MicroRNA dysregulation in gastric cancer: a new ...

Oncogene (2010), 1–11

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REVIEW

MicroRNA dysregulation in gastric cancer: a new player enters the game WKK Wu1, CW Lee1, CH Cho1,2, D Fan3, K Wu3, J Yu1 and JJY Sung1 1

Institute of Digestive Diseases, LKS Institute of Health Sciences and Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, PR China; 2School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, PR China and 3State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an, PR China

Gastric carcinogenesis is a multistep process involving genetic and epigenetic alteration of protein-coding protooncogenes and tumor-suppressor genes. Recent discoveries have shed new light on the involvement of a class of noncoding RNA known as microRNA (miRNA) in gastric cancer. A substantial number of miRNAs show differential expression in gastric cancer tissues. Genes coding for these miRNAs have been characterized as novel protooncogenes and tumor-suppressor genes based on findings that these miRNAs control malignant phenotypes of gastric cancer cells. In this connection, miRNA dysregulation promotes cell-cycle progression, confers resistance to apoptosis, and enhances invasiveness and metastasis. Moreover, certain polymorphisms in miRNA genes are associated with increased risks for atrophic gastritis and gastric cancer, whereas circulating levels of miRNAs may serve as biomarkers for early diagnosis. Several miRNAs have also been shown to correlate with gastric cancer progression, and thus may be used as prognostic markers. Elucidating the biological aspects of miRNA dysregulation may help us better understand the pathogenesis of gastric cancer and promote the development of miRNAdirected therapeutics against this deadly disease. Oncogene advance online publication, 30 August 2010; doi:10.1038/onc.2010.352 Keywords: microRNA; gastric cancer; signaling pathway; proliferation; apoptosis; metastasis

Introduction Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death in the world (Danaei et al., 2005). Both histological subtypes of gastric cancer, namely, the intestinal type and the diffuse type are associated with Helicobacter pylori infection that contributes to more than 80% of cases (Houghton and Wang, 2005). Interactions among host, Correspondence: Dr J Yu or Professor JJY Sung, Institute of Digestive Diseases, LKS Institute of Health Sciences and Department of Medicine and Therapeutics, The Chinese University of Hong Kong, 7/F, LKS Medical Sciences Building, Prince of Wales Hospital, Shatin, NT, Hong Kong, PR China. E-mails: [email protected] or [email protected] Received 14 June 2010; accepted 13 July 2010

environmental and bacterial factors also influence the disease outcome. For instance, strong inflammatory response as a result of cytokine gene polymorphisms (Zambon et al., 2004; Lee et al., 2005; Sakuma et al., 2005), infection with the CagA-positive strain of H. pylori (Blaser et al., 1995) and diet high in salt and nitrate are synergistic risk factors for gastric cancer (Yamaguchi and Kakizoe, 2001). Clinically, the absence of specific symptoms renders early diagnosis of this deadly disease difficult. Gastrectomy remains the mainstay treatment for gastric cancer, but the prognosis for advanced stage patients is still very poor (Catalano et al., 2009). The complex interaction among different etiological factors leads to genetic and epigenetic alterations of proto-oncogenes and tumor-suppressor genes, which underlie the pathogenesis of cancer. It has long been believed that dysregulation of these genes results in abnormal function or expression of oncogenic and tumor-suppressor proteins. Increasing interest in the function of nonprotein-coding genomic sequences, however, has recently led to the discovery that a class of regulatory RNA known as microRNA (miRNA) is involved in the pathogenesis of many types of cancer (Balch et al., 2009; Faber et al., 2009; Mott, 2009; Wang et al., 2009; Rachagani et al., 2010), including gastric cancer (Ueda et al., 2010). miRNA carries out its biological functions by repressing the expression of its target genes through base-pairing with endogenous mRNAs. In this connection, miRNA genes have been characterized as novel proto-oncogenes or tumorsuppressor genes in gastric carcinogenesis (Inui et al., 2010). In this review, the function of miRNA and its relation to signaling pathways governing cancer-related phenotypes, such as proliferation, resistance to apoptosis and invasiveness in gastric cancer will be discussed. On the basis of recent data, we also describe the possible use of miRNA to improve the diagnosis, prognosis and treatment of gastric cancer. Function and biogenesis of miRNA miRNAs are important post-transcriptional regulators of gene expression. They are small noncoding RNAs that are 18–25 nucleotides in length. To date, more than 900 miRNAs have been identified in humans. A single miRNA can downregulate multiple targets, which often belong to the same metabolic or signaling pathway, through its ability to bind to the 30 untranslated regions

