suppressor miRNAs including the Let-7family and the miR-200 family through a mechanism thatis independent of latent protein expression It seems thatEBV miR-BART6 can repress the expression level of miRNAprocessing human DICER1 enzyme in EBV-infected cellscompared to noninfected cells playing an important role inthe progression of EBV-associated tumors [102] ThereforeEBV-encoded miRNAs may be an aetiological factor incancer development in immunocompetent individuals thatcould bypass the requirement for viral protein expression


and the consequential recognition by the immune systemSurprisingly not all miR-BARTs inhibit apoptosis or promotecell growth For example Choi et al [103] reported the tumorsuppressive effect of miR-BART15 in gastric carcinogenesisThe authors demonstrated that miR-BART15 inhibited cellgrowth and induced early apoptosis inAGS gastric carcinomacell in part by targeting the BRUCE gene which encodes theBRUCE protein an inhibitor of apoptosis proteins (IAPs)In addition miR-BART15 might also indirectly contribute tocancer development by inducing its proapoptotic activity inthe adjacent immune cells by targeting BRUCE [103] Theseresults collectively suggest that some EBV-encoded miRNAsmay regulate the expression of several key cancer-relatedproteins including those involved in latency maintenanceimmune suppression and tumor promotion Additional datawould warrant a more solid conclusion on this issue

6 Conclusions

Accumulating evidence suggests that miRNAs can beencoded not only by eukaryotes but also by certain virusesThe vast majority of viral miRNAs are encoded by the her-pesviruses family including pathogens such as EBV herpessimplex viruses (HSVs) human cytomegalovirus (HCMV)Kaposirsquos sarcoma herpes virus (KSHV) andMHV 68 Specif-ically EBV expresses multiple noncoding RNAs during alltypes of latency including two clusters of miRNAs BARTmiRNAs andBHRF1miRNAs EBV-transformed cells encodeat least 44 mature viral miRNAs that target viral and cellulargenes Some viralmiRNAs including thosewhich are analogsto host miRNAs and those which are virus-specific seemto exert an important role in the establishment of a latentviral infection by suppression of an effective host immuneresponse or through blockade of apoptotic processes in theinfected cells [105 106] In all EBV-associated cancers theviral infection also promotes important changes in the cellu-larmiRNAs profile It has been shown thatmany cell miRNAsin cell lines or in tumor tissues are deregulated upon EBVinfection The presently available information suggests thatviral miRNAs also contribute to induction or maintenance ofthe transformed phenotype In fact latent infectionwith EBVis considered responsible for several malignancies includinga big amount of all gastric carcinomas EBVaGC is charac-terized by unique clinical and pathologic features includingmale predominance the presence of EBV genomes and EBV-encoded small RNA (EBER) in gastric carcinoma cell linesand monoclonal proliferation of EBV-infected carcinomacells [4 9 57 104] In addition it also presents elevatedlevels of serum antibodies against EBV early antigen and EBVcapsid antigen as well as a lymphoepithelioma-like histologyand a relatively favourable prognosis [4 9 57 104] EBVaGCalso shows abnormal hypermethylation of several tumorsuppressor gene promoter regions causing downregulationof their expression In addition EBVaGC is distinctive owingto the limited number of EBV latent genes expressed incancer cells [96] It is classified as latency type I becausethe expressed latent genes are restricted to BARF0 EBERsEBNA1 and LMP2A excluding EBNA2 or LMP1 which are

essential for its transforming ability [107ndash109] All four ofthese genes play an important role in the downregulation ofmature miR-200 and the subsequent upregulation of tran-scription repressors ZEB1ZEB2 resulting in the inhibitionof E-cadherin expression and induction of the EMT whichis a crucial step in the carcinogenesis of EBVaGC Notablydownregulation of mature miR-200 may be mediated byaberrant DNAmethylation due to overexpression of DNMTsfollowing EBV infection EBER1may also increase the expres-sion of IGF-1 an autocrine growth factor which acceleratescell proliferation in EBVaGC [78] Another important aspectunderlined by Marquitz et al [101] is that in several EBV-infectedAGSgastric carcinoma cell lines a significant fractionof the changes in cellular expression likely reflect(s) theexpression of the viral noncoding RNAs such as the BARTRNAs and not the latent protein expression These changescomprise a decrease in host tumor suppressor miRNAs levelsand increased expression of viral miRNAs with putativeoncogenic potential However the precise role of EBV in themultifactorial aetiology of gastric carcinoma is still not wellknown In particular it will be necessary to identify moretargets of viral and deregulated cellular miRNAs A generalproblem in EBV-encoded miRNAs research is the lack ofan animal model The advancement of humanized miceshould facilitate the eventual development of EBV infectionmouse models Such models will ultimately be necessary toevaluate viralmiRNAs as potential therapeutic cancer targetsThey might be also used to evaluate different chemopre-ventive agents In this respect scientific evidence suggeststhat natural bioactive agents such as green tea catechinscould be useful for the modulation of the epigenome [110]and the subsequent inhibition of viral infections and virus-associated malignancies [111ndash114] Furthermore green teacatechins may also inhibit chronic inflammation involved inviral oncogenesis by regulating the Nrf2 and NF-120581B signalingpathways [115ndash117]

