Cancer Gene Therapy (2014) 21, 305–316 & 2014 Nature America, Inc. All rights reserved 0929-1903/14 www.nature.com/cgt

REVIEW MicroRNA and signaling pathways in gastric cancer

Z Zhang1,ZLi1,YLi2 and A Zang1

MicroRNAs (miRNAs) function as either oncogenes or tumor suppressors by inhibiting the expression of target genes, some of which are either directly or indirectly involved with canonical signaling pathways. The relationship between miRNAs and signaling pathways in gastric cancer is extremely complicated. In this paper, we determined the pathogenic mechanism of gastric cancer related to miRNA expression based on recent high-quality studies and then clarified the regulation network of miRNA expression and the correlated functions of these miRNAs during the progression of gastric cancer. We try to illustrate the correlation between the expression of miRNAs and outcomes of patients with gastric cancer. Understanding this will allow us to take a big step forward in the treatment of gastric cancer.

Cancer Gene Therapy (2014) 21, 305–316; doi:10.1038/cgt.2014.37; published online 25 July 2014

INTRODUCTION THE miRNA BIOGENESIS AND FUNCTION Gastric cancer is the fourth most common cancer and the second The miRNAs are small noncoding RNAs that are B22 nucleotides leading cause of cancer-related death in the world.1 There are in length. To date, more than 2000 miRNAs have been identified in B1 million new patients diagnosed with gastric cancer every humans. In the nucleus, a primary miRNA of several kilobases is year.2 Gastrectomy is the mainstay treatment for gastric cancer; transcribed from the miRNA gene by RNA polymerase II/III.8,9 After however, early diagnosis is very difficult in clinics and the pro- transcription, the ribonuclease enzyme Drosha, coupled with its gnosis for advanced-stage patients is still very poor.1 There will be binding partner DGCR8, excises one or more B70-nucleotide a significant improvement in the diagnosis and treatment of stem-loop structures from the primary miRNA to form precursor gastric cancer if the relationships between oncogenesis, develop- miRNA.10 The precursor miRNA is then transported into the ment and prognosis are much better understood. cytoplasm by the Ran-GTP-dependent nuclear export factor, The complex interactions that take place among different Exportin-5.11 In the cytoplasm, Dicer processes the precursor etiological factors lead to genetic and epigenetic alterations of miRNA and generates a B22-nucleotide RNA duplex that contains proto-oncogenes and tumor-suppressor genes; a basic paradigm both the mature miRNA strand and its anti-strand.12 The mature underlying the pathogenesis of any cancer. Dysregulation of these stranded miRNA is incorporated into the RNA-induced silenc- genes results in abnormal function or expression of oncogenic ing complex that targets complementary sequences in the and tumor-suppressor proteins. Accumulating evidence indicates 30-untranslated region of mRNAs, thereby functioning as a means that microRNAs (miRNAs) are involved in important biological to cleave mRNA or inhibit its translational function.13,14 Afterward, processes related to apoptosis, proliferation, differentiation, the complementary strand is usually rapidly degraded15 (Figure 1). metastasis, angiogenesis and the immune response, dysregulation of which is crucial to cancer initiation, progression and treatment outcomes. Expression of miRNAs has been shown to be related to DYSREGULATED miRNAs IN GASTRIC CANCER gastric cancer as well as other cancers and plays important roles in There are an increasing number of studies demonstrating the regulating cancer-related genes.3,4 overexpression or downregulation of specific miRNAs in gastric The miRNAs act as posttranscriptional regulators of gene cancer, and these are listed in Table 1. There have been many studies expression that could regulate 430% of the protein-coding genes regarding miRNA functions in gastric cancer. Some miRNAs exhibit in the human genome.5 This class of RNAs was identified through downregulation as tumor suppressors (listed in Table 2), whereas investigation of Caenorhabditis elegans.6,7 Up till now, 1872 pre- others exhibit upregulation as oncogenes (listed in Table 3). cursors and 2578 mature forms of human miRNAs have been Furthermore, some miRNAs have displayed conflicting functions in discovered in accordance with the publication of miRBase version different studies (listed in Table 4), thereby indicating that more 20.0 (http://www.mirbase.org/). It is strongly believed that the research is needed. amount of miRNAs and the ways in which they function are much more complex than expected. We try to determine the progress of gastric cancer research THE miRNAs AND SIGNALING PATHWAYS IN GASTRIC CANCER related to miRNA expression based on studies performed in In the process of tumorigenesis and the development of gastric recent years and reviews of the regulation network and corre- cancer, miRNAs function as either oncogenes or tumor suppres- lated functions of these miRNAs. A better understanding of sors by inhibiting the expression of target genes, some of which miRNAs in gastric cancer may be achieved through the review are involved in canonical signaling pathways either directly or of this literature that will thus give rise to new avenues of indirectly. The results of recent studies regarding these activities research. are reviewed herein.

