**6. Epigenetic abnormalities of EBV-associated gastric carcinoma: microRNAs**

MicroRNAs (miRNAs) are small (~22 nt) noncoding RNAs and fundamental in posttranscriptional regulation of a broad range of biological processes of different organisms. This fundamental regulation is achieved through specific complementary binding to the 3′ untranslated region (3′UTR) of one or more target mRNAs, allowing regulation of multiple genes simultaneously [80] (**Figure 7**). Increasing evidence has shown that dysregulation of specific miRNAs has a crucial role in tumorigenesis. In fact, microRNAs involved in this process are usually called oncomiRs and anti-oncomiRs, acting similar to onco- and tumorsuppressor genes [11]. Particularly, in gastric cancer, cellular miRNAs have gained special interest because they have shown differential expression between distinct cancer subtypes and have been related to progression and prognosis of the disease (for a review, see [80]). Viral microRNAs were first described in EBV [81]. It is now known that diverse virus families

characteristic of the microsatellite instability (MSI) GC subtype [21, 27, 66–71]. Individual methylation of p14ARF and p16INK4a, alternative reading frames of CDKN2A locus, has also been described, and many studies have proved a significant association between their methylation and EBV positivity [72, 73]. Ushiku et al. [74] observed a uniform methylation of all CpG sites on promoter regions of both genes in EBVaGC, whereas it was variable in EBV-negative tumors. In addition, methylation frequency of p16INK4a appears to be about three times higher in EBVaGC than in EBV-negative tumors [65, 75]. CDH1, p15 and p73 tumor suppressor genes are also frequently methylated in EBVaGC, representing one of the

**Figure 6.** DNA methylation. (A) CpG methylation mechanism is mediated by DNA methyltransferases and consists in the addition of a methyl group to the carbon in the 5th position of cytosines that precedes guanine nucleotides. (B) CpG islands are DNA sequences rich in CpG sites (>50% CpG sites within a 200bp sequence). Methylation of CpG islands

Despite EBV-induced host gene methylation in EBVaGC is well established, the exact underlying mechanisms are not entirely understood. It has been proposed that when the host cell detects the viral genome, it defends itself by starting a host-driven extensive methylation of the foreign genome, which may trigger the subsequent host genome methylation [75–77]. However, based on the specific methylation patterns observed, a possible participation of EBV in maintenance and de novo methylation has been proposed [72, 74]. Several studies have shown that EBV can modulate the expression of DNA methyltransferases (DNMT), which catalyze the transfer of methyl groups to DNA. Specifically, LMP-2A, EBV latent gene, has been shown to upregulate DNMT1 and DNMT3b expression in gastric cancer cell lines, which further induced methylation of several tumor suppressor genes, such as PTEN [78, 79]. Therefore, LMP-2A may play an essential role in the epigenetic abnormalities in host

most common abnormalities described in this tumor [65, 74].

inside a promoter region may control gene expression.

16 Gastric Cancer

cells and in the development and maintenance of EBV-associated cancer.

**Figure 7.** Canonical pathway of miRNA biogenesis in human. miRNAs are transcribed by RNA polymerase II (RNAP II) from intergenic, intronic or polycistronic loci to long primary transcript, called primary miRNA (pri-miRNA), which consists in a stem, a terminal loop and single-stranded RNA segments at both the 5′- and 3′-UTR sides. Microprocessor complex (Drosha and DGCR8 cofactor) cleaves the stem-loop and releases a small hairpin-shaped RNA, called precursor miRNA (pre-miRNA). Following, pre-miRNA is exported into the cytoplasm by the transport complex formed by protein exportin 5 (EXP5) and GTP-binding nuclear protein RAN•GTP. Subsequently, pre-miRNAs are cleaved by a ternary complex formed by dicer, TAR RNA-binding protein (TRBP) and protein activator of PKR (PACT), producing a small RNA duplexes (miRNA-miRNA\*). Next, these are loaded onto an Argonaute protein (AGO) to form an immature RNA-induced silencing complex (RISC) or pre-RISC, in a process mediated for heat shock cognate 70 (Hsc70)-heat shock protein (Hsp90) chaperone complex. AGO protein separates the two strands to generate a mature RISC effector. Finally, RISC binds the target mRNA through complementary binding of six to eight base pairs of the miRNA, with a specific sequence of the target resulting in the gene silencing. Taken from [80] with permission.

encode miRNAs and that they are capable of targeting both cellular and viral genes [82]. EBV-miRNA expression is dependent on the host cellular miRNA processing machinery for its biogenesis. EBV miRNAs are encoded in two clusters within the EBV genome. As shown in **Figure 1**, the first cluster is localized in the Bam HI fragment H rightward open reading frame 1 (BHRF1) gene and originates four mature miRNAs, which express only during lytic infection and in latency type IIb/III [83]. The second cluster is localized among the Bam HI-A region rightward transcript (BART) gene, encoding 40 mature miRNAs [84], which are expressed in all EBV latency types [83]. However, variable expression patterns of BART miRNAs have been reported in different EBV-associated malignancies or cell types [83, 85]. Additionally, discrepant reports exist concerning specific BART miRNAs' relative expression within the same cellular context, which could be in part a result of different EBV strains studied [86–89]. Most BART miRNAs host targets, identified so far, are involved in proapoptotic and immune response pathways, suggesting a crucial role in promoting host cell survival [90]. For instance, EBVaGC highly expressed miRBART4-5p and miRBART5-5p that have been shown to target and downregulate the BH3-interacting domain death agonist (BID) protein [91] and the p53-upregulated modulator of apoptosis (PUMA) [92], respectively. Furthermore, EBV not only expresses its own miRNAs but also alters miRNAs' expression of the host cells. Particularly, miR-200 family has been shown to be consistently downregulated both in GC cell lines and in EBVaGC tumor samples compared to normal adjacent mucosa and other GC subtypes [93, 94]. Aberrant DNA methylation following EBV infection and viral proteins such as BRAF0, EBER, and LMP-2A have been proposed as the main mechanisms of downregulation of these miRNAs [93, 95].