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to Dr Alessandra Trocino Librarianat the Scientific Library of the NCI ldquoG Pascalerdquo of NaplesItaly for bibliographic assistance

References

[1] M A Epstein B G Achong and Y M Barr ldquoVirus particlesin cultured lymphoblasts from burkittrsquos lymphomardquoTheLancetvol 283 no 7335 pp 702ndash703 1964

[2] S Maruo Y Wu S Ishikawa T Kanda D Iwakiri andK Takada ldquoEpstein-Barr virus nuclear protein EBNA3C isrequired for cell cycle progression and growth maintanance oflymphoblastoid cellsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 103 no 51 pp19500ndash19505 2006


[3] H H Niller H Wolf and J Minarovits ldquoRegulation anddysregulation of Epstein-Barr virus latency implications for thedevelopment of autoimmune diseasesrdquo Autoimmunity vol 41no 4 pp 298ndash328 2008

[4] M Fukayama R Hino and H Uozaki ldquoEpstein-Barr virusand gastric carcinoma virus-host interactions leading to car-cinomardquo Cancer Science vol 99 no 9 pp 1726ndash1733 2008

[5] L S Young and A B Rickinson ldquoEpstein-barr virus 40 yearsonrdquo Nature Reviews Cancer vol 4 no 10 pp 757ndash768 2004

[6] M P Thompson and R Kurzrock ldquoEpstein-Barr virus andcancerrdquo Clinical Cancer Research vol 10 no 3 pp 803ndash8212004

[7] A Jemal F Bray M M Center J Ferlay E Ward and DForman ldquoGlobal cancer statisticsrdquo CAmdashA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011

[8] Y Qu S Dang and P Hou ldquoGene methylation in gastriccancerrdquo Clinica Chimica Acta vol 424 pp 53ndash65 2013

[9] J Van Beek A zur Hausen E K Kranenbarg et al ldquoEBV-positive gastric adenocarcinomas a distinct clinicopathologicentity with a low frequency of lymph node involvementrdquoJournal of Clinical Oncology vol 22 no 4 pp 664ndash670 2004

[10] T O Yau C-M Tang and J Yu ldquoEpigenetic dysregulation inEpstein-Barr virus-associated gastric carcinoma disease andtreatmentsrdquo World Journal of Gastroenterology vol 20 no 21pp 6448ndash6456 2014

[11] K Matsusaka A Kaneda G Nagae et al ldquoClassification ofEpstein-Barr virus-positive gastric cancers by definition ofDNA methylation epigenotypesrdquo Cancer Research vol 71 no23 pp 7187ndash7197 2011

[12] D N Kim and S K Lee ldquoBiogenesis of Epstein-Barr virusmicroRNAsrdquoMolecular and Cellular Biochemistry vol 365 no1-2 pp 203ndash210 2012

[13] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[14] H P Bogerd H W Karnowski X Cai J Shin M Pohlersand B R Cullen ldquoA mammalian herpesvirus uses noncanon-ical expression and processing mechanisms to generate viralmicroRNAsrdquoMolecular Cell vol 37 no 1 pp 135ndash142 2010

[15] K W Diebel D J Claypool and L F van Dyk ldquoA conservedRNA polymerase III promoter required for gammaherpesvirusTMER transcription andmicroRNAprocessingrdquoGene vol 544no 1 pp 8ndash18 2014

[16] K W Diebel A L Smith and L F van Dyk ldquoMature and func-tional viral miRNAs transcribed from novel RNA polymeraseIII promotersrdquo RNA vol 16 no 1 pp 170ndash185 2010

[17] E R Feldman M Kara C B Coleman et al ldquoVirus-encodedmicroRNAs facilitate gammaherpesvirus latency and pathogen-esis in vivordquomBio vol 5 no 3 Article ID e00981-14 2014

[18] A W Whisnant T Kehl Q Bao et al ldquoIdentification of novelhighly expressed retroviral MicroRNAs in cells infected bybovine foamy virusrdquo Journal of Virology vol 88 no 9 pp 4679ndash4686 2014