1Department of Oncological Surgery, Affiliated Hospital of Hebei University, Baoding, China and 2Department of Gastric Cancer, Fourth Hospital of Hebei Medical University, Shijiazhuang, China. Correspondence: Dr A Zang, Department of Oncological Surgery, Affiliated Hospital of Hebei University, 212, Yuhua East Road, Boding 071000, China. E-mail: [email protected] Received 20 February 2014; revised 19 June 2014; accepted 20 June 2014; published online 25 July 2014 MiRNA and signaling pathways in gastric cancer Z Zhang et al 306

Figure 1. Mechanism of microRNA action. pre-miRNA, precursor microRNA; pri-miRNA, primary microRNA; RISC, RNA-induced silencing complex.

PI3K/Akt pathway Expression of MIF has been shown to be targeted by miRNA- The phosphatidylinositide 3-kinase (PI3K)/Akt pathway has been 451, the expression of which is downregulated in gastric cancer. implicated in cancer since the discovery of its enzymatic activity Restoration of miRNA-451 expression downregulates MIF and associated with viral oncoproteins 20 years ago. However, in the decreases expression of reporter genes with MIF target sequences past 10 years, it has become apparent that this pathway is one of in gastric cancer cells that is accompanied by a reduction in cell proliferation and an enhancement of cell death in response to the most frequently mutated pathways in all spontaneous human 31 tumors. PI3Ks belong to a conserved family of lipid kinases that irradiation. MiRNA-375 suppresses the PI3K/Akt pathway phosphorylate the 30-hydroxyl group of phosphoinositides.133 through direct targeting of PDK1, a kinase that phosphorylates There are three classes of PI3Ks grouped according to their sub- Akt. This is yet another miRNA that regulates the activity of the strate preference and sequence homology, but only class IA PI3Ks PI3K/Akt pathway. Ectopic expression of miRNA-375 substantially reduces cell viability through induction of the caspase-dependent are implicated in human cancers. The most well-characterized apoptotic pathway23; microarray analysis has revealed that product of this reaction is phosphatidylinositol-3,4,5-trisphosphate miRNA-375 is one of the most downregulated miRNAs in gastric or PIP3, a critical second messenger that recruits AKT for activation 134 cancer. Finally, other research has indicated that miRNA-143 is of growth, proliferation and survival signaling. PIP3 is negatively involved in the regulation of cell function through the PI3K/Akt regulated through dephosphorylation by the tumor-suppressor pathway because its target gene is Akt itself.35 phosphatase and tensin homolog (PTEN). Class IA PI3Ks are heterodimers comprising a regulatory subunit (p85a, p55a, p50a,p85b, p55g) and a catalytic subunit (p110a, p110b, p110d) Ras/Raf/MEK/ERK pathway 135 and are activated downstream of receptor tyrosine kinases. The Ras/Raf/MEK/extracellular-signal-regulated kinase (ERK) path- PTEN is a tumor-suppressor gene and its role in tumor biology is 136 way is another important pathway that plays a fundamental role well characterized. Inactivation of PTEN leads to the activation in the regulation of cell proliferation and survival as well as in of AKT through accumulation of PIP3. Moreover, pAkt is a crucial human tumorigenesis. The Ras/Raf/MEK/ERK pathway is a central protein involved in the regulation of cell cycle progression, cell signal transduction pathway that transmits signals from multiple survival, apoptosis, invasion and radiosensitivity. The PTEN gene is cell surface receptors to transcription factors in the nucleus.138 an important functional target of the miRNA-221/222 cluster in This pathway is frequently referred to as the MAP kinase pathway gastric cancer cells. Modulation of miRNA-221/222 expression by as MAPK stands for mitogen-activated protein kinase, thereby antisense or overexpression strategies directly affects PTEN indicating that this pathway can be stimulated by mitogens, cyto- expression.89 PTEN is also one of the target genes of miRNA-21 kines and growth factors. The pathway is activated by membrane that increases the proliferation and invasion of gastric cancer translocation of Raf stimulated by Ras. Three members of the RAS cells.92 MiRNA-214 has displayed similar effects.109 family (HRAS, KRAS and NRAS) are found to be activated by Macrophage migration inhibitory factor (MIF) promotes gastric mutation in human tumors.139 Hashimoto et al.48 demonstrated epithelial cell proliferation through the PI3K/Akt pathway.137 that reduced expression of miR-181c may play an important role

