Micrornas in Tumorigenesis, Metastasis, Diagnosis and Prognosis of Gastric Cancer

REVIEW MicroRNAs in tumorigenesis, metastasis, diagnosis and prognosis of gastric cancer

C Jiang1, X Chen2, M Alattar3,JWei4 and H Liu5

MicroRNAs (miRNAs), which are small single-stranded RNA molecules composed of 18–23 nucleotides, act as oncogenes or tumor suppressor genes playing important roles in tumor formation, infiltration and metastasis. Subsequently, miRNAs expression contributes to cancer diagnosis and prognosis. Gastric cancer currently has high morbidity and mortality among all malignant tumors, yet it lacks early specific diagnostic markers and effective treatments. In gastric cancer, many studies have detected abnormal expression forms of miRNAs and confirmed their involvement in its tumorigenesis, progression, invasion and metastasis. They may become valuable diagnostic markers and therapeutic targets for gastric cancer. Studying the role of miRNAs in gastric cancer and its relationship with diagnostic and prognostic parameters might help to improve the sensitivity of diagnosis as well as the efficacy of gastric cancer treatment. This review aims to highlight the advancements which might provide new methods for early clinical diagnosis and effective therapeutic options, along with predict response to treatment for gastric cancer.

Cancer Gene Therapy (2015) 22, 291–301; doi:10.1038/cgt.2015.19; published online 22 May 2015

Gastric cancer is the most common gastrointestinal malignant act as a tumor suppressor, whereas targeting tumor suppressor tumor which poses a serious threat to human health. According genes could be a potential oncogene. At the same time, the to statistical data in CA2010, there are 934 000 newly diagnosed expression and function of microRNAs were regulated by various cases of gastric cancer patients each year, and two-thirds of them factors, such as gene sequence, phosphorylation level, miRNAs are located in Eastern Europe, South America and Asia. The inci- modification enzyme and so on. Many recent studies on miRNA dence of gastric cancer in Asian countries is higher than revealed its involvement in pathogenesis of different tumors others, furthermore, Chinese account for 42% of Asian patients.1 including gastric carcinoma. The prognosis of gastric cancer depends on the stage when diagnosed, and the later stage carries poor prognosis. In general, about 50% of these patients have had metastasis at miRNAs AND TUMORIGENESIS OF GASTRIC CANCER the time of diagnosis. Subsequently, the long-term curative effect As a post-transcriptional gene regulator, miRNAs are involved in is poor. Though great progress has been made in the examination many cellular processes, including the development, differentia- and treatment, it could only improve curative effect of early tion, proliferation, apoptosis and stress reaction and so on. Human detection, but not the curative effect in case of middle and late malignant tumors including gastric cancer are usually character- stages. Current treatment still cannot significantly prolong the ized by abnormal expression of a large number of genes. Some of survival time of gastric cancer patients. them may lead to tumorgenesis as a consequence.3 MicroRNAs (miRNAs) are endogenous non-coding small miR-106-25b family including miR-25, miR-106 and miR-93b molecule RNAs, a subset of 19–24 ribonucleotide in length. shows higher expression in primary tumors of stomach and in miRNA was firstly discovered in nematode Caenorhabditis elegans, all of gastric cancer cell lines, which may take part in the process and then a variety of such molecules were found in plants of the occurrence of gastric cancer.4 In a study on 365 cases of and animals. In living cells, the miRNA molecules are modified gastric cancer, 15 miRNAs and 24 families of miRNAs were isolated by Drosha and Dicer enzyme to induce degradation or silence by application of 133 labeled-single nucleotide polymorphisms. of target mRNAs after transcription, thus inhibiting protein The results suggest that miR-25, miR-29, miR-93 and miR-106-b synthesis.2 may have an important role in the genetic susceptibility and the It is predicted that about 30% of the mRNAs in the human body molecular mechanisms of gastric cancer.5 miR-181-c is critical to are the miRNA targets, and each miRNA can regulate hundreds of the occurrence of gastric cancer through regulating oncogenes genes. They have a role on the organ development, differentiation NOTCH4 and KRAS.6 When exposed to N-nitrose compound and function, as well as tumor formation, progression, invasion (MNNG (N-methyl-N-nitro-N'-nitrosoguanidine)) in vivo or in vitro, and metastasis. Studies on function of miRNA proved that it could miR-21 is activated during the process of malignant transforma- be an oncogene or a tumor suppressor gene. The role of miRNA tion and tumorigenesis, which could induce gastric tumor. After depends on its target genes. MicroRNA targeting oncogenes could activation, miR-21 expression level is time- and dose-dependent