Dysregulated miRNA in gastric cancer WKK Wu et al

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Figure 1 Schematic diagram depicting the biogenesis of miRNA and its dysfunction in gastric cancer. miRNA gene is transcribed as primary miRNA (pri-miRNA), which is processed by Drosha to produce precursor miRNA (pre-miRNA) in the nucleus. Pre-miRNA is subsequently exported to the cytoplasm by exportin 5, and further processed by Dicer to form the miRNA:miRNA*duplex. The mature miRNA is incorporated into RNA-induced silencing complex (RISC) to induce gene silencing. The mode of action is determined by the sequence complementarity between the ‘seed region’ of miRNA and binding sites on the 30 untranslated region (30 -UTR) of the target mRNA. In gastric cancer, the biogenesis and gene-silencing function of miRNA are impaired at several points, which are marked by ‘X’.

(UTRs) of target mRNAs, and thus exerts it effects as a master switch for regulation of gene expression. The regulatory function of miRNA depends on the complementarity between its seed region (nucleotide positions 2–8) and the target mRNA. Partial and perfect complementarity results in translational repression and mRNA degradation, respectively. It is estimated that up to 30% of genes in the human genome are regulated by miRNA (Carthew and Sontheimer, 2009). Most miRNA genes are located in intergenic regions or in antisense orientation of certain genes, which contains their own promoters and regulatory elements. However, as much as a quarter of miRNA genes are intronic, sharing their promoters with their host genes. miRNA gene is transcribed by RNA polymerase II in the nucleus, producing primary miRNA that contains a 50 cap, at least one B70-nucleotide hairpin loop structure and a 30 poly(A) tail. However, primary miRNA may be polycistronic, containing up to sevenhairpin loop structures that give rise to different mature miRNAs. After transcription, primary miRNA is processed by a complex consisting of Drosha and its co-factor DGCR8 into precursor miRNA that is Oncogene

deprived of the 50 cap, the 30 poly(A) tail and sequences flanking the hairpin loop structure (Lee et al., 2006a). Precursor miRNAs are subsequently exported by exportin 5 from nucleus to cytoplasm (Lund et al., 2004), where they are further processed by the RNAse Dicer to produce a miRNA:miRNA*duplex of around 20–25 nucleotides in size, which contains the mature miRNA strand and its complementary miRNA*strand (Bernstein et al., 2001). The mature miRNA strand is then incorporated into the RNA-induced silencing complex with the help of Dicer and its co-factors TRBP and PACT (Chendrimada et al., 2005; Lee et al., 2006b). The mature miRNA then guides the RNA-induced silencing complex to its target mRNAs to induce silencing (Tijsterman and Plasterk, 2004; Figure 1). Dysregulation of biogenesis and function of miRNA in gastric cancer There are emerging evidence showing that the biogenesis of miRNA may be dysregulated in cancer. This can be exemplified by the observation that the processing of precursor miRNAs to mature miRNAs is in general


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impaired in cancer cell lines (Lee et al., 2008). Moreover, contact inhibition (Hwang et al., 2009) and major tumor-suppressing pathways, such as p53 (Suzuki et al., 2009) and transforming growth factor-b (TGFb)/bone morphogenetic protein (Davis et al., 2008) promote the maturation of precursor miRNAs, whereas there is a widespread downregulation of miRNAs caused by a failure at the Drosha processing step in some cancer types (Thomson et al., 2006). Moreover, impaired miRNA processing by conditioned deletion of Dicer1 enhances cellular transformation and tumorigenesis (Kumar et al., 2007). Dicer is also downregulated in lung adenocarcinomas (Chiosea et al., 2007). It is therefore generally believed that the miRNAome as a whole has an inhibitory role in carcinogenesis. However, it is noteworthy that Dicer is upregulated in lung squamous cell carcinomas (Chiosea et al., 2007) and prostate carcinomas (Chiosea et al., 2006), indicating a tissue-specific role. In gastric cancer, the mRNA and protein expression of Dicer1 are significantly reduced during disease progression (Zheng et al., 2007). In a subset of gastric cancer with high microsatellite instability, Ago2 and TNRC6A, both of which are related to the execution of the gene-silencing function of miRNA, are also mutated and downregulated (Kim et al., 2010). Ago2 is a major component of RNAinduced silencing complex, whereas TNRC6A associates with Ago proteins and the target mRNA to induce gene silencing (Lazzaretti et al., 2009). These findings indicate that the biogenesis and function of miRNA may be impaired in gastric cancer (Figure 1).