Cancer Gene Therapy (2014), 305 – 316 & 2014 Nature America, Inc. & 04Ntr mrc,Inc. America, Nature 2014 Table 1. Blue miRNAs (miR) are commonly dysregulated, and pink miRNAs (miR) are identified as being oppositely regulated in different studies

Method Sample Upregulation Downregulation Reference

Microarray 20 Cases of gastric carcinoma and 21 miR-223, miR-21, miR-103-2, miR-92-2, miR-25, miR-191, miR-221, miR-125b-2, miR-218-2, miR-136, miR-212prec, miR-96, miR-138-2, 16 (NA) corresponding cases of normal gastric miR-103-1, miR-214, miR-222, miR-125b-1, miR-100, miR-107,miR-92-1, miR- miR-33b tissue 192, miR-23a, miR-215, miR-7-2, miR-24-1, miR-99b, miR-24-2 Microarray Three cases of gastric cancer samples miR-17, miR-18a, miR-18b, miR-19a, miR-20a, miR-20b, miR-21, miR-106a, miR- miR-31, miR-133b, miR-139-5p, miR-195, miR-378, 17 (NA) stored in liquid nitrogen, clinical stage III 106b, miR-340* ,miR-421, miR-658 (all of these were moderately different in miR-497, miR-768-3p intestinal-type gastric adenocarcinoma. Nontumorous tissues were selected 1.5 cm from the tumor and contained no obvious tumor cells, as evaluated by a pathologist). qRT-PCR (72) 42 Cases of undifferentiated carcinoma miR-34b, miR-34c, miR-128a miR-128b, miR-129, miR-148a 18 specimens and corresponding normal tissues Microarray Gastric cancer tissues and corresponding miR-7, miR-10a, miR-15a, miR-15b, miR-17-3p, miR-17-5p, miR-18a, miR-17B18aB19aB20aB19b-1B92-1 cluster, miR- 19 (NA) normal tissues, but the number of cases miR-19a, miR-19b, miR-20a, miR-20b, miR-21, miR-23a, miR-25, miR-27a, miR- 106bB93B25 cluster, were not indicated 29b, miR-33, miR-34a, miR-34b, miR-92, miR-93, miR-99b, miR-106a, miR-106b, miR-222B221 cluster miR-135b, miR-146a, miR-146b, miR-155, miR-182, miR-183, miR-188, miR-194, miR-196b, miR-198, miR-199a, miR-199a*, miR-200a*, miR-200b, miR-203, miR- 210, miR-214, miR-215, miR-221, miR-222, miR-223, miR-338, miR-370, miR-425-5p, miR-429, miR-494, miR-575, miR-629, miR-630, miR-663, miR-765, miR-801 Microarray Four pairs of gastric cancer tissues and miR-9-1, miR-23a, miR-27a , miR-191 miR-181b-1, miR-181c, miR-182, miR-183, miR-210 20 corresponding normal tissues Microarray Three normal gastric tissues and 24 miR-518b, miR-26b, miR-212, miR-320, miR-409-3b, miR-30a-5b, miR-379 miR-9, miR-433, miR-490, miR-155, miR-188, miR-630, 21 (NA) malignant tissues (2 in early phase and miR-503, miR-611, miR-545, miR-567, miR-575, miR-197, 22 in late phase of gastric cancer) miR-649, miR-19b, miR-338, miR-383, miR-652, miR-551a, miR-370 Microarray Three pairs of gastric cancer tissues and miR-223, miR-106b, miR-147, miR-34a, miR-130b*, miR-106a, miR-18a, miR-17, miR-638, miR-378 22 (847) corresponding normal tissues miR-98, miR-616*, miR-181a-2*, miR-185, miR-1259, miR-601,