1Department of Thoracic Surgery & Tumor Research Institute, Tianjin Union Medicine Centre, Tianjin, China; 2Department of Microbiology & Infectious Disease Center, School of Basic Medical Science, Peking University Health Science Center, Beijing, China; 3Department of Cardiothoracic Surgery, Zagazig University Hospital, Faculty of Medicine, Zagazig University, Sharkia, Egypt; 4Department of Basic Medical Sciences, Medical College, Xiamen University, Xiamen, China and 5Office of Clinical Drug Trial Institution, The Affiliated Tumor Hospital of Xinjiang Medical University, Urumqi, China. Correspondence: Dr. H Liu, Office of Clinical Drug Trial Institution, The Affiliated Tumor Hospital of Xinjiang Medical University, Urumqi 830011, China. E-mail: [email protected] Received 8 January 2015; revised 15 March 2015; accepted 16 March 2015; published online 22 May 2015 Role of MicroRNAs in gastric cancer C Jiang et al 292 and it may have a role in chemical tumorigenesis through the miR-223 acts as a proto-oncogene through post-transcription regulation of death receptor Fas ligand and BTG2 (B-cell trans- regulation of its target gene FBXW7/hCdc4 (a cancer suppressor location gene 2).7 Genetic variations of miR-25, miR-29, miR-93 gene regulating apoptosis, proliferation and invasion of gastric and miR-106-b may induce increased genetic susceptibility of cancer cells).21 miR-301a can directly suppress RUNX3 (Runt- gastric cancer, and participate in the molecular mechanisms of related transcription factor 3) expression and accordingly advance gastric tumorigenesis.5 cell growth, migration and invasion.22 miR-363, via raising the Disturbance of pro-oxidant–antioxidant balance can enhance expression of MBP-1 (c-Myc promoter binding protein 1) in gastric the oxidative damage to gastric tissues and then promote the cancer SC-M1 cells, causes cell growth, progression, epithelial– occurrence of cancer. Reactive oxygen species induced by raising mesenchymal transformation and tumor formation. Endogenous miR-21 expression level in gastric cells, which cut the expression of miR-363 knockout in SC-M1 cells, by downregulating MBP-1, PDCD4 (programmed cell death protein 4), may eventually bring inhibits tumorigenesis and progression.23 about tumorigenesis.8 Both the overexpression of miR-21 and low The reduced level of miR-365 in human gastric cancer tissue expression of PDCD4 are significantly related to the degree of related to poorly differentiated tumor tissues, infiltration depth gastric cancer differentiation, clinical stage, as well as local and and progress stages, and also to the deficiency of PTEN, distant lymph node metastasis. phosphorylated Akt, p53, cyclin D1 and cdc25A. The PTEN–Akt– The gene polymorphism of G/A in miR-27a suppresses the p53–miR-365–cyclin D1/cdc25A axis serves as a new mechanism expression of mature miR-27a, which could reduce the genetic underlying gastric tumorigenesis.24 miR-372 produces its tumor- susceptibility to gastric cancer.9 In addition, in eight kinds of promoting effect by downregulating the tumor suppressor gene gastric cancer cell lines and human gastric cancer tissues, the LATS2 (large tumor suppressor 2), which can regulate gastric obviously downregulated miR-212 suppresses its target gene cancer cell cycle and apoptosis, and thus contributes to gastric MeCP2 (methyl-CpG-binding protein) expression which is asso- oncogenesis.25 ciated with occurrence of gastric cancer.10 miR-544 is another cancerogenic miRNA which can promote gastric cancer cell proliferation and cell cycle progression in vitro. Tumor-promoting effects of miRNAs Overexpression of miR-544 can inactivate the tumor suppressor gene IRX1 (iroquois homeobox 1) and diminish its expression level The aberrant expressions of miR-17-5p, miR-20a, miR-21, miR-92, in gastric cancer.26 After combining with the 3ʹ untranslated miR-106-a and miR-155 are associated with tumorigenicity, and region of GC-associated CLDN18 (claudin-18 gene) mRNA, the their elevated levels can be detected in many types of cancers. expression of miR-1303 is reduced, which will accordingly ’ In addition, inhibition of miR-27a s expression can depress the promote the occurrence and migration of gastric cancer. In growth of gastric adenocarcinoma, which proved that it could be 11 parallel, suppressed miR-1303 can inhibit proliferation, migration a gastric oncogene. and invasion of gastric cancer cells.27 Some miRNAs can promote the development of gastric cancer The gastric tumorigenesis promoting effect for miRNAs may through direct or indirect regulation of oncogenes or tumor be associated with gene polymorphism. One study on single suppressor genes. Increased expressions of miR-17, miR-18a/b, nucleotide polymorphisms of miR-196a-2 shows that the variant miR-19a, miR-20a/b, miR-21, miR-106-a/b, miR-340, miR-421 and homozygote CC can markedly increase the risk of gastric cancer, miR-658 in gastric cancer are related to downregulated levels which clearly indicates that miR-196a-2 gene mutation may of tumor suppressor genes Rb and PTEN (phosphatase and play an important role in the occurrence and progression of tensin homolog). These miRNAs may have a role in accelerating 28 12 gastric cancer. miR-372 also has a key role in the occurrence the development of gastric cancer. Furthermore, excep- of gastric cancer by regulating the mRNA level of TNFAIP1 (tumor tional expression of miR-19a dramatically facilitates gastric cell necrosis factor-alpha-induced protein 1) which controls cell proliferation and tumorigenesis in vitro and in vivo by inhibi- growth.29 ting tumor suppressor genes SOCS1 (suppressor of cytokine Cell apoptosis and cell cycle regulation are important mechan- 13 signaling 1). isms of how miRNAs participate in initiation of gastric cancer. Moreover, the overexpression of miR-21 in gastric cancer is High expression of miR-106a in both gastric cancer tissues directly correlated with higher degree of differentiation, local and cell lines shows significant negative correlation with the level invasion and lymph node metastasis. While lowering the of Fas gene (cell surface death receptor encoding gene), which expression of miR-21 could significantly restrain these biological thereby weakens Fas-induced apoptosis.17 The overexpression behaviors by raising the expression of tumor suppressor gene of miR-125b in gastric cancer HGC-27 cells can distinctly increase 14 PTEN in BGC-823 cells. Except for PDCD4 and PTEN, miR-21, as cell proliferation and decreases apoptosis. This is a possible an oncogenic factor, could target many other tumor suppressor mechanism involved in its tumorigenicity.30 SOX2 (Sex deter- genes including TPM1 (tropomyosin 1) and BTG2 (BTG family mining region Y-box 2) has positive effect on the growth 15 member 2). miR-25 is obviously overexpressed both in human inhibition by regulating the cell cycle and apoptosis. Both the gastric cancer tissues and in cell lines, as well as plays a pivotal excessive expression of miR-126 and the decreased expression of role in the tumorigenesis and metastasis. And it may promote SOX2 were found in some gastric cancer cell lines in vitro as well gastric carcinoma cell growth by targeting and regulating RECK as primary gastric cancer cells, which is found that the (reversion-inducing cysteine-rich protein with Kazal motifs) phenomenon usually concomitants to gastric tumorigenesis.31 gene.16 The expression of miR-146a in gastric cancer tissues was Suppressing the expression of miR-106b can activate its target upregulated, moreover, by regulating target genes SMAD family gene PTEN and subsequently depress invasion and metastasis of member 4 (SMAD4 (small mothers against decapentaplegic 4)), it gastric cancer cell, which indicated that miR-106b, as a oncogene, could promote cell proliferation and inhibit apoptosis.32 miR-148a could become a therapeutic target gene.17,18 Overexpression of can conduce to gastric cancer cell proliferation by reducing miR-150 in gastric cancer tissues and various gastric cancer cell the expression of p27 (kip1), while its knockout can produce lines promotes tumorigenesis and cell proliferation through the opposite effect.33 High expression of miR-196a in gastric negative regulation of tumor suppressor gene EGR2 (early cancer tissue is directly correlated with tumor size, higher clinical growth response 2).19 miR-181a can promote proliferation, stage and shortened survival time. Futhermore, p27 (kip1)- aggregation, invasion and migration of gastric cancer cells mediated repression in cell proliferation was reverted by SGC-7901, as well as restrain apoptosis by direct inhibition of exogenous miR-196a expression.34 Studies were summarized in tumor suppressor KLF6.20 Table 1.