Dysregulated miRNAs in gastric cancer There is an increasing number of studies showing the overexpression or downregulation of specific miRNA in H. pylori-infected gastric mucosa and gastric cancer. The dysregulated miRNAs and their confirmed targets are listed in Supplementary Table 1 (Volinia et al., 2006; Zhang et al., 2007a, 2008, 2009, 2010; Kim do et al., 2007; Chan et al., 2008; Choy et al., 2008; Petrocca et al., 2008; Ando et al., 2009; Bandres et al., 2009; Du et al., 2009; Guo et al., 2009; Katada et al., 2009; Kim et al., 2009; Liu et al., 2009; Saito et al., 2009; Takagi et al., 2009; Chen et al., 2010; Fassi Fehri et al., 2010; Gao et al., 2010; Hashimoto et al., 2010; Jiang et al., 2010; Lai et al., 2010; Matsushima et al., 2010; Motoyama et al., 2008, 2010; Shen et al., 2010; Shinozaki et al., 2010; Tsujiura et al., 2010; Tsukamoto et al., 2010; Ueda et al., 2010; Wan et al., 2010; Wu et al., 2010a; Xia et al., 2008; Xiao et al., 2009; Zhu et al., 2010). miRNA and cell cycle Increased cell proliferation is a common feature of malignancy. Progression through the cell cycle requires an orchestrated expression of cyclins, which results in sequential activation of cyclin-dependent kinases (CDKs) and transcription of phase-specific genes.

Cyclin D and cyclin E, which bind to CDK4/6 and CDK2, respectively, are important gap 1 (G1) cyclins that are rate-limiting for (S) phase entry. After entering the S phase, the expression of cyclin E reduces, but increasing levels of cyclin A maintain the activity of CDK2. Cyclin A/CDK2 complex is active during both S and gap 2 (G2) phases, whereas an elevation in cyclin B/CDK1 activity at the late G2 phase triggers progression to mitosis (M) phase (Malumbres and Barbacid, 2009). Aside from cyclins, CDK activities are regulated by a class of molecules known as CDK inhibitors. Depending on their binding affinities, CDK inhibitors can be categorized into p16 family (p15, p16, p18 and p19; Canepa et al., 2007) and p21 family (p21, p27, p28 and p57; Shou and Dunphy, 1996; Pateras et al., 2009). In general, the former binds to and inhibits the activities of CDK4/6, whereas the latter binds to a broad range of cyclin/CDK complexes, leading to inhibition of all phases of the cell cycle. In addition, the activity of cyclin B/CDK1 and thus G2–M progression are enhanced by CDC25A/B/C and antagonized by Wee1. CDC25A also enhances the activity of cyclin E/A– CDK2 and thereby facilitating the S phase entry and progression (Niida and Nakanishi, 2006). It has been shown that miRNA dysregulation promotes cell-cycle progression by downregulating the expression of CDK inhibitors in gastric carcinogenesis. For example, TGFb is known to suppress gastric cancer cell proliferation through transcriptional upregulation of p21 (Yoo et al., 1999). In this respect, miR-106b and miR-93, both of which are upregulated in gastric cancer and are downstream targets of the oncogenic transcription factor E2F-1, directly target p21 and thus impair the tumor-suppressive activity of TGFb (Petrocca et al., 2008). The miRNAs in two clusters (miR-106b-93-25 and miR-222-221) have also been reported to suppress the p21 family of CDK inhibitors (Kim et al., 2009). Kim et al. reported that miR-25 targets p57 through the 30 -UTR. In addition, miR-106b and miR-93 control p21, whereas miR-222 and miR-221 downregulate both p27 and p57. Ectopic expression of these miRNAs also enhances CDK2 activity and facilitates the G1–S transition. Consistent with their biological roles, both clusters are upregulated in gastric cancer tissues. Enforced expression of cluster miR-222-221 also enhances the growth of gastric cancer xenograft in nude mice (Kim et al., 2009). In addition to direct targeting of CDK inhibitors, miRNA has been shown to affect the expression of a CDK inhibitor-interacting protein known as anion exhanger-1 (AE1). AE1 is an erythroid-specific membrane protein, which is unexpectedly expressed in gastric cancer cells. AE1 sequesters p16 in the cytoplasm and thus promotes cell proliferation (Shen et al., 2007). The expression of AE1 is modulated by miR-24, which directly binds to its 30 -UTR. Transfection of miR-24 leads the return of AE1sequestered p16 to the nucleus and inhibition of cell proliferation (Wu et al., 2010b). In addition to p16 and p21 families, Wee1 expression may also be altered by miRNA dysregulation in gastric cancer. The expression of Wee1 is regulated by the Skp1/cullin/F-box complex Oncogene