[19] R P Kincaid Y Chen J E Cox A Rethwilm andC S SullivanldquoNoncanonical microRNA (miRNA) biogenesis gives rise toretroviral mimics of lymphoproliferative and immunosuppres-sive host miRNAsrdquo mBio vol 5 no 2 Article ID e00074-142014

[20] J M Burke C R Bass R P Kincaid and C S SullivanldquoIdentification of tri-phosphatase activity in the biogenesisof retroviral microRNAs and RNAP III-generated shRNAsrdquoNucleic Acids Research vol 42 no 22 pp 13949ndash13962 2014

[21] R P Kincaid J M Burke and C S Sullivan ldquoRNA virusmicroRNA that mimics a B-cell oncomiRrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 109 no 8 pp 3077ndash3082 2012

[22] S Pfeffer A Sewer M Lagos-Quintana et al ldquoIdentification ofmicroRNAs of the herpesvirus familyrdquo Nature Methods vol 2no 4 pp 269ndash276 2005

[23] D Cazalla M Xie and J A Steitz ldquoA primate herpesvirus usesthe integrator complex to generate viral microRNAsrdquoMolecularCell vol 43 no 6 pp 982ndash992 2011

[24] M Landthaler A Yalcin and T Tuschl ldquoThe human DiGe-orge syndrome critical region gene 8 and its D melanogasterhomolog are required for miRNA biogenesisrdquo Current Biologyvol 14 no 23 pp 2162ndash2167 2004

[25] V Ambros ldquoThe functions of animal microRNAsrdquo Nature vol431 no 7006 pp 350ndash355 2004

[26] A Grundhoff and C S Sullivan ldquoVirus-encoded microRNAsrdquoVirology vol 411 no 2 pp 325ndash343 2011

[27] S Pfeffer M Zavolan F A Grasser et al ldquoIdentification ofvirus-encodedmicroRNAsrdquo Science vol 304 no 5671 pp 734ndash736 2004

[28] X Cai A Schafer S Lu et al ldquoEpstein-Barr virus microRNAsare evolutionarily conserved and differentially expressedrdquo PLoSPathogens vol 2 no 3 article e23 2006

[29] R H Edwards A R Marquitz and N Raab-Traub ldquoEpstein-Barr virus BART microRNAs are produced from a large intronprior to splicingrdquo Journal of Virology vol 82 no 18 pp 9094ndash9106 2008

[30] H Chen J Huang F Y Wu G Liao L Hutt-Fletcher and S DHayward ldquoRegulation of expression of the Epstein-Barr virusBamHI-A rightward transcriptsrdquo Journal of Virology vol 79 no3 pp 1724ndash1733 2005

[31] D N Kim H-S Chae S T Oh et al ldquoExpression of viralmicroRNAs in Epstein-Barr virus-associated gastric carci-nomardquo Journal of Virology vol 81 no 2 pp 1033ndash1036 2007

[32] S M Jun Y S Hong J S Seo Y H Ko C W Yang and S KLee ldquoViral microRNA profile in Epstein-Barr virus-associatedperipheral T cell lymphomardquo British Journal of Haematologyvol 142 no 2 pp 320ndash323 2008

[33] C M Tsang and S W Tsao ldquoThe role of Epstein-Barr virusinfection in the pathogenesis of nasopharyngeal carcinomardquoVirologica Sinica vol 30 no 2 pp 107ndash121 2015

[34] R P Kincaid andC S Sullivan ldquoVirus-encodedmicroRNAs anoverview and a look to the futurerdquo PLoS Pathogens vol 8 no12 Article ID e1003018 2012

[35] S M Tugizov J W Berline and J M Palefsky ldquoEpstein-Barr virus infection of polarized tongue and nasopharyngealepithelial cellsrdquoNatureMedicine vol 9 no 3 pp 307ndash314 2003

[36] C Shannon-Lowe and M Rowe ldquoEpstein-Barr virus infectionof polarized epithelial cells via the basolateral surface bymemory B cell-mediated transfer infectionrdquo PLoS Pathogensvol 7 no 5 Article ID e1001338 2011

[37] M Fukayama and T Ushiku ldquoEpstein-Barr virus-associatedgastric carcinomardquo Pathology Research and Practice vol 207 no9 pp 529ndash537 2011

[38] M Sugiura S Imai M Tokunaga et al ldquoTranscriptionalanalysis of Epstein-Barr virus gene expression in EBV-positivegastric carcinoma unique viral latency in the tumour cellsrdquoBritish Journal of Cancer vol 74 no 4 pp 625ndash631 1996

[39] G Niedobitek A Agathanggelou H Herbst L WhiteheadD H Wright and L S Young ldquoEpstein-Barr virus (EBV)


infection in infectious mononucleosis virus latency replicationand phenotype of EBV-infected cellsrdquo Journal of Pathology vol182 no 2 pp 151ndash159 1997