Table 1. miRNAs with experimental data supporting a tumor oncogene function in gastric carcinoma

miRNA Source Expression in Conﬁrmed targets Experimental data Function References patients

miR-17, miR-18a/b, miR-20b, Gastric carcinoma tissue, serum, Increased Rb, PTEN Increase tumorigenicity, diagnosis OG 12, 112 miR-340, miR-421, miR-658 plasma miR-17-5p Gastric carcinoma tissue, serum, Increased Increase tumorigenicity OG 11, 68 plasma miR-19a Gastric carcinoma tissue, serum, Increased Rb, PTEN, SOCS1 Increase tumorigenicity, promote cell proliferation OG 12, 13 plasma miR-20a Gastric carcinoma tissue, serum Increased Increase tumorigenicity, metastasis, bad prognosis OG 11, 132 miR-21 Gastric carcinoma tissue and cell Increased Rb, PTEN, PDCD4, Tumor promoter, invasion, metastasis, inhibit OG 11, 12, 14, 15, lines, serum, plasma, gastric juice TPM1, BTG2 apoptosis, diagnosis, bad prognosis 68, 91, 103, 109, 116, 123, 131 miR-25 Gastric carcinoma tissue and Increased RECK Promote cell proliferation, invasion, metastasis, OG 4–6, 16, 132 cell lines inhibit apoptosis, bad prognosis miR-27a Gastric carcinoma cell lines Increased Promote cell proliferation OG 9, 11 miR-92 Gastric carcinoma tissue Increased Increase tumorigenicity OG 11 miR-106a/b Gastric carcinoma tissue, serum, Increased Rb, PTEN, Fas Inhibit apoptosis, diagnosis, bad prognosis OG 11, 12, 17, 18,

plasma, gastric juice 68, 120, 123, 132 cancer Jiang gastric C in MicroRNAs of Role miR-125b Gastric carcinoma tissue, Increased Promotes cell proliferation, inhibit apoptosis, OG 30, 122 HGC-27cell line metastasis, bad prognosis

miR-126 Gastric carcinoma cell lines Increased/ SOX2 Promotes cell proliferation, inhibit apoptosis, OG/TS 31, 73 al et decreased invasion, metastasis miR-146a Gastric carcinoma tissue Increased SMAD4 Promotes cell proliferation, inhibit apoptosis OG 32 miR-148a Gastric carcinoma cell lines Increased/ p27 Promotes cell proliferation and cell cycle OG/TS 33, 77, 78 decreased progression, metastasis miR-196a Gastric carcinoma tissue and cell Increased p27 Promotes cell proliferation and cell cycle OG 34 lines progression miR-223 Gastric carcinoma tissue and cell Increased FBXW7/hCdc4 Promote cell proliferation, invasion, metastasis, OG 21, 95, 116, 131 lines diagnosis, bad prognosis miR-301a Gastric carcinoma tissue and cell Increased RUNX3 Promote cell proliferation, invasion, metastasis OG 22