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Figure 2 Regulation of apoptosis by miRNA in gastric cancer. Several miRNAs have been shown to directly or indirectly regulate the expression or activity of proapoptotic and antiapoptotic members of Bcl-2 family. miRNAs also alter the threshold of gastric cancer cells to the effect of apoptotic stimuli by regulating the activity of PI3k/Akt and NF-kB signaling.

that polyubiquitinates Wee1 for proteasome-dependent degradation (Watanabe et al., 2004). let-7, an miRNA frequently downregulated in gastric cancer (Zhang et al., 2007a), has been shown to target CDC34, the ubiquitinconjugating enzyme of the Skp1/cullin/F-box complex in primary fibroblasts, and thereby promoting the half-life and expression of Wee1 protein (Legesse-Miller et al., 2009). Whether a similar regulatory mechanism exists in gastric cancer cells, however, requires further elucidation. These findings suggest that aberrant miRNA expression may enhance cell-cycle progression through direct or indirect regulation of CDK inhibitors and other cell-cycle regulators. miRNA and apoptosis Evasion of apoptosis is a hallmark of cellular transformation (Cotter, 2009). Apoptosis can be biochemically categorized into two pathways, namely, mitochondriaand death receptor-mediated apoptotic pathways. The execution of mitochondria-mediated apoptosis is mainly governed by opposing activities of proapoptotic (Bax, Bak, Bik, Bim, Bad, Bid, HRK, NOXA, PUMA, BNIP3 and BNIP3L) and antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, A1 and Mcl-1) members of the Bcl-2 family, which regulate mitochondrial membrane permeability. Tilting the balance toward proapoptotic signaling leads to cytochrome c release from mitochondria, generation of reactive oxygen species and Apaf-1-dependent Oncogene

activation of caspase-9 (Cory and Adams, 2002). Conversely, ligand-dependent stimulation of death receptors (Fas, TNF-R1, DR3, TRAIL-R1 (DR4), TRAIL-R2 (DR5), DR6, p75-NGFR and EDAR) triggers FADDdependent activation of caspase-8. Activation of mitochondria- and death-receptor-mediated apoptotic pathways leads to activation of a common set of effector caspases (caspases-3, -6 and -7) that are responsible for the biochemical and morphological features observed in apoptotic cells. The execution of apoptosis can be blocked by a class of proteins known as inhibitors of apoptosis (cIAP-1, XIAP and survivins) that suppresses the effector caspases. Moreover, the prosurvival PI3K/ Akt and nuclear factor-kB (NF-kB) signaling have been shown to regulate apoptosis threshold by altering the activity of Bad and transcriptional activation of XIAP gene, respectively (Qiao and Wong, 2009). miRNA dysregulation has been shown to regulate apoptosis by altering the expression of Bcl-2 family members in gastric cancer (Figure 2). For instance, TGFb is known to induce RUNX3, which interacts with FoxO3a/FKHRL1 to activate the proapoptotic protein Bim and induce apoptosis in gastric cancer cells (Vogiatzi et al., 2006; Yamamura et al., 2006). In this regard, miR-106b and miR-93 impair TGFb-induced apoptosis in gastric cancer cells by inhibiting Bim expression (Petrocca et al., 2008). Overexpression of miR-130b has also been reported to suppress TGFbinduced Bim expression and apoptosis by targeting