[40] L Frappier ldquoRole of EBNA1 in NPC tumourigenesisrdquo Seminarsin Cancer Biology vol 22 no 2 pp 154ndash161 2012

[41] N Sivachandran F Sarkari and L Frappier ldquoEpstein-Barrnuclear antigen 1 contributes to nasopharyngeal carcinomathrough disruption of PML nuclear bodiesrdquo PLoS Pathogensvol 4 no 10 Article ID e1000170 2008

[42] V H J Wood J D OrsquoNeil WWei S E Stewart CW Dawsonand L S Young ldquoEpstein-Barr virus-encoded EBNA1 regulatescellular gene transcription andmodulates the STAT1 and TGF120573signaling pathwaysrdquo Oncogene vol 26 no 28 pp 4135ndash41472007

[43] G T-Y Chung W P-K Lou C Chow et al ldquoConstitutiveactivation of distinctNF-120581B signals in EBV-associated nasopha-ryngeal carcinomardquoThe Journal of Pathology vol 231 no 3 pp311ndash322 2013

[44] B Luo Y Wang X-F Wang et al ldquoExpression of Epstein-Barr virus genes in EBV-associated gastric carcinomasrdquo WorldJournal of Gastroenterology vol 11 no 5 pp 629ndash633 2005

[45] C W Dawson R J Port and L S Young ldquoThe role of theEBV-encoded latentmembrane proteins LMP1 and LMP2 in thepathogenesis of nasopharyngeal carcinoma (NPC)rdquo Seminars inCancer Biology vol 22 no 2 pp 144ndash153 2012

[46] R Hino H Uozaki Y Inoue et al ldquoSurvival advantage of EBV-associated gastric carcinoma survivin up-regulation by virallatent membrane protein 2Ardquo Cancer Research vol 68 no 5pp 1427ndash1435 2008

[47] F Scholle K M Bendt and N Raab-Traub ldquoEpstein-Barr virusLMP2A transforms epithelial cells inhibits cell differentiationand activates Aktrdquo Journal of Virology vol 74 no 22 pp 10681ndash10689 2000

[48] M Fukuda and R Longnecker ldquoEpstein-Barr virus latent mem-brane protein 2Amediates transformation through constitutiveactivation of the RasPI3-KAkt pathwayrdquo Journal of Virologyvol 81 no 17 pp 9299ndash9306 2007

[49] J A Morrison and N Raab-Traub ldquoRoles of the ITAM and PYmotifs of Epstein-Barr virus latent membrane protein 2A in theinhibition of epithelial cell differentiation and activation of 120573-catenin signalingrdquo Journal of Virology vol 79 no 4 pp 2375ndash2382 2005

[50] B A Barber and M Rastegar ldquoEpigenetic control of Hoxgenes during neurogenesis development and diseaserdquo Annalsof Anatomy vol 192 no 5 pp 261ndash274 2010

[51] S Sharma T K Kelly and P A Jones ldquoEpigenetics in cancerrdquoCarcinogenesis vol 31 no 1 pp 27ndash36 2009

[52] M Rijnkels E Kabotyanski M B Montazer-Torbati et alldquoThe epigenetic landscape ofmammary gland development andfunctional differentiationrdquo Journal of Mammary Gland Biologyand Neoplasia vol 15 no 1 pp 85ndash100 2010

[53] Y Sukawa H Yamamoto K Nosho et al ldquoAlterations inthe human epidermal growth factor receptor 2-phosphatidy-linositol 3-kinase-v-Akt pathway in gastric cancerrdquoWorld Jour-nal of Gastroenterology vol 18 no 45 pp 6577ndash6586 2012

[54] K Wang J Kan S T Yuen et al ldquoExome sequencing identifiesfrequent mutation of ARID1A in molecular subtypes of gastriccancerrdquo Nature Genetics vol 43 no 12 pp 1219ndash1223 2011

[55] Q Liang X Yao S Tang et al ldquoIntegrative identification ofEpstein-Barr virus-associated mutations and epigenetic alter-ations in gastric cancerrdquo Gastroenterology vol 147 no 6 pp1350ndash1362e4 2014

[56] The Cancer Genome Atlas Research Network ldquoComprehensivemolecular characterization of gastric adenocarcinomardquoNaturevol 513 no 7517 pp 202ndash209 2014

[57] A Shinozaki-Ushiku A Kunita and M Fukayama ldquoUpdateon epstein-barr virus and gastric cancer (review)rdquo InternationalJournal of Oncology vol 46 no 4 pp 1421ndash1434 2015

[58] R Feil and M F Fraga ldquoEpigenetics and the environmentemerging patterns and implicationsrdquo Nature Reviews Geneticsvol 13 no 2 pp 97ndash109 2012