acrGn hrp 21) 291 (2015), Therapy Gene Cancer lines miR-363 SC-M1 cell line Increased MBP-1 Increase tumorigenicity, promote cell proliferation OG 23 miR-372 Gastric carcinoma tissue and Increased LATS2, TNFAIP1 Promotes cell proliferation, increase OG 25, 29 cell lines tumorigenicity, inhibit apoptosis miR-544 Gastric carcinoma tissue Increased IRX1 Promotes cell proliferation and cell cycle progression OG 26 miR-1303 Gastric carcinoma tissue and Increased CLDN18 Increase tumorigenicity, metastasis OG 27 cell lines Abbreviations: miRNA, microRNA; OG, oncogene; TS, tumor suppressor. – 301 293 Role of MicroRNAs in gastric cancer C Jiang et al 294 Tumor suppressor effects of miRNAs of miR-218 in gastric tissues can promote carcinogenesis as a Let-7 family miRNAs exert tumor-suppression effects through result of excessive activation of the target gene NF-κB, which may 56 targeting and regulating RAS and HMGA2 oncogenes.35 miR-9 is be originally a consequence of chronic Helicobacter pylori. another tumor suppressor gene which can inhibit tumor cell miR-219-2-3p, by modulating ERK1/2 related signaling pathways, 57 growth by adjusting NF-κB (nuclear factor kappa B) expression.36 can inhibit progression and metastasis of gastric cancer. in addition, it can suppress RAB34 (member RAS oncogene family) The occurrence of gastric cancer is often characterized by the expression and tumor cell migration.37 On the other hand, disorder of transcription factor E2F1 activities. miR-331-3p, by opposite results suggest that miR-9 may bind to 3ʹ untranslated interfering with the activity of E2F1, inhibits cellular growth in vitro region sites of CDX2 to inhibit its expression, and eventually and thus could be regarded as a tumor suppressor gene in gastric 38 cancer. Enhancing the expression level of miR-331-3p may be a induce gastric cancer cell proliferation. Hypermethylation signals 58 in the upstream region of miR-181C were associated with potential treatment of gastric cancer. miR-370 can directly reduction in its expression. The treatment with 5-mixed nitrogen decrease the expression of protein transcription factor FoxM1 (Forkhead box M1) in gastric cancer cells, so overexpression of DNA cytosine nucleoside in gastric cancer cell can increase 59 miR-181C’s level and inhibit cell proliferation by modulating miR-370 may depress cell proliferation and gastric oncogenesis. NOTCH4 and Kras.39 Reduced miR-204 expression in gastric cancer Gene polymorphism and changes in methylation level regu- could repress RAS activation, cell growth and migration. Down- lated by miRNAs also have a big role in the process of tumor regulation of miR-204 and abnormal activation of RAS participate progression. The G/A polymorphism of miR-27a gene can in the mechanisms of gastric tumorigenesis.40 miR-212 expression decrease its expression and reduce the risk of gastric canceration.9 The overexpression of miR-10b in MGC-803 and levels in gastric cancer tissues and cell lines (BGC-823, SGC-7901 fi and MKN-45) are significantly lowered due to excessive hyper- HGC-27 cells can signi cantly induce apoptosis and inhibit cell methylation of CpG islands. The proto-oncogene Myc is one of proliferation, migration and invasion. However, miR-10b was obviously downregulated in gastric cancer tissues due to target genes of miR-212 and downregulation of miR-212 induced excessive DNA methylation, which could cause its partial Myc activation has a role in the progress of gastric cancer.41 silencing.60 In gastric cancer, miR-338-3p is classified as a tumor suppressor In 13 gastric cancer cell lines, downregulation of miR-34b/c can gene and the oncogene SSX2IP is one of its target genes.42 The be seen as a result of the CpG island methylation of them. obviously reduced level of miR-338-3p in gastric cancer is directly Transfection of miR-34b and miR-34c precursor into gastric cancer related to the aggressiveness of the clinical pathologic features cells can halt the growth of cells. In gastric cancer tissue samples, of gastric cancer patients. Restoring the expression of miR-338-3p miR-34b/c, miR-34b and miR-34c as new tumor-inhibitory in gastric cancer SGC-7901 cells can inhibit cell proliferation, 61 – molecules were often silenced by DNA methylation. In some migration, invasion and tumorigenicity. miR-375 can block JAK2 gastric cancer cell lines, DNA methylation levels of miR-124a-1/-2/- STAT3 (signal transducer and activator of transcription 3) signaling 3 are higher than those of normal gastric cells, at the same time pathway and cell proliferation by inhibiting the expression of lowering their expression can consequently reduce their inhibitory oncogene JAK2 (Janus kinase 2), and then prevent gastric cancer 62 43,44 effect on gastric cancer cell proliferation. The levels of miR-195 occurrence as a tumor suppressor gene. and miR-378 were obviously lessened in gastric cancer tissues and It is clear that many miRNAs with tumor suppressor effect work fi cell lines and this was closely related to DNA hypermethylation of via modulating speci c target genes. The tumor suppressive effect their promoter CpG island. Their upregulated expression can of miR-10b should be owed to direct regulation of MAPRE1 significantly restrain the growth of gastric cancer cells and boost (microtubule-associated protein RP/EB family member 1) during 63 45 the proliferation of normal gastric epithelial cells. gastric oncogenesis. A similar effect produced by miR-24 may be Some miRNAs inhibit gastric tumors by modulating cell cycle due to inhibition of its target gene RegIV (Regenerating gene type 46 and apoptosis. Low expression of Let-7a was detected in human IV). miR-29 may serve as tumor suppressor in gastric tissue by gastric cancer tissue and cell lines and its level was directly suppressing gene expression of CCND2 and MMP2 (matrix correlated with the degree of cell differentiation. Overexpressed metalloproteinase-2).47 Similarly, miR-29c can directly target 48 Let-7a decreases cell proliferation and leads to G1 arrest, which ITGB1. The inhibitory action of miR-34a against gastric tumor- finally depresses the growth of gastric cancer.64 In the cells with igenesis is mediated through the PI3K/Akt pathway by targeting 49 p53 mutation, miR-34 can target Bcl-2, Notch and HMGA2, and PDGFR (platelet-derived growth factors' receptors) and MET. enhance chemotherapy sensitivity by cell cycle arrest in G1 phase COX-2 (cyclooxygenase 2) is a key regulatory factor in the induced growth inhibition. In addition, it can promote apoptosis occurrence and progression of gastric cancer. miR-101 can inhibit by increasing the active form of caspase-3.65 Downregulated gastric cancer cell proliferation and promote apoptosis in vitro and miR-137 expression in gastric cancer tissues correlates with the also has anti-proliferative activity in vivo. These inhibitory effects level of the target gene-encoding Cell Division Cycle 42 (Cdc42). 50 are tightly related to downregulation of its target gene COX-2. In On revival of miR-137 expression, the functional inactivation of addition to COX-2, miR-101 can inhibit tumor cell growth, invasion Cdc42 can induce gastric cancer cell apoptosis and cell cycle arrest 51 and metastasis by acting on EZH2, Mcl-1 and Fos. in G1 phase.66 Downregulation of miR-205 is bound up with the Upregulated SPHK1 expression is associated with poor survival depth of invasion of gastric carcinoma. Inhibiting miR-205 in gastric cancer patients. miR-124, by downregulating SPHK1, expression significantly promotes the progression of gastric 52 inhibits gastric cancer cell proliferation. On targeting CCKBR, cancer through cell cycle regulation.67 miR-148b exerts its antineoplastic effect by depressing gastric Results of large-scale microarray assays showed that the cancer cell proliferation.53 miR-200 family members include expression of miR-218 was obviously downregulated in gastric miR-200a and miR-200b. Their expression was obviously declined cancer tissue, while overexpression of miR-218 can inhibit human after Epstein Barr Virus (EBV) infection in gastric cancer cell lines gastric epithelial AGS cells proliferation and enhance apoptosis.56 (MKN74–EBV, MKN7–EBV and NUGC3–EBV), which accordingly miR-331-3p can prevent G1/S transition in gastric cancer SGC-7901 resulted in downregulation of E-cadherin protein (which mediates and AGS cells by interfering with transcriptional factor E2F1 along calcium-dependent intercellular adhesion) expression and it is with repressing gastric cancer cell clone formation and cell regarded as a key step of EBV-related gastric cancerogenesis.54 growth.58 miRNA microarray platform covering a total of 470 miR-212 can directly target the 3ʹ untranslated region of RBP2 human miRNAs revealed that the expressions of six miRNAs (Retinoblastoma Binding Protein 2) and inhibit its expression, and including miR-375 were significantly lowered in gastric cancer finally arrest the growth of gastric cancer cells.55 Low expression tissues while ectopic expression of miR-375 could remarkably