miR-196a*, miR-221*, miR-302f, miR-340*, miR-337-3p, miR-520c-3p, cancer gastric Zhang in Z pathways signaling and MiRNA miR-575,miR-138 Microarray 22 Gastric cancer tissues, representing 14 miR-550, miR-18a*, miR-196a, miR-615, miR-425-3p, miR-181a*, miR-431, miR- miR-204, miR-551b, miR-133a, miR-139, miR-133b, miR- 23 (470) of the intestinal type and 8 of the diffuse 196b, miR-224, miR-92b, miR-135b, miR-18a, miR-106a, miR-17-5p, miR-146a, 375, miR-30a-3p, miR-29c, miR-363, miR-582, miR-148a, type, as well as 5 nonneoplastic gastric miR-301, miR-93, miR-19a, miR-20a, miR-18b, miR-20b, miR-583, miR-221, miR- miR-30a-5p, miR-30e-5p, miR-497, miR-572, miR-638, miR- al et epithelia and 5 control 335, miR-25, miR-324-5p, miR-15b, miR-425-5p, miR-92, miR-194, miR-361, 195 miR-10a, miR-222 Microarray 184 Gastric cancer tissues and 169 non- miR-181d, miR-181a-1, miR-181a-2, miR-181c, miR-181b-1, miR-181b-2, miR- miR-148a, miR-148b, miR-375, miR-29b-1, miR-29b-2, miR- 24 (326) tumor mucosae 21, miR-25, miR-92-1, miR-92-2, miR-93, miR-17-5p, miR-106a, miR-20b, miR- 29c, miR-152, miR-218-2, miR-451, miR-30d, miR-30a-5p, 135a-1, miR-135a-2, miR-425-5p, miR-106b, miR-20a, miR-19b-1, miR-19b-2, miR-30b, miR-30c-1, miR-30c-2, miR-422b miR-224, miR-18a, miR-135b, miR-19a, miR-345, miR-191 qRT-PCR (NA) 30 Pairs of gastric cancer tissues and miR-125a-3p, miR-133b, miR-143, miR-195, miR-212 (selected 60 miRNAs that miR-30b 25 corresponding normal tissues had been reported to participate in the regulation of cell growth, cell proliferation, cell differentiation, cell apoptosis, tumor cell invasion, migration

acrGn hrp 21) 0 316 – 305 (2014), Therapy Gene Cancer in other cancers) Microarray 40 Pairs of gastric cancer tissues and miR-21, let-7i, miR-16, miR-20a, let-7f, miR-199b-3p, miR-214, miR-15a, miR-17, miR-623, miR-134, miR-181c(a), miR-654-5p, miR-936, 26 (799) corresponding normal tissues miR-142-3p, let-7a, let-7d, miR-18a, miR-27a, miR-146b-5p, miR-192, miR-23a, miR-939, kshv-miR-K12-3, miR-550, miR-486-5p, miR-575, let-7e, miR-107, miR-103, miR-19a, miR-199b-5p, miR-451, miR-200a, miR-27b, hcmv-miR-UL70-3p, miR-638, hsv1-miR-H1, miR-139-3p, miR-223, miR-18b, miR-29b, miR-224, let-7g, miR-135b, miR-200b, miR-34a, miR-202, miR-378, hsv1-miR-LAT, miR-596, miR-188-5p, miR-199a-5p, miR-125b, miR-215, miR-885-5p, miR-28-3p, miR-30d, miR-572, miR-1225-5p, miR-345, miR- miR-331-3p, miR-28-5p, miR-301a 30a, miR-671-5p, hiv1-miR-H1, miR-148a(a), miR-222, miR- 10b(a), miR-564, miR-193b(a), miR-125a-3p, miR-370, miR- 375, miR-923, miR-513c, miR-513a-5p, miR-494, miR-513b Microarray 20 Gastric primary tumors of the miR-21, miR-223, miR-25, miR-17-5p, miR-125b, miR-181b, miR-106a, miR-107, miR-136, miR-218, miR-212, miR-96, miR-339 27 intestinal type and corresponding miR-92,miR-103, miR-221, miR-93, miR-100, miR-106b normal tissues Abbreviations: miRNA, microRNA; NA, not available; qRT-PCR, quantitative reverse transcriptase-PCR. The number of probes is indicated in parentheses in the first column. 307 MiRNA and signaling pathways in gastric cancer Z Zhang et al 308 Table 2. MiRNAs act as a tumor suppressor