[59] T Ushijima and E Okochi-Takada ldquoAberrant methylations incancer cells where do they come fromrdquo Cancer Science vol96 no 4 pp 206ndash211 2005

[60] J Zhao Q Liang K-F Cheung et al ldquoGenome-wide identifica-tion of Epstein-Barr virus-driven promotermethylation profilesof human genes in gastric cancer cellsrdquoCancer vol 119 no 2 pp304ndash312 2013

[61] A Kaneda K Matsusaka H Aburatani and M FukayamaldquoEpstein-Barr virus infection as an epigenetic driver of tumori-genesisrdquo Cancer Research vol 72 no 14 pp 3445ndash3450 2012

[62] K Matsusaka S Funata M Fukayama and A Kaneda ldquoDNAmethylation in gastric cancer related to helicobacter pylori andEpstein-Barr virusrdquo World Journal of Gastroenterology vol 20no 14 pp 3916ndash3926 2014

[63] MKusanoMToyotaH Suzuki et al ldquoGenetic epigenetic andclinicopathologic features of gastric carcinomas with the CpGisland methylator phenotype and an association with Epstein-Barr virusrdquo Cancer vol 106 no 7 pp 1467ndash1479 2006

[64] L Zong and Y Seto ldquoCpG island methylator phenotype Heli-cobacter pylori Epstein-Barr virus andmicrosatellite instabilityand prognosis in gastric cancer a systematic review and meta-analysisrdquo PLoS ONE vol 9 no 1 Article ID e86097 2014

[65] T Okada M Nakamura J Nishikawa et al ldquoIdentification ofgenes specifically methylated in Epstein-Barr virus-associatedgastric carcinomasrdquo Cancer Science vol 104 no 10 pp 1309ndash1314 2013

[66] M Saito J Nishikawa T Okada et al ldquoRole of DNA methyla-tion in the development of Epstein-Barr virus-associated gastriccarcinomardquo Journal of Medical Virology vol 85 no 1 pp 121ndash127 2013

[67] M-S Chang H Uozaki J-M Chong et al ldquoCpG islandmethylation status in gastric carcinoma with and withoutinfection of Epstein-Barr virusrdquo Clinical Cancer Research vol12 no 10 pp 2995ndash3002 2006

[68] GHKang S LeeWHKim et al ldquoEpstein-Barr virus-positivegastric carcinoma demonstrates frequent aberrant methyla-tion of multiple genes and constitutes CpG island methylatorphenotype-positive gastric carcinomardquoTheAmerican Journal ofPathology vol 160 no 3 pp 787ndash794 2002

[69] R Hino H Uozaki N Murakami et al ldquoActivation of DNAmethyltransferase 1 by EBV latent membrane protein 2A leadsto promoter hypermethylation of PTEN gene in gastric carci-nomardquo Cancer Research vol 69 no 7 pp 2766ndash2774 2009

[70] S Ohfuji M Osaki S Tsujitani M Ikeguchi T Sairenji andH Ito ldquoLow frequency of apoptosis in Epstein-Barr virus-associated gastric carcinoma with lymphoid stromardquo Interna-tional Journal of Cancer vol 68 no 6 pp 710ndash715 1996

[71] J Zhao Q Liang K-F Cheung et al ldquoSomatostatin receptor 1 anovel EBV-associated CpG hypermethylated gene contributesto the pathogenesis of EBV-associated gastric cancerrdquo BritishJournal of Cancer vol 108 no 12 pp 2557ndash2564 2013


[72] J Kim H S Lee S I Bae Y M Lee and W H KimldquoSilencing and CpG island methylation of GSTP1 is rare inordinary gastric carcinomas but common inEpstein-Barr virus-associated gastric carcinomasrdquo Anticancer Research vol 25 no6 pp 4013ndash4019 2005

[73] G A Calin C D Dumitru M Shimizu et al ldquoFrequentdeletions and down-regulation of micro-RNA genes miR15 andmiR16 at 13q14 in chronic lymphocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 99 no 24 pp 15524ndash15529 2002

[74] G A Calin C Sevignani C D Dumitru et al ldquoHumanmicroRNA genes are frequently located at fragile sites andgenomic regions involved in cancersrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 9 pp 2999ndash3004 2004

[75] R Feederle S D Linnstaedt H Bannert et al ldquoA viralmicroRNA cluster strongly potentiates the transforming prop-erties of a human herpesvirusrdquo PLoS Pathogens vol 7 no 2Article ID e1001294 2011

[76] M J Strong G Xu J Coco et al ldquoDifferences in gastric car-cinoma microenvironment stratify according to EBV infectionintensity implications for possible immune adjuvant therapyrdquoPLoS Pathogens vol 9 no 5 Article ID e1003341 2013

