**4. The dysregulated miRNAs involved in GC**

**3. Molecular classification and pathogenies of GC**

10 Stomach Disorders

therapeutic potential for this kind of molecular subtype.

Up to 90% of stomach malignancies are adenocarcinomas. Non-Hodgkin's lymphomas and gastrointestinal stromal tumor (GIST) make up most of the remaining 10% [31]. Even though infrequently, adenosquamous, squamous, and undifferentiated carcinomas also occur. In regard to clinical diagnosis, several pathological characterization varied from time to time. Several histological classification systems for gastric adenocarcinoma have been described, but the most frequently used are those of the World Health Organization (WHO) and Lauren [32]. In the World Health Organization (WHO) classification, there are 10 histological types [33]. The Lauren classification is commonly applied and it makes the distinction between intestinal and diffuse types. The intestinal GC consists of cohesive neoplastic cells forming gland-like structures while the diffuse type has lost cell cohesion and resulting in diffuse discohesive cellular infiltration [31]. Men and elderly are more likely to suffer intestinal type, whereas diffuse type carcinomas are relatively more common among the younger population with an equal male-tofemale ratio [32]. Recently, a project named The Cancer Genome Atlas (TCGA) has proposed a brand new classification, in which GC is grouped by four subtypes: EBV-positive (EBV), micro-

satellite instability (MSI), genomically stable [34], and chromosomal instability [35, 36].

According to previous studies, about 9% of GC cases are infected by EBV [37]. All the EBVpositive GCs harbor the property of CpG island methylator phenotype (CIMP) [36, 38, 39]. EBV-positive tumors exhibited a higher incidence of whole-genomic DNA hypermethylation than any molecular subtypes. The genes with promoter hypermethylation showed most differentially silenced expression in EBV-associated GC [36]. Moreover, PI(3)-kinase inhibition was also strongly detected in EBV-positive GC, which offered a new method for the evaluation of this subtype [36]. The most highly transcribed EBV viral, message RNAs (mRNAs) and microRNAs (miRNAs), fell within the BamH1A region of the viral genome and showed similar expression patterns across tumors [36]. The mutation rate of PIK3CA is exclusively high in EBV-positive gastric cancer compared with other molecular subtypes. The mutation rate of PIK3CA in this subtype is about 80 and 68% of the mutations belongs to recurrent mutation in this dataset. In contrast, in other molecular subtypes, the mutation rates of PIK3CA are from 3 to 42%. So, this result provides a hint that using PI3K inhibitor might have the clinical

The next subtypes of GC are abundant in MSI, which display increased mutation rates (in major histocompatibility complex class I genes, including B2M and HLA-B) and hypermethylation (containing hypermethylation at the *MLH1* promoter). The most obvious difference between EBV-CIMP (CpG island methylator phenotype) and MSI-associated gastric-CIMP methylation profiles is that all EBV-positive gastric tumors show promoter hypermethylation of *CDKN2A* (*p16INK4A*), but the *MLH1* hypermethylation was only detected in MSI-associated CIMP [38].

In genomically stable subtype, *RHOA* mutation was detected [36]. When binding with Guanosine-5'-triphosphate (GTP), RHOA behaves through a great number of downstream effectors, such as ROCK1, mDIA, and protein kinase N. This will lead to actin-myosin-dependent cell contractility and cellular motility [40, 41] and activation of STAT3 to promote carcinogenesis [42, 43]. Except from activating mDia or ROCK1, the *RHOA* mutation Y42C has been confirmed to attenuate the MicroRNAs (miRNAs) are one predominant category of small (roughly 20–30 nucleotides) non-coding RNAs that participate in gene expression and control [44]. Their effects are mostly lead to the degradation of message RNAs (mRNAs) or inhibitory of the translations, and subsequently affect a series of biological behaviors of cells, such as inflammation, cell proliferation, apoptosis and differentiation. In the nucleus, together with its cofactor Pasha (DGCR8), the RNase III enzyme Drosha cut out primary miRNA transcripts into a fragment of approximately 60 nucleotides named precursor miRNAs (pre-miRNAs), and initiate the biogenesis of miRNAs [45]. A cytoplasmic RNase III called Dicer will be responsible for the further processing of the pre-miRNAs and makes them mature after they are transported to the cytoplasm [46, 47]. A mature miRNA, with the length of about 18–24 nucleotides, is singlestranded, which can sometimes aim at multiple targets. These mature miRNAs always bind to the complementary sequences of targeted mRNAs directly to make mRNAs degrade or bind directly to 3′-untranslated regions (3′-UTR) of mRNAs to decrease their translation, so that miRNAs can exert their effects on regulating certain gene expression [44, 48]. Accordingly, miRNAs regulate at least 30% of genes of human as it is estimated [49].

In other words, miRNAs are capable of acting as a switch to control genes related to cell proliferation and apoptosis under pathogenic circumstances, consequently, they may have a chance to be involved in both cancer initiation and progression. It seems that no matter how clear the mechanism of malignancy behaviors or an effective therapy that might prevent tumorigenesis from the beginning, an increasing knowledge of these miRNAs is crucial. During the physiological periods, miRNAs present or absent in proper time of different stage of lives. However, they are produced abnormally in tumors, that the levels of some miRNAs are highly detected while some are lower or even none. Hence, those which are upregulated called onco-miRs, whereas the downregulated ones called tumor-suppressive miRNAs. As the names suggested, genes controlled by onco-miRs are oncogenes whose products may promote tumor cells in many aspects, whereas the opposite site of genes is tumor suppressive, which plays a role of inhibitor among the initiation or development of tumors (e.g., miR-15a and miR-16-1, which target a member of Hippo pathway called YAP1, are downregulated in GC) [50]. Thus, identifying the target genes of these miRNAs is crucial and it may lead us to a better understanding of the miRNAs themselves.

For example, miR-21 was the first miRNA which was influenced by *H. pylori*. In tissues of both GC and *H. Pylori* infection, it was highly detected [51, 52]. Several data have reported that this miRNA can be used as a biomarker in GC diagnosis in the clinic [53–55]. And most recently, a research conducted in China suggested this miR-21 to be a GC biomarker in both diagnosis and prognosis, for the reason that, besides the high levels found in tumor tissues compared with the normal ones, miR-21 was revealed to be associated with poor survivals in clinical patients [56]. Behind the phenomena, the molecular mechanism is still unclear in GC. In other types of cancer, however, such as colorectal cancer, miR-21 decreased the tumor suppressor protein programmed cell death 4 (PDCD4) and exhibited an oncogenic function [57]. Additionally, in nonsmall cell lung carcinoma, miR-21 was found to deregulate PTEN, a tumor suppressor, to promote carcinogenesis [58].