Name Study materials Methods Target gene Mechanism of action Reference

miRNA-34 Cell line Luciferase reporter Bcl-2, Notch, HMGA2 Cell proliferation, cell cycle, 28 apoptosis, chemosensitization let-7 family Tissue and cell line qRT-PCR HMGA2 Cell invasion 29 miRNA-15b Cell line Luciferase reporter BCL2 Apoptosis 30 and miRNA-16 miRNA-451 Tissue and cell line Luciferase reporter MIF Cell proliferation 31 miRNA-9 Tissue and cell line Luciferase reporter RAB34, NF-kB1, CDX2 Cell proliferation, cell cycle 21,32,33 miRNA-433 Tissue and cell line Luciferase reporter GRB2 NA 21 miRNA-512-5p Cell line Luciferase reporter Mcl-1 Cell proliferation, apoptosis 34 miRNA-143 Cell line Western blot Akt, IRS-1, b-actin, COX-2 Cell proliferation, apoptosis 35,36 and miRNA-145 miRNA-141 Tissue and cell line NA FGFR2 Cell proliferation 37 miRNA-152 Tissue and cell line Western blot CCKBR Cell proliferation, invasion 38 miRNA-375 Tissue and cell line Luciferase reporter JAK2, PDK1, 14-3-3z Cell proliferation, apoptosis 23,39 miRNA-218 Tissue and cell line Luciferase reporter ECOP, Robo1 Cell proliferation, apoptosis, 40,41 invasion, migration miRNA-331-3p Cell line Luciferase reporter E2F1 Cell proliferation 42 miRNA-29 Cell line NA Cdc42 Cell proliferation, migration, invasion 43 miRNA-129-2 Tissue Immunohistochemical SOX4 Migration 44 study, qRT-PCR miRNA-212 Tissue and cell line Luciferase reporter MeCP2, Myc Cell proliferation 45,46 miRNA-101 Tissue and cell line qRT-PCR EZH2, Cox-2, FOS, Mcl-1 Cell proliferation, migration, invasion 47 miRNA-181c Tissue and cell line Luciferase reporter NOTCH4, KRAS Cell proliferation 48 miRNA-449a Cell line qRT-PCR, western blot cyclin D1, BCL2 Cell cycle, apoptosis 49 miRNA-449b Cell line Luciferase reporter GMNN, MET, CCNE2, SIRT1 Cell proliferation 50 miRNA-137 Tissue and cell line Luciferase reporter CDC42 Apoptosis 51 miRNA-29a Tissue and cell line Luciferase reporter p42.3 Cell proliferation, cell cycle 52 let-7f Tissue and cell line Luciferase reporter MYH9 Invasion, migration 53 miRNA-125a-5p Tissue and cell line Luciferase reporter ERBB2 Cell proliferation 54 miRNA-486 Tissue and cell line Luciferase reporter OLFM4 Cell proliferation 26 miRNA-148b Tissue and cell line Luciferase reporter CCKBR Cell proliferation 55 miRNA-429 Tissue and cell line Luciferase reporter c-Myc Cell proliferation 56 miRNA-148a Tissue and cell line Luciferase reporter PAI-1, VAV2, ITGA5, ITGB8, Cell proliferation, invasion, 38,57–60 ROCK1, SMAD2, MMP7 migration, adhesion miRNA-610 Tissue and cell line Luciferase reporter VASP Invasion, migration 61 let-7a Tissue and cell line Luciferase reporter RAB40C Cell proliferation, cell cycle 62 miRNA-409 Tissue and cell line Luciferase reporter RDX Invasion, migration 63 miRNA-409-3p Tissue and cell line Luciferase reporter PHF10 Cell proliferation, apoptosis 64 miRNA-516a-3p Tissue and cell line Luciferase reporter SULF1 Migration 65 miRNA-10b Tissue and cell line Luciferase reporter MAPRE1 Cell proliferation 66 miRNA-497 Cell line Luciferase reporter BCL2 Apoptosis 67 miRNA-101 Tissue and cell line qRT-PCR, western blot COX-2 Cell proliferation, apoptosis 68 miRNA-144 Tissue and cell line Luciferase reporter ZFX NA 69 miRNA-223 Tissue and cell line Luciferase reporter STMN1 Cell proliferation, apoptosis 70 miRNA-135a Tissue and cell line Luciferase reporter JAK2, ROCK1 Cell proliferation 71,72 miRNA-133b Tissue and cell line Luciferase reporter FGFR1 Cell proliferation 73 miRNA-206 Tissue and cell line Luciferase reporter CyclinD2 (CCND2) Cell proliferation 74 miRNA-145 Tissue and cell line Luciferase reporter N-cadherin Invasion, migration 75 miRNA-338-3p Tissue and cell line Luciferase reporter SSX2IP, P-REX2a Cell proliferation, invasion, 76,77 migration miRNA-7 Tissue and cell line Luciferase reporter IGF1R, EGFR Invasion, migration 78,79 miRNA-22 Tissue and cell line Luciferase reporter Sp1 Invasion, migration 80 miRNA-204 Tissue and cell line Luciferase reporter Bcl-2 Apoptosis 81 miRNA-874 Tissue and cell line Luciferase reporter AQP3 Cell proliferation, invasion, 82 migration miRNA-129 family Cell line Western blot CDK6 Cell proliferation 83 miRNA-199a-3p Tissue and cell line Luciferase reporter mTOR Cell proliferation, apoptosis 84 miRNA-146a/b Tissue and cell line Luciferase reporter UHRF1 Invasion, migration 85 miRNA-124 Tissue and cell line Luciferase reporter EZH2 Cell proliferation, apoptosis 86 miRNA-363 Tissue and cell line Luciferase reporter MBP-1 Cell proliferation, migration 87 Abbreviations: miRNA, microRNA; NA, not available; qRT-PCR, quantitative reverse transcriptase-PCR.