[77] A R Marquitz and N Raab-Traub ldquoThe role of miRNAs andEBV BARTs in NPCrdquo Seminars in Cancer Biology vol 22 no 2pp 166ndash172 2012

[78] D Iwakiri Y Eizuru M Tokunaga and K Takada ldquoAutocrinegrowth of Epstein-Barr virus-positive gastric carcinoma cellsmediated by anEpstein-Barr virus-encoded small RNArdquoCancerResearch vol 63 no 21 pp 7062ndash7067 2003

[79] A Nanbo and K Takada ldquoThe role of Epstein-Barr virus-encoded small RNAs (EBERs) in oncogenesisrdquo Reviews inMedical Virology vol 12 no 5 pp 321ndash326 2002

[80] A S Banerjee A D Pal and S Banerjee ldquoEpstein-Barr virus-encoded small non-coding RNAs induce cancer cell chemore-sistance and migrationrdquo Virology vol 443 no 2 pp 294ndash3052013

[81] E Y-W Choy K-L Siu K-H Kok et al ldquoAn Epstein-Barrvirus-encoded microRNA targets PUMA to promote host cellsurvivalrdquoThe Journal of Experimental Medicine vol 205 no 11pp 2551ndash2560 2008

[82] A R Marquitz A Mathur C S Nam and N Raab-Traub ldquoTheEpstein-Barr Virus BART microRNAs target the pro-apoptoticprotein Bimrdquo Virology vol 412 no 2 pp 392ndash400 2011

[83] L Dolken G Malterer F Erhard et al ldquoSystematic analysis ofviral and cellular microRNA targets in cells latently infectedwith human 120574-herpesviruses by RISC immunoprecipitationassayrdquo Cell Host amp Microbe vol 7 no 4 pp 324ndash334 2010

[84] E Lomonosova and G Chinnadurai ldquoBH3-only proteins inapoptosis and beyond an overviewrdquo Oncogene vol 27 supple-ment 1 pp S2ndashS19 2008

[85] R L Skalsky D L Corcoran E Gottwein et al ldquoThe viraland cellular microRNA targetome in lymphoblastoid cell linesrdquoPLoS Pathogens vol 8 no 1 Article ID e1002484 2012

[86] G Bellot P-F Cartron E Er et al ldquoTOM22 a core componentof the mitochondria outer membrane protein translocationpore is a mitochondrial receptor for the proapoptotic proteinBaxrdquo Cell Death and Differentiation vol 14 no 4 pp 785ndash7942007

[87] R W-M Lung J H-M Tong and K-F To ldquoEmerging roles ofsmall Epstein-Barr virus derived non-coding RNAs in epithelial

malignancyrdquo International Journal of Molecular Sciences vol 14no 9 pp 17378ndash17409 2013

[88] D N Kim M K Seo H Choi et al ldquoCharacterization ofnaturally Epstein-Barr virus-infected gastric carcinoma cell lineYCCEL1rdquo Journal of General Virology vol 94 no 3 pp 497ndash506 2013

[89] J Qiu K Cosmopoulos M Pegtel et al ldquoA novel persistenceassociated EBV miRNA expression profile is disrupted inneoplasiardquo PLoS Pathogens vol 7 no 8 Article ID e10021932011

[90] A Shinozaki-Ushiku A Kunita M Isogai et al ldquoProfilingof virus-encoded microRNAs in Epstein-Barr virus-associatedgastric carcinoma and their roles in gastric carcinogenesisrdquoJournal of Virology vol 89 no 10 pp 5581ndash5591 2015

[91] H Kim H Choi and S K Lee ldquoEpstein-Barr virus miR-BART20-5p regulates cell proliferation and apoptosis by target-ing BADrdquo Cancer Letters vol 356 no 2 part B pp 733ndash7422015

[92] T Kanda M Miyata M Kano S Kondo T Yoshizaki and HIizasa ldquoClustered microRNAs of the Epstein-Barr virus coop-eratively downregulate an epithelial cell-specific metastasissuppressorrdquo Journal of Virology vol 89 no 5 pp 2684ndash26972015

[93] K Jiang Z Shen Y Ye X Yang and S Wang ldquoA novel molec-ular marker for early detection and evaluating prognosis ofgastric cancer N-myc downstream regulated gene-1 (NDRG1)rdquoScandinavian Journal of Gastroenterology vol 45 no 7-8 pp898ndash908 2010

[94] Q Fu C He and Z-R Mao ldquoEpstein-Barr virus interactionswith the Bcl-2 protein family and apoptosis in human tumorcellsrdquo Journal of Zhejiang University Science B vol 14 no 1 pp8ndash24 2013