Cancer Gene Therapy (2015), 291 – 301 © 2015 Nature America, Inc. Role of MicroRNAs in gastric cancer C Jiang et al 295 reduce cell vitality by caspase mediated apoptosis, which Robo1, the ligand for neural axon guidance factor Slit, is demonstrated its tumor suppressor function.68 negatively modulated by miR-218. Once the expression of Robo1 In gastric cancer BGC-823 cells, miR-429 obviously restrained is reduced, miR-218 can activate Slit-Robo1 signal pathway and the proliferation of tumor cells serving as a tumor suppressor.69 cause gastric cancer cell metastasis. On the contrary, recovering miR-449 also has anti-gastric tumor effect by activating p53, the inhibitory effect on Robo1 in vitro or in vivo, miR-218 can inducing apoptosis and inhibiting cell proliferation.70 In gastric restrain invasion and metastasis of gastric cancer cells.82 miR-335 cancer cell lines (BGC-823 and SNU5), downregulation of miR-663 can inhibit gastric cancer cell invasion and metastasis via can cause cell proliferation and tumor formation.71 regulation of both Bcl-w and specificity protein 1. Low expression In addition, the expression of miR-7 is negatively correlated with of miR-335 in gastric cancer cells particularly related to aggres- interleukin-1β and TNF-α levels in human gastric cancer. In other siveness of lymph node metastasis, tumor-node metastasis stage words, decreased expression of miR-7 is closely related to severe and lymphatic vessel invasion.83 inflammatory response. Downregulation of the tumor suppressor Through direct combination with 3′-translated area of pro- miR-7 induced by inflammation is another new mechanism of metastatic gene radixin (RDX), miR-409-3p restrains gastric cancer gastric tumorigenesis.72 Significantly reduced miR-126 expression cell invasion and metastasis. Downregulated miR-409-3p also in gastric cancer tissues was relevant to the progress of facilitates lymph node metastasis.84 Even for the highly metastatic clinicopathological features, including tumor size, lymph node 44As3 cells, miR-516-3p can block their metastasis.85 Interestingly, metastasis, local invasion and tumor-node metastasis stage.73 the reduced level of miR-574-3p occurred mainly in the early Studies were summarized in Table 2. stages of gastric cancer or in cancers with high level of differentiation. miR-574-3p obviously inhibits cell proliferation, invasion and metastasis after transfected into gastric cancer miRNAs AND THE INVASION AND METASTASIS OF GASTRIC SGC-7901 cells. This is a powerful evidence that miR-574-3p does CANCER its work as a tumor suppressor gene in gastric carcinoma.86 The An increasing body of studies showed that miRNAs can control prominent reduction of miR-625 in gastric cancer tissues and cell many important tumor-related genes involved in tumor occur- lines also shows negative correlation with lymph node metastasis. rence and progression, and can also regulate the invasion and Studies in vivo and in vitro have demonstrated that miR-625 could metastasis of tumor (see Figure 1). obviously inhibit gastric cancer cell invasion and metastasis, which may be owed to direct modification of integrin-linked kinase expression.87 miRNAs inhibiting gastric cancer metastasis Overexpressed Let-7f in gastric cancer tissues can directly target tumor metastasis-related gene MYH9 and inhibit tumor invasion miRNAs enhancing gastric cancer metastasis and metastasis.74 On targeting cancer gene-encoding insulin like Abnormal expression of miR-9 can promote gastric cancer cell growth factor 1 receptor, miR-7 can significantly reverse proliferation, invasion and metastasis. It often shows lower epithelial–mesenchymal transition process and inhibit metastasis expression level in gastric cancer tissues than that of normal 88 of gastric cancer cells in vivo.75 The oncogene PDGFR is directly counterparts. The expression level of miR-10a in the gastric targeted by miR-34a, which suppresses gastric cancer cell cancer cells is 10 times higher than those of normal proliferation, migration and invasion.49 miR-101 can not only gastric mucosa. Compared with early stage, in late stages of gastric cancer, those patients with lymph node metastasis, inhibit multiplication, invasion and migration of gastric cancer 89 cells in vitro but also inhibit their growth in vivo.51 miR-145 can showed notably raised expression of miR-10a. also attenuate gastric cancer metastasis by inhibiting N-cadherin miR-20a potentiates gastric cancer cells growth, migration and (CDH2) protein translation, and indirectly by downregulating its invasion. Moreover, and it can enhance their resistance to 76 chemotherapy (cisplatin and taxol). The role of miR-20a in gastric downstream effector MMP9. 90 The reduction of miR-148a, a tumor suppressor gene, con- tumorigenesis is a result of regulating EGR2 signaling pathways. tributes to gastric cancer metastasis and progression.77,78 The Excessive expression of miR-21 in human gastric cancer tissues overexpression of miR-155 in the gastric cancer SGC-7901 and and cell lines has been reported. The increased proliferation and MKN-45 cell lines can obviously hinder cell migration and invasion were found in human gastric adenocarcinoma AGS cells, while knockout of miR-21 in AGS cells distinctly reduced cell adhesion. This function may be associated with reduced expres- 91 sion of its target gene Smad2.79 The decreased expression of invasion and metastasis abilities. miR-27 also has a pivotal role in miR-206 could be detected in gastric cancer cell lines and gastric regulating gastric cancer metastasis and has potential for clinical treatment. Upregulated miR-27 may encourage metastasis of AGS cancer tissues especially in those with lymphatic metastasis or 92 local invasion. Forced expression of miR-206 can inhibit tumor- cells and vice versa. igenesis, proliferation, invasion and metastasis in xenograft Overexpression of miR-107 can promote gastric cancer cell vaccinated with gastric cancer SCG-7901 cell. Therefore, miR-206 metastasis by suppressing miRNA-processing enzyme DICER1. Silencing of miR-107 inhibits invasion and metastasis of gastric in gastric cancer acts as a tumor suppressor as well as has a role in 93 regulating tumor cell metastasis.80 cancer cells in vivo and in vitro. Transfection of miR-181b in In 80 gastric cancer tissue samples and 4 gastric cancer cell gastric cancer cells progressively facilitates their proliferation, invasion and migration. It may be another possible molecular lines, the expression levels of miR-214 were considerably lower 94 than in normal tissues and control cells. There is a remarkably target of gastric cancer treatment. negative correlation between the expression level of miR-214 and In several kinds of human gastric cancer cell lines, overexpression of miR-223 was a characteristic feature in metastatic gastric cancer lymph node metastasis along with tumor size. 95 Ectopic expression of miR-214 in gastric cancer cell lines SGC-7901 gastric cancer cells. Ectopic expression of miR-622 promotes and MKN-45 may weaken tumor migration and invasion abilities. invasion and metastasis of gastric cancer cells in vivo and in vitro by regulation of ING1 (inhibitor of growth 1) gene.96 Knockout of miR-214 can largely stimulate the MKN28, BGC-823 and GES-1 cells proliferation, migration and invasion. Colony stimulating factor 1 (CSF-1) is one of the miR-214 target genes and miRNAs associated with lymph node metastases in gastric cancer lower expression of miR-214 can prompt gastric cancer cell Lymphatic metastasis is the most common pathway of metastatic proliferation, migration and metastasis, which was inversely spread for malignant tumors and is known to have serious related to CSF-1 expression.81 influence on the quality of life and prognosis of patients. Now it's