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RUNX3 in gastric cancer cells (Lai et al., 2010). Moreover, several miRNAs, including miR-15b, miR16, miR-34 and miR-181b, have been shown to target directly the antiapoptotic protein Bcl-2 and positively regulate apoptosis (Xia et al., 2008; Zhu et al., 2010). miR-15b, miR-16 and miR-181b are downregulated in the multidrug-resistant human gastric cancer cell line SGC7901/VCR in which restoration of their expression sensitizes SGC7901/VCR cells to chemotherapeuticdrug-induced apoptosis (Xia et al., 2008; Zhu et al., 2010). miR-34 is a downstream target of p53 (He et al., 2007), the guardian of human genome. Overexpression of miR-34 increases caspase-3 activation and impairs tumorsphere formation and growth in p53-mutant gastric cancer cells (Ji et al., 2008). In addition, 5-aza20 -deoxycytidine (a DNA methylation inhibitor) and 4-phenylbutyric acid (a histone deacetylase inhibitor) restore the expression of the epigenetically silenced miR512-5p in human gastric cancer cells in which miR-5125p induces apoptosis by suppressing the antiapoptotic Mcl-1 expression (Saito et al., 2009). EGR2 is a tumorsuppressive transcription factor, whose activation induces apoptosis by direct transactivation of BNIP3L and Bak (Unoki and Nakamura, 2003). To this end, the oncogenic miR-150 has been shown to target EGR2 to promote gastric cancer growth (Wu et al., 2010b). miR-129-2 also regulates apoptosis probably through regulating the relative abundance of proapoptotic and antiapoptotic members of Bcl-2 family. MiR-129-2 is epigenetically silenced in gastric cancer in which restoration of its expression downregulates SOX4 and triggers apoptosis in gastric cancer cells (Shen et al., 2010). Macrophage migration inhibitory factor (MIF) is a lymphokine whose expression is increased in H. pyloriinfected mucosa and gastric cancer (He et al., 2006). Serum levels of MIF are also elevated in gastric cancer patients (He et al., 2006). It has been demonstrated that MIF binds to CD74 to reduce apoptosis in gastric epithelial cells through downregulation of p53 phosphorylation and upregulation of Bcl-2 expression (Beswick et al., 2006). MIF also promotes gastric epithelial cell proliferation through transactivation of epidermal growth factor receptor (Beswick and Reyes, 2008) and the PI3K/Akt pathway (Li et al., 2009). The expression of MIF has been shown to be targeted by miR-451, whose expression is downregulated in gastric cancer. Restoration of miR-451 expression downregulates MIF and decreases expression of reporter genes with MIF target sequences in gastric cancer cells, which is accompanied by reduction in cell proliferation and enhancement of cell death in response to irradiation. Moreover, there is a significant inverse correlation between miR-451 and MIF expression in gastric cancer biopsies, suggesting that miR-451 functions as a tumor suppressor by repressing MIF (Bandres et al., 2009). Epstein–Barr virus (EBV) infection is associated with 6–16% of gastric cancer worldwide (Shah and Young, 2009). It is the first human virus found to express miRNA (Pfeffer et al., 2004). In this regard, EBVencoded miRNAs can be detected in EBV-infected

gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model (Kim do et al., 2007). An EBV miRNA known as miR-BART5 has been shown to reduce the sensitivity of EBV-infected gastric cancer cells to the effects of various proapoptotic agents by targeting PUMA, a proapoptotic member of Bcl-2 family. Depletion of miR-BART5 or induction of PUMA also induces apoptosis in EBV-infected gastric cancer cells, suggesting that miR-BART5 promotes gastric cancer cell survival by repressing PUMA expression (Choy et al., 2008). Aside from the effect on Bcl-2 family, miRNA dysregulation has been demonstrated to alter apoptosis by regulating prosurvival signaling pathways. Phosphatase and tensin homologue is a tumor suppressor and a negative regulator of the PI3K/Akt pathway through its ability to dephosphorylate phosphatidylinositol (3,4,5)trisphosphate (Diaz-Meco and Abu-Baker, 2009). It has been demonstrated that phosphatase and tensin homologue is the direct target of miR-21 (Meng et al., 2006, 2007; Yamanaka et al., 2009) whose expression is elevated in gastric cancer tissues and cell lines, as well as H. pylori-infected gastric mucosa. Knockdown of miR-21 causes a significant increase in apoptosis (Zhang et al., 2008). miR-375 is another miRNA that regulates the activity of PI3K/Akt pathway. This miRNA suppresses the PI3K/Akt pathway through direct targeting of PDK1, a kinase that phosphorylates Akt. Microarray analysis has revealed that miR-375 is one of the most downregulated miRNAs in gastric cancer in which ectopic expression of miR-375 substantially reduces cell viability through induction of caspasedependent apoptotic pathway (Tsukamoto et al., 2010). In addition to PDK1, miR-375 targets 14-3-3zeta, an antiapoptotic protein that mediates prosurvival signals by binding to phosphoserine-containing proteins. Moreover, downregulation of PDK1 or 14-3-3zeta phenocopies the effect of miR-375, indicating that miR-375 may exert its proapoptotic effect, at least in part, through downregulating these proteins (Tsukamoto et al., 2010). Similar to the PI3K/Akt pathway, NF-kB signaling inhibits apoptosis (Shen and Tergaonkar, 2009). Importantly, this pathway is induced during H. pyloriinduced gastritis and is constitutively active in gastric cancer tissues (Sasaki et al., 2001). miR-218 has been shown to induce apoptosis in gastric cancer cells through direct targeting of epidermal growth factor receptor-co-amplified and overexpressed protein, a positive regulator of NF-kB transcriptional activity (Gao et al., 2010). Importantly, miR-218 is found to be downregulated in gastric cancer tissues and H. pyloriinfected gastric mucosa. Overexpression of miR-218 also inhibits NF-kB transcriptional activation and transcription of cyclooxygenase-2, a proliferative and antiapoptotic gene regulated by NF-kB, in gastric cancer cells (Gao et al., 2010). NF-kB has also been shown to be directly targeted by miRNA in gastric cancer. miR-9, an miRNA downregulated in gastric cancer, binds to the 30 -UTR of NF-kB1 and thereby suppressing NF-kB transcriptional activity. Restoration of miR-9 expression suppresses the proliferation of cultured gastric Oncogene