[95] A Shinozaki T Sakatani T Ushiku et al ldquoDownregulation ofmicroRNA-200 in EBV-associated gastric carcinomardquo CancerResearch vol 70 no 11 pp 4719ndash4727 2010

[96] Y Du Y Xu L Ding et al ldquoDown-regulation of miR-141 ingastric cancer and its involvement in cell growthrdquo Journal ofGastroenterology vol 44 no 6 pp 556ndash561 2009

[97] A R Marquitz A Mathur K H Y Shair and N Raab-TraubldquoInfection of Epstein-Barr virus in a gastric carcinoma cell lineinduces anchorage independence and global changes in geneexpressionrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 109 no 24 pp 9593ndash95982012

[98] P M Borralho A E S Simoes S E Gomes et al ldquomiR-143 overexpression impairs growth of human colon carcinomaxenografts inmice with induction of apoptosis and inhibition ofproliferationrdquo PLoS ONE vol 6 no 8 Article ID e23787 2011

[99] X Chen X Guo H Zhang et al ldquoRole of miR-143 targetingKRAS in colorectal tumorigenesisrdquoOncogene vol 28 no 10 pp1385ndash1392 2009

[100] Y Ni L Meng L Wang et al ldquoMicroRNA-143 functions as atumor suppressor in human esophageal squamous cell carci-nomardquo Gene vol 517 no 2 pp 197ndash204 2013

[101] A R Marquitz A Mathur P E Chugh D P Dittmer and NRaab-Traub ldquoExpression profile of microRNAs in Epstein-Barrvirus-infected AGS gastric carcinoma cellsrdquo Journal of Virologyvol 88 no 2 pp 1389ndash1393 2014

[102] H Iizasa B-E Wulff N R Alla et al ldquoEditing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicertargeting and consequently affects viral latencyrdquo The Journal ofBiological Chemistry vol 285 no 43 pp 33358ndash33370 2010


[103] H Choi H Lee S R Kim Y S Gho and S K LeeldquoEpstein-Barr virus-encoded microRNA BART15-3p promotescell apoptosis partially by targetingBRUCErdquo Journal of Virologyvol 87 no 14 pp 8135ndash8144 2013

[104] M Tokunaga Y Uemura T Tokudome et al ldquoEpstein-Barrvirus related gastric cancer in Japan a molecular patho-epidemiological studyrdquo Acta Pathologica Japonica vol 43 no10 pp 574ndash581 1993

[105] B R Cullen ldquoViruses andmicroRNAsrdquoNatureGenetics vol 38no 1 pp S25ndashS30 2006

[106] C S Sullivan ldquoNew roles for large and small viral RNAs inevading host defencesrdquo Nature Reviews Genetics vol 9 no 7pp 503ndash507 2008

[107] J L Kutok and F Wang ldquoSpectrum of Epstein-Barr virus-associated diseasesrdquoAnnual Review of Pathology vol 1 pp 375ndash404 2006

[108] L S Young and A B Rickinson ldquoEpstein-Barr virus 40 yearsonrdquo Nature Reviews Cancer vol 4 no 10 pp 757ndash768 2004

[109] L S Young and P G Murray ldquoEpstein-Barr virus and oncoge-nesis from latent genes to tumoursrdquo Oncogene vol 22 no 33pp 5108ndash5121 2003

[110] V S Thakur K Gupta and S Gupta ldquoThe chemopreventiveand chemotherapeutic potentials of tea polyphenolsrdquo CurrentPharmaceutical Biotechnology vol 13 no 1 pp 191ndash199 2012

[111] C Cabrera R Artacho and R Gimenez ldquoBeneficial effectsof green teamdasha reviewrdquo Journal of the American College ofNutrition vol 25 no 2 pp 79ndash99 2006

[112] E C H Yiannakopoulou ldquoRecent patents on antibacterialantifungal and antiviral properties of teardquo Recent Patents onAnti-Infective Drug Discovery vol 7 no 1 pp 60ndash65 2012

[113] H K Tae H L Jin K S Chung et al ldquoEpigallocatechin-3-gallate enhances CD8+ T cell-mediated antitumor immunityinduced by DNA vaccinationrdquo Cancer Research vol 67 no 2pp 802ndash811 2007

[114] A B Rickinson ldquoCo-infections inflammation andoncogenesisfuture directions for EBV researchrdquo Seminars in Cancer Biologyvol 26 pp 99ndash115 2014

[115] T BDeramaudt CDill andMBonay ldquoRegulation of oxidativestress by Nrf2 in the pathophysiology of infectious diseasesrdquoMedecine et Maladies Infectieuses vol 43 no 3 pp 100ndash1072013