Table 2. miRNAs with experimental data supporting a tumor suppressor function in gastric carcinoma

miRNA Source Expression in Conﬁrmed targets Experimental data Function References –

301 patients oeo irRA ngsrccancer gastric in MicroRNAs of Role Let-7 family Gastric carcinoma tissue and cell lines, Decreased RAS, HMGA2, MYH9 Inhibit cell proliferation, cell cycle progression, invasion TS 35, 64, 68, 74, serum, plasma and metastasis, prognosis 122, 131 miR-7 Gastric carcinoma tissue Decreased IL-1β, TNF-α Inhibit tumorigenicity TS 72 miR-9 Gastric carcinoma tissue and cell lines Decreased/ NF-κB1, RAB34, CDX2 Inhibit or promote cell proliferation, invasion and TS/OG 4, 36–38, 89, increased metastasis, prognosis 132 miR-10b Gastric carcinoma tissue and cell lines Decreased MAPRE1 Inhibit cell proliferation, invasion and metastasis, TS 45, 60, 131 induce apoptosis, prognosis miR-24 Gastric carcinoma tissue and cell lines Decreased RegIV Inhibit tumorigenicity, cell proliferation, invasion and TS 46 metastasis Jiang C miR-29, miR-29c Gastric carcinoma tissue and cell lines Decreased CCND2, MMP2, ITGB1 Inhibit invasion and metastasis TS 47, 48 miR-34 miR-34a Gastric carcinoma tissue and cell lines Decreased Bcl-2, Notch, HMGA2, Inhibit cell proliferation, cell cycle progression, invasion TS 49, 65 PDGFR, MET and metastasis, induce apoptosis al et miR-101 Gastric carcinoma tissue and cell lines Decreased COX-2, EZH2, Mcl-1, Fos Inhibit cell proliferation, invasion and metastasis, induce TS 50, 51 apoptosis miR-124 Gastric carcinoma tissue and cell lines Decreased SPHK1 Inhibit cell proliferation, prognosis TS 52, 62 miR-137 Gastric carcinoma tissue and cell lines Decreased Cdc42 Inhibit cell cycle progression, induce apoptosis TS 66 miR-148b Gastric carcinoma tissue and cell lines Decreased CCKBR Inhibit cell proliferation TS 53 miR-181c Gastric carcinoma tissue Decreased NOTCH4, KRAS Inhibit cell proliferation TS 6, 39 miR-204 Gastric carcinoma tissue Decreased RAS Inhibit cell growth, invasion and metastasis TS 40 miR-205 Gastric carcinoma tissue and cell lines Decreased — Inhibit cell proliferation TS 67 miR-212 Gastric carcinoma tissue and cell lines Decreased MECP2, Myc, RBP2 Inhibit cell growth, invasion and metastasis TS 10, 41, 55, 103 miR-218 Gastric carcinoma tissue and cell lines Decreased NF-κB Inhibit tumorigenicity and cell proliferation, induce TS 56 apoptosis miR-219-2-3p Gastric carcinoma tissue and cell lines Decreased ERK1/2 Inhibit cell proliferation, invasion and metastasis TS 57 miR-331-3p Gastric carcinoma tissue and cell lines Decreased E2F1 Inhibit cell growth TS 58 miR-338-3p Gastric carcinoma tissue and cell lines Decreased SSX2IP Inhibit cell proliferation, invasion and metastasis, TS 42 prognosis miR-370 Gastric carcinoma tissue and cell lines Decreased FoxM1 Inhibit cell proliferation TS 59 miR-375 Gastric carcinoma tissue and cell lines, Decreased JAK2 Inhibit cell proliferation, induce apoptosis, diagnosis TS 43, 44, 68, 120 serum, plasma miR-449 Gastric carcinoma tissue and cell lines Decreased p53 Inhibit cell proliferation, induce apoptosis TS 70