in gastric carcinogenesis through KRAS overexpression. There are phosphatases as well as the GTP/GDP exchange protein Ras and at least three Raf gene families that have been confirmed to be members of the Rho family of GTPases including RhoA, Rac1 and present in eukaryotic cells: Raf-1, A-Raf and B-Raf. Activation and Cdc42.140 A group of Chinese researchers demonstrated that miRNA- inactivation of Raf-1 is complex and involves multiple kinases and 29s could effectively inhibit protein expression/phosphorylation of

Cancer Gene Therapy (2014), 305 – 316 & 2014 Nature America, Inc. MiRNA and signaling pathways in gastric cancer Z Zhang et al 309 Table 3. MiRNAs act as an oncogene

Name Study materials Methods Target gene Mechanism of action Reference

miRNA-421 Tissue and cell line Western blot CBX7, RBMXL1, Cell proliferation, apoptosis, 88 CNTN-1 cell adhesion miRNA-106b-25 Cluster Cell line Luciferase reporter E2F1, Bim Cell proliferation, apoptosis 27 miRNA-106bB93B 25 Tissue and cell line Luciferase reporter p57, p21 Cell proliferation, cell cycle 19 miRNA-222 B 221 Tissue and cell line Luciferase reporter p27, p57 Cell proliferation, cell cycle 19 miRNA-221/222 cluster Cell line Luciferase reporter PTEN Cell proliferation, cell 89 cycle, apoptosis, invasion, enhances cell radiosensitivity miRNA-21 Tissue and cell line Luciferase reporter RECK, PDCD4, PTEN Cell proliferation, cell cycle, 90–92 metastasis, apoptosis miRNA-21 Tissue and cell line Luciferase reporter Serpini1 NA 93 miRNA-372 Cell line Luciferase reporter LATS2 Cell proliferation, cell 94 cycle, apoptosis miRNA-372 Cell line Luciferase reporter TNFAIP1 Cell proliferation, apoptosis 95 miRNA-27a Tissue and cell line Luciferase reporter, PHB (Prohibitin), Cell proliferation, migration 20,96 qRT-PCR ZBTB10 miRNA-27a Tissue and cell line Luciferase reporter HOXA10 NA 97 miRNA-27 Tissue and cell line Luciferase reporter APC Migration 98 miRNA-106a Tissue Immunohistochemical Rb1,E2F1 Cell cycle 99 study miRNA-181b Tissue and cell line Luciferase reporter BCL2 Apoptosis 100 miRNA-130b Tissue and cell line qRT-PCR, RUNX3 Apoptosis 101 immunohistochemical study miRNA-199a Tissue and cell line Luciferase reporter MAP3K11 Cell proliferation, 102,103 migration, apoptosis miRNA-150 Cell line Luciferase reporter EGR2 Cell proliferation 104 miRNA-650 Tissue and cell line Luciferase reporter ING4 Cell proliferation, migration 105 miRNA-23a Tissue and cell line Luciferase reporter IL6R Cell proliferation 106 miRNA-192 Tissue and cell line Luciferase reporter ALCAM Invasion, migration 107 and miRNA -215 miRNA-191 Tissue and cell line Luciferase reporter NDST1 Cell proliferation 108 miRNA-214 Cell line Immunohistochemical PTEN Cell cycle, apoptosis 109 study, western blot