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cancer cells and the growth of gastric cancer xenograft in severe combined immunodeficiency mice. Importantly, overexpression of NF-kB1 rescues growth inhibition caused by miR-9 (Wan et al., 2010). These findings suggest that aberrant activation of NF-kB signaling as a result of miRNA dysregulation may be an important molecular event in gastric carcinogenesis. miRNA and metastasis Invasiveness and metastasis are essential aspects of cancer cells (Geiger and Peeper, 2009). To metastasize, malignant cells must become motile and invasive to break apart from the primary tumor and degrade the surrounding extracellular matrix. These cells then travel to distal organs through vasculature or lymphatic vessels, during which they have to withstand the hematogenous sheering force and avoid anoikis, an apoptotic program activated on detachment of epithelial cells from the substratum and neighboring cells. Finally, these cells are arrested by receptor-mediated adhesion or mechanical trapping by narrow capillary beds in the secondary site, in which they must acquire an ability to proliferate, despite the local immunological response and the change of microenvironment (Geiger and Peeper, 2009). Several intracellular signaling pathways, such as TGFb (Romano, 2009) and hepatocyte growth factor /c-Met (Benvenuti and Comoglio, 2007) signaling, have been shown to promote metastasis. The metastatic potential of cancer cells is also regulated by mechanisms that control cell survival, cytoskeleton remodeling, the activity of extracellular matrix-degrading proteinases (MMPs), as well as the expression of homing receptors and their ligands (Geiger and Peeper, 2009). MiR-21 is overexpressed in H. pylori-infected gastric mucosa and gastric cancer tissues (Zhang et al., 2008). Enforced expression of miR-21 increases the invasiveness of cultured gastric cancer cells, whereas knockdown experiment shows an opposite result. Zhang et al. have identified RECK as the direct target of miR-21. RECK is a tumor-suppressor gene in gastric cancer, which inhibits tumor metastasis and angiogenesis through modulating MMPs, including MMP9, MMP2 and MMP14. Activation of PI3K/Akt pathway by miR-21 through downregulation of phosphatase and tensin homologue may also confer gastric cancer cells with the ability to avoid detachment-induced anoikis. In addition, miR-21 has been shown to target PDCD4, a tumor suppressor, in gastric cancer in which low levels of PDCD4 mRNA are correlated with lymph node metastasis and venous invasion (Zhang et al., 2008; Motoyama et al., 2010). Similarly, miR-21, the oncogenic miRNA miR-106a is upregulated and its expression correlates with invasion as well as lymphatic and distant metastasis (Xiao et al., 2009). Downregulation of miR-218 is also implicated in gastric cancer metastasis (Tie et al., 2010). It has been demonstrated that reduced expression of miR-218 in gastric cancer results in derepression of its target Robo1, a transmembrane receptor for Slit, and thereby enhancing Slit/Robo1 signaling. Originally identified as a molecular mechanOncogene