05Ntr mrc,Inc. America, Nature 2015 © Abbreviations: miRNA, microRNA; OG, oncogene; TS, tumor suppressor. Role of MicroRNAs in gastric cancer C Jiang et al 297 and their target genes may be helpful to ﬁnd new diagnostic and therapeutic methods of cancer lymphatic metastasis.

miRNAs IN DIAGNOSIS OF GASTRIC CANCER In the field of molecular markers for tumor diagnosis at the level of DNA and RNA, miRNAs showed unique superiority because of their stability and specificity. So, they have gained widespread attention. At present, finding miRNAs associated with early diagnosis and the prognosis of gastric cancer is becoming an actively growing area of research, but yet their validity as reliable tool in identification of benign and malignant tumors still remains to be further confirmed. Some results suggested that expression of miRNAs in circulating blood of patients with gastric cancer significantly differed from that of healthy people and may even be consistent with miRNAs expression in gastric cancer tissue. Circulating miRNAs can be used as potential gastric cancer markers for diagnosis and monitoring of disease status.107,108 It was reported that miR-17-5p, miR-21 and miR-106a/b in the blood of the gastric cancer patients were significantly higher than the control group, while Let-7a was significantly reduced.68 Figure 1. microRNAs and the invasion and metastasis of gastric Circulating miR-21 level in gastric cancer patients are positively cancer. Some specific microRNAs can regulate many important tumor-related genes involved in tumor progression to inhibit or correlated with tumor lymph node metastasis, tumor-node promote invasion and metastasis of gastric cancer cells. Certain metastasis stage, tumor size and histological type, so it may be 109 microRNAs were associated with tumor lymphatic metastasis. used as a marker for gastric cancer detection. miRNA, microRNA. In addition, miR-17 and miR-106a levels in peripheral blood of patients with gastric cancer were closely related to the number of tumor cells. Multivariate analysis confirmed that miR-143 and miR-195 could be associated with some clinicopathologic para- been confirmed that there is a relation between miRNAs and meters including depth of invasion and lymph node metastasis.110 invasion and metastasis of malignant tumors.97–100 Wu et al101 It was found that expressions of miR-17 and miR-106a have have found that there was a significant difference in expression obvious relevance to the number of gastric cancer cells as levels among 38 kinds of miRNAs in gastric cancer tissues and in indicators and to the detection of peripheral circulating tumor 111 their adjacent normal tissues. Further research found that 5 kinds cells as molecular tools. of miRNAs were associated with tumor lymphatic metastasis and miR-195-5p is a newly discovered tumor suppressor which may those tumors with high has-miR-195 level and low miR-212 level have an impact on gastric tumorigenesis. In a study on 740 kinds may be prone to lymphatic metastasis. of miRNAs from the serum, the researchers found that only The expression level of miR-21 in gastric cancer tissue miR-195-5p expression level in serum of gastric cancer patients fi 112 associated with lymph node metastasis was significantly higher was signi cantly lower than control group. Both miR-221 and miR-376c in sera of gastric cancer patients showed obviously than those with no lymph node metastasis.102 In human gastric positive correlation with low differentiation of their cancer cells, cancer, miR-650 was also correlated with lymph node and distant while miR-744 was reated to high proliferation rate of these cells. metastasis. Ectopic expression of miR-650 promotes gastric All these three miRNAs may serve as early biomarkers for non- tumorigenesis and cellular proliferation through regulation of 113 103 invasive diagnosis of gastric carcinoma. the tumor suppressor ING4. In 37 cases of tumor tissues and pericarcinoma tissues from gastric cancer patients, miR-21 expression level measured using miRNAs and lymphangiogenesis quantitative PCR uncovered that miR-21 was overexpressed in It is now generally accepted that the formation of lymphatic 92% of these gastric cancer samples. So it may be used as an 114 vessels is the initial step of lymphatic metastasis as it provides effective marker for gastric cancer diagnosis. Overexpressed 104 miR-21 binding to tumor suppressor gene PDCD4 by way of base many channels for the inevitable spread. One study found that 115 upregulated miR-1236 in human lymphatic endothelial cells pairing can inhibit PDCD4 protein expression. miR-21 and fi PDCD4 are directly relevant to tumor size, invasion depth, lymph signi cantly reduces expression of VEGFR-3 but not VEGFR-2 and fi that overexpression of miR-1236 may weaken migration and tube node metastasis and vascular invasion but not bene cial in determining the clinical prognosis of gastric cancer patients. formation abilities of these cells by affecting generation of new In gastric cancer tissues, concomitant changes of three miRNAs, lymphatic vessels.105 increase of both miR-21 and miR-223 combined with decrease Another comparative study of miRNA target genes shows that it of miR-218, may be potential molecular markers for gastric cancer is involved in lymph vessels generation in endothelial cells of diagnosis.116 Researchers have also found that downregulation of normal lymphatics and in those of lymphatics derived from miR-31, which is associated with gastric tumorigenesis, could fi T-241/VEGF-C brosarcoma tumor metastasis model. Geno- serve as a molecular marker for gastric cancer diagnosis.117 typing microarray methods preliminarily detected three kinds In the intestinal-type of gastric cancer, three molecules involved of lymphangiogenesis-related genes including endothelial cell- in expression disorders including miR-32, miR-182 and miR-143, selective adhesion molecule, TGFβ1/3 receptor Endoglin and are generally associated with the pathological stage of tumor and angiogenesis related to Leptin receptor.106 may be potential diagnostic biomarkers for this type.118 In another Relationship between miRNAs and lymphangiogenesis is study, the miR-106a levels in 55 specimens of gastric cancer and becoming the hotspot in the research of tumor lymphatic 17 of non-gastric tumor tissues were determined. The result metastasis. Further study on differential expression of miRNAs showed that miR-106a in gastric cancer tissue was 1.625 times