[116] Z Yan T Yong-Guang L Fei-Jun T Fa-Qing T Min and CYa ldquoInterference effect of epigallocatechin-3-gallate on targetsof nuclear factor 120581B signal transduction pathways activated byEB virus encoded latent membrane protein 1rdquoThe InternationalJournal of Biochemistry amp Cell Biology vol 36 no 8 pp 1473ndash1481 2004

[117] C Di Lorenzo M DellrsquoAgli E Sangiovanni et al ldquoCorrelationbetween catechin content and NF-120581B inhibition by infusions ofgreen and black teardquo Plant Foods for Human Nutrition vol 68no 2 pp 149ndash154 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Gastric carcinoma andother malignancies

DNMTsinduction

Viral mRNAdegradation or

translationinhibition

Degradation DegradationTranslation Translation

Host cell mRNAdegradation or

translation inhibition

Aberrant DNA methylation andinactivation of tumor suppressor

genes

Host cell genome

Increase persistentlatent infection in the

host cells

Reduce hostimmune

surveillance

Regulate cell signalingcell division and

apoptosis

RiscViral miRNAs

Viral proteins(LMP2A)

ViralDNA

genome

Figure 1 Graphical representation of possible mechanisms by which viral miRNAs and viral proteins might contribute to EBV-associatedgastric carcinogenesis This model indicates that EBV-encoded miRNAs (eg BART miRNAs and BHRF-1 miRNAs) target viral genes tomediate immune evasion or maintenance of latency whereas some viral proteins (eg LMP2A) promote aberrant host DNA methylationand subsequent inactivation of tumor suppressor genes via DNA methyltransferases (DNMTs) induction In addition these viral miRNAsincorporated into RISC complex can also interact directly with specific host genes involved in immune surveillance cell proliferation andapoptosis playing a crucial role in the aetiology of diverse diseases including EBVaGC

most common cancer in the world and the second leadingcause of cancer-related death Globally gastric cancer posesa significant public health burden both economically andsocially [7 8] Risk factors of gastric cancer aremultifactorialhence genes diet age and chronic inflammation need tobe evaluated in connection with infectious agents (EBVHelicobacter pylori) and environmental factors (eg alcoholand smoking) [8] Notably EBV-associated gastric carcinoma(EBVaGC) represents almost 10 of all gastric carcinomacases and expresses restricted EBV latent genes (Latency I)[4 9] In recent years it has become increasingly evident thatEBV may contribute to gastric carcinogenesis through theexpression of viral proteins and microRNAs (miRNAs) [10](Figure 1) A growing body of scientific evidence also suggeststhat in addition to genetic alterations epigenetic alterationsincluding aberrant DNA methylation of CpG islands andposttranslational modifications of histones are involved inthe development and progression of EBVaGC [10 11] Thisreview briefly summarizes remarkable advancements in ourunderstanding of the functions and mechanisms of action ofherpesviral miRNAs in gastric carcinoma in recent years Inparticular it discusses how the expression of viral proteins andepigenetic alterations contribute to EBVaGC and the rolesof EBV-encoded and virally modulated cellular miRNAs in

the respective viral life cycles and in EBV-associated gastriccarcinoma

2 Biogenesis of EBV-Encoded miRNAs

The generation of viral miRNA and selection of targets aretotally dependent on the host molecular miRNA apparatusinvolved in the maturation and silencing Most viruses utilizea strategy similar to that of the host cell to produce the viralpri-miRNAs by using the host RNA polymerase II (RNAP II)[12 13] Exceptionally certain viral miRNAs (such as thosefrom mouse 120574-herpesvirus 68 MHV68) are produced fromtRNA-like genes transcribed by host RNA polymerase III(RNAP III) and processed Drosha independently by hosttRNase Z [14] Aminority of viruses from the herpesvirus andretrovirus families also utilize noncanonical pathways otherthan ldquotRNA-likerdquo to generate pre-miRNAmolecules [15ndash22]For instance herpesvirus saimiri (HVS) an oncogenic 120574-herpesvirus that infects NewWorldmonkeys expresses smallnuclear RNAs (snRNAs) of the Sm-class called HSURs whichare processed by the integrator complex to produce viralpre-miRNAs Subsequently both the MHV68 and HVS pre-miRNAs are processed by Dicer to generate miRNAs [23]Additionally some retroviruses such as foamy viruses (FVs)























Review [57]







Kanda et al2015





Tokunaga et al1993










6 Conclusions





Acknowledgments


References

































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































Review [57]







Kanda et al2015





Tokunaga et al1993










6 Conclusions





Acknowledgments


References

































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















6 Conclusions





Acknowledgments


References

































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article role of viral mirnas and epigenetic...

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