ism of axon guidance, Slit/Robo1 signaling is now recognized to have a paradoxical role in carcinogenesis. Both oncogenic and tumor-suppressing functions of Slit/ Robo signaling have been reported (Wang et al., 2003; Schmid et al., 2007; Marlow et al., 2008; Stella et al., 2009; Yuasa-Kawada et al., 2009). In this respect, Tie et al. (2010) reported that activation of Slit/Robo1 signaling induced by miR-218 downregulation triggers metastasis, whereas restoration of miR-218 expression inhibits invasion and metastasis of gastric cancer cells in vitro and in vivo. miRNA and transcriptional regulation The process by which the genetic code is transferred from DNA to mRNA is regulated by a myriad of factors, including promoter sequences, cis-acting regulatory elements, basal and gene-specific transcription factors and co-regulators, DNA methylation and chromatin structure. In gastric cancer, it has been postulated that miRNA dysregulation may modulate transcription through alteration of chromatin architecture. The high mobility group A2 (HMGA2) is a nonhistone chromosomal protein, which by itself has no transcriptional activity, but can promote the assembly of regulatory protein complexes at sites of transcription (Pfannkuche et al., 2009). This gene is expressed abundantly during early development, but at very low levels in adult tissues. HMGA2 overexpression is a hallmark of various benign and malignant tumors, including gastric cancer, in which HMGA2 overexpression is correlated with serosal invasion and is an independent prognostic factor for poor clinical outcome (Motoyama et al., 2008). It has been demonstrated that HMGA2 is negatively regulated by the let-7 miRNA family. In this regard, there is an inverse relationship between the expression of let-7 and HMGA2 in gastric cancer cell lines and primary gastric cancer tissues (Motoyama et al., 2008). These findings suggest that the loss of inhibition by let-7 contributes to HMGA2 overexpression and probably enhances transcription in gastric cancer tissues. Aside from genome-wide modulation of gene transcription, alteration of miRNA expression may affect transcriptional activities of various oncogenic/tumorsuppressing transcription factors through regulating the expression of prohibitin (Liu et al., 2009). This transcriptional co-regulator has been reported to enhance the transcriptional activity of p53 (Fusaro et al., 2003), but have opposite effects on E2F-1 (Rastogi et al., 2006) and NF-kB (Theiss et al., 2009). MiR-27a, an miRNA upregulated in gastric cancer, has been shown to directly bind to the 30 -UTR of prohibitin mRNA and downregulate its protein expression. Suppression of miR-27a also impairs gastric cancer cell proliferation, suggesting that miR-27a may function as an oncogene by targeting prohibitin (Liu et al., 2009). In addition to HGMA2 and prohibitin, miRNA dysregulation has been reported to alter the expression of a methyl-CpG-binding protein known as MeCP2 that is involved in epigenetic silencing of genes whose


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promoters are hypermethylated (Wada et al., 2009). In this connection, both in silico prediction and luciferase assay have identified MeCP2 as the direct target of miR-212, an miRNA frequently downregulated in gastric cancer (Wada et al., 2009). Increased expression of MeCP2 is associated with enhanced epigenetic silencing of various important tumor suppressors, such as STAT5A (Zhang et al., 2007b), TIMP-2 (Pulukuri et al., 2007) and C/EBPa/d (Tada et al., 2006; Agrawal et al., 2007) in other types of cancer. Moreover, downregulation of MeCP2 induces senescence in mesenchymal stem cells (Squillaro et al., 2010), whereas its expression is required for prostate cancer cell proliferation (Bernard et al., 2006). It is therefore speculated that derepression of MeCP2 expression by downregulation of miR-212 may promote carcinogenesis by enhancing epigenetic silencing of tumor-suppressor genes.

Clinical implication of miRNA in the management of gastric cancer miRNA as disease susceptibility markers One of the major goals of personalized medicine is to assess disease risk based on the genetic make-up of an individual. Polymorphisms of various genes, such as CDH1 (Humar et al., 2002), GSTT1 (Saadat, 2006), IL1RN (Rocha et al., 2005), MTHFR (Sun et al., 2008), TP53 (Zhou et al., 2007) and TNF-A (Gorouhi et al., 2008), have been shown to be associated with gastric cancer. Aside from these candidate genes, some recent studies suggest that polymorphisms in the miRNA genes may serve as novel risk predictors for gastric cancer. Arisawa et al. (2007) first reported that a polymorphism of miR-27a genome region is associated with a higher risk for the development of gastric mucosal atrophy in Japanese men. Peng et al. (2010) later reported an association of miRNA-196a-2 gene polymorphism with gastric cancer risk in a Chinese population. It is anticipated that more polymorphisms in miRNA genes will be discovered. With this information, there will be more accurate assessment to determine the genetic susceptibility of an individual to gastric cancer. miRNA as diagnostic markers Early detection of cancer allows administration of curative treatment before it progresses to the late inoperable stage. Identification of serum biomarkers for early cancer diagnosis is a major focus of current investigative efforts. In clinical practice, there is no reliable biomarker for diagnosing gastric cancer. Most of the biomarkers of interest (for example, pepsinogens I and II, gastrin-17, interleukin-8, antibodies against H. pylori, CagA and parietal cells, and ghrelin) tend to be associated with atrophic or inflammatory conditions of gastric mucosa, but not specific to gastric cancer (di Mario and Cavallaro, 2008). Novel methods such as serum proteomic fingerprinting (Poon et al., 2006) and circulating miRNA profiling (Tsujiura et al., 2010) have been suggested as useful tools for noninvasive diagnosis