© 2015 Nature America, Inc. Cancer Gene Therapy (2015), 291 – 301 Role of MicroRNAs in gastric cancer C Jiang et al 298 higher than the control. What’s more, level of miR-106a was of patients.131 A similar research has further confirmed that closely related to tumor size, differentiation degree, lymph node overexpression of miR-223 in primary gastric cancer was affection and distant metastasis, so it can act as a useful molecular associated with less survival without metastasis.95 marker for gastric cancer diagnosis.119 A study involving 353 case of gastric cancer has found that In comparison to the distal tissues, expression of miR-375 in miR-125b, miR-199a and miR-100 were associated with progres- gastric adenocarcinoma was significantly lowered. At the same sion of gastric cancer. In addition, low expression of both Let-7g time, downregulation of miR-375 in peripheral blood of these and miR-433 along with high expression of miR-214 were patients could also demonstrate its diagnostic role of gastric fl 120 connected with the poor prognosis of patients, which re ected carcinoma. In 60 cases of early gastric cancer, miR-421 in tumor infiltration depth, lymph node metastasis and tumor 73.33% of gastric cancer tissues was overexpressed. Furthermore, stage.122 compared with carcinoembryonic antigen, serum miR-421 has In tissues of advanced gastric cancer, the high expressions of significantly higher detection rate of gastric cancer.121 122 miR-20a, miR-25, miR-93, miR-103, miR-106a, miR-106b, miR-130, Ueda et al. have employed microarray technology to assess miR-155, miR-221 and miR-222 carried high risk for invasion the expression spectrum of 353 kinds of miRNAs in gastric cancer through serous membrane, lymph node metastasis and distant samples compared with normal gastric tissues and to evaluate the metastasis and brought about poor long-term survival.132 relationship between the progression and prognosis of gastric cancer. It was found that 22 kinds of miRNAs were upregulated and 13 kinds of miRNAs were downregulated in 160 paired gastric SUMMARY cancer samples and diagnostic accuracy of gastric cancer was 83% miRNAs have a vital role in the occurrence and development of by using the differential expressions of these miRNAs. These experimental results fully demonstrated that the utilization of gastric cancer. Accompanied with further research, this prelimin- miRNAs as new markers for gastric cancer diagnosis is promising ary understanding uncovers an essential characteristics and and worthy of further exploration. biological function of miRNAs during the process of the formation Beside peripheral blood and tumor tissues, differential expres- and progression of gastric cancer. There is a growing need for sion of miRNAs in gastric juice from gastric cancer patients also distinguishing miRNAs between interacting with oncogenes or began to draw the attention of the researchers. Levels of miR-21 tumor suppressor genes and affecting invasion and metastasis. and miR-106a in gastric juice have significant difference between Some of these miRNAs may inhibit oncogenicity by combining patients with gastric cancer and patients with benign gastric with target genes, or suppress tumor growth, invasion and diseases.123 Research on miR-421 also showed similar findings. For metastasis by raising the expression of tumor suppressor genes. the diagnosis of early gastric cancer, detection of miR-421 in Expression levels of miRNAs may vary during different devel- gastric juice is gaining more improvement than single use of opmental stages of cancer tissues. The advancing technology of serum carcinoembryonic antigen.124 miRNA profiling microarray chips can speed up the study of the relationship between gastric cancer and miRNAs. In recent years, such researches have gained great progress and provided some miRNAs AND THE PROGNOSIS OF GASTRIC CANCER new methods for clinical diagnosis and treatment, which may The prognosis of gastric cancer depends mainly on the probability illuminate the risk of specific population and response to the of recurrence and metastasis. A large number of studies have treatment and thus assist in the prognosis assessment and cancer shown that expression patterns of some miRNAs can be used for prevention. judging prognosis of gastric cancer. A case in a point is that the In the meantime, miRNAs may become valuable diagnostic high expression of miR-20 and miR-150b, or the low expression of markers and therapeutic targets for numerous diseases including miR-451, is associated with poor prognosis in patients with gastric 125,126 gastric carcinoma. In addition, it may be one of the promising cancer. Expression of tumor suppressor miR-29 family anti-cancer therapies by modulating the expression of prompting- members may also be used as new biomarkers for prognosis fi 47 cancer miRNAs using ef cient gene knockout, antisense nucleo- and therapeutic targets of gastric cancer patients. tide and miRNA modification in the future. miR-106b is another tumor promoter associated with poor prognosis of gastric cancer patients.18 The study of 87 gastric cancer cases found that lower expression of miR-125a-5p often CONFLICT OF INTEREST correlated with tumor size, invasion, liver metastasis and poor The authors declare no conflict of interest. prognosis. Multivariate analysis showed that its downregulation is an independent prognostic factor for survival. So, miR-125a-5p 127 could be a reliable prognostic marker. The reduced miR-206 ACKNOWLEDGEMENTS level in gastric cancer tissues is associated with higher degree of This work was supported by grant from the Tianshan Talent Project of Xinjiang (No. lymph node metastasis, distant metastasis and poor clinical stage. Y0382067). miR-206 has been proved to be an independent influence factor for the prognostic evaluation of patients with gastric cancer.128 Studies of microRNA expression profiles suggested that variety REFERENCES of miRNAs were differentially expressed. Most of their expression 1 Power DG, Kelsen DP, Shah MA. Advanced gastric cancer-slow but steady patterns were associated with the prognosis of patients with progress. Cancer Treat Rev 2010; 36: 384–392. gastric cancer. 2 Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J et al. 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