of gastric cancer. It has been demonstrated that the plasma concentrations of various miRNAs, such as miR-17-5p, miR-21, miR-106a, miR-106b, are higher whereas let-7a is lower in gastric cancer patients. The value of the area under the receiver-operating characteristic curve can be achieved as high as 0.879 for the miR-106a/let-7a ratio assay (Tsujiura et al., 2010). High levels of miR-17 and miR-106a in peripheral blood of gastric cancer patients have also been confirmed in another study in which the value of the area under the receiver-operating characteristic curve for combined miR-17/miR-106a assay was 0.741 (Zhou et al., 2010). These findings suggest that miRNAs are useful biomarkers for early diagnosis of gastric cancer. It is expected that the incorporation of miRNA into current panels of biomarkers may enhance the sensitivity and specificity of noninvasive diagnostic tests for gastric cancer. miRNA as prognostic markers Prognosis of gastric cancer patients is heterogeneous, with overall 5-year survival rates of approximately 20% (Catalano et al., 2009). Efforts have been put forth to predict disease outcome and response to treatment. For gastric cancer, the degree of tumor penetration through the gastric wall and the presence of lymph node involvement and distant metastasis are major determinants of a poor disease outcome. The amount of residual malignant tissues left after tumor resection also predicts the likelihood of disease recurrence (Catalano et al., 2009). Aside from these conventional prognostic factors, increasing interest in the molecular pathology of gastric cancer has led to discoveries that specific proteins expressed by the tumor may serve as biological prognostic markers for outcome prediction in gastric cancer. For instance, p21 expression is associated with a better 5-year survival rate in p53-negative gastric cancer (Xiangming et al., 2000). Vascular endothelial growth factor expression and microvessel count density also predict metastasis and shorter survival time (Araya et al., 1997; Maeda et al., 1998). Other biological prognostic factors reported in the literature include overexpression of epidermal growth factor receptor (Jonjic et al., 1997), cyclin D2 expression (Takano et al., 2000), urokinase-type plasminogen activator and its inhibitor PAI-1 (Nekarda et al., 1994), the serum level of soluble receptor for IL-2 (Saito et al., 1999), and proliferative markers such as Ki-67 (Kikuyama et al., 1998) and proliferating cell nuclear antigen (Yonemura et al., 1994). miRNAs have recently been used to predict the outcome of patients with gastric cancer. For example, a seven-miRNA signature (miR-10b, miR-21, miR-223, miR-338, let-7a, miR-30a-5p and miR-126) is closely associated with relapse-free and overall survival among patients with gastric cancer (Li et al., 2010). High expression levels of miR-20b or miR-150 (Katada et al., 2009) or downregulation of miR-451 (Bandres et al., 2009) or miR-218 (Tie et al., 2010) are also associated with poor survival, whereas there is a correlation between miR-27a and lymph node metastasis (Katada et al., 2009). In addition, Ueda et al. (2010) recently Oncogene


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reported that miR-125b, miR-199a and miR-100 represents a progression-related signature, whereas low expression of let-7g and high expression of miR-214 are associated with shorter overall survival independent of depth of invasion, lymph-node metastasis and stage (Ueda et al., 2010). These prognostic miRNAs could be applicable to future decisions concerning treatment. Concluding remarks and future perspectives Dysregulation of miRNA occurs in gastric cancer as well as other malignant diseases. The mechanisms by which miRNA takes part in tumor promotion and progression are complex and numerous. However, most of them converge on common signaling mechanisms that govern cell proliferation, apoptosis and invasiveness. The relevance of some isolated cell studies to in vivo situation, however, should be assessed with caution for the possible use of nonphysiological levels of miRNA for transfection experiments. Moreover, the significance of specific miRNAs in gastric carcinogenesis should be interpreted in appropriate biological contexts as miRNA interacts widely with other signaling cascades and may behave differently in different histological subtypes of gastric cancer. Population-based differences in miRNA dysregulation, and thus the diagnostic or prognostic use of miRNAs in different ethnic groups are also key considerations. Despite these limitations, recent advances in the development of in vivo RNA delivery

system may open the window for use of miRNA as cancer therapeutics. For instance, adeno-associated virus has been used to deliver therapeutic miRNA that suppress tumorigenesis in a mouse model of liver cancer (Kota et al., 2009). Nonpathogenic bacteria have also been used to express and deliver therapeutic shorthairpin RNA to the large intestine to inhibit colon carcinogenesis (Xiang et al., 2006). Similar strategies may be devised for gastric cancer. In addition to restoring the expression of downregulated miRNAs, overexpressed miRNAs may be targeted by a novel class of chemically engineered oligonucleotides known as antagomirs that silence endogenous miRNAs (Krutzfeldt et al., 2005). It is anticipated that, with a more comprehensive understanding of miRNA dysregulation and the associated abnormalities in cellular signaling in gastric cancer, novel therapeutics will emerge. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by research grant from the National Basic Research Program of China (973 Program, 2010CB529305). CUHK Group Research Scheme (3110043) and CUHK Focused Investments Scheme-Scheme C.

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