miRNA-200bc/429 cluster Tissue and cell line Luciferase reporter BCL2 and XIAP Apoptosis 110 miRNA-43c Tissue and cell line Luciferase reporter VEZT Cell cycle 111 miRNA-222 Cell line Luciferase reporter RECK Cell proliferation 112 miRNA-10b Tissue and cell line Luciferase reporter HOXD10 Invasion 113 miRNA-196a Tissue and cell line Luciferase reporter p27kip1 Cell proliferation, apoptosis 114 miRNA-181a Tissue and cell line Luciferase reporter MTMR3, ATM Cell proliferation, apoptosis 115,116 miRNA-18a Tissue and cell line Luciferase reporter PIAS3 Cell proliferation, apoptosis 117 miRNA-301a Tissue and cell line Luciferase reporter RUNX3 Cell proliferation, migration, 118 invasion, apoptosis miRNA-200b Tissue and cell line Luciferase reporter ZEB1 and ZEB2 Cell proliferation, 119 migration, invasion miRNA-296-5p Tissue and cell line Luciferase reporter Caudal-related Cell proliferation 120 homeobox 1 (CDX1) miRNA-17-5p/20a Tissue and cell line Luciferase reporter p21 and TP53INP1 Cell proliferation 121 miRNA-1303 Tissue and cell line Luciferase reporter CLDN18 Cell proliferation, 122 migration, invasion Abbreviations: miRNA, microRNA; NA, not available; qRT-PCR, quantitative reverse transcriptase-PCR.

Cdc42 and its downstream molecule PAK1,43 thereby influencing levels of cyclin A2 maintain CDK2 activity. The CDK2-Cyclin A2 the Ras/Raf/MEK/ERK pathway. complex is active during both S and gap 2 (G2) phases and an In research regarding blood diseases, such as leukemia, it has elevation in CDK1-cyclin B activity in the late G2 phase triggers been found that the Ras/Raf/MEK/ERK pathway exerts its effects progression to mitosis.144 CDK6 is a cyclin-D1-dependent kinase on cell cycle progression through the induction of cell cycle that phosphorylates the retinoblastoma protein, thereby removing regulatory proteins including CDKs, cyclins and p21Cip1, and repression of E2F transcription factor activity and influencing cell transcription factors including NF-kB, c-Myc, Ets, CREB and cycle progression.145 AP-1.141–143 Previous research has demonstrated that many of Feng et al.126 demonstrated that CDK6 is regulated by miRNA- the factors in the Ras/Raf/MEK/ERK pathway are miRNA target 107 in gastric cancer. The expression of miRNA-107 is significantly genes and can interact with miRNAs. Cyclin D and cyclin E that decreased in gastric cancer and re-expression of miRNA-107 in bind to CDK4/6 and CDK2, respectively, are important gap 1 (G1) gastric cancer cells significantly decreases proliferation. In other cyclins that are rate-limiting for S-phase entry. After entering the S research, it was found that miRNA-206 is involved in gastric cancer phase, expression of cyclin E becomes reduced but increased proliferation, potentially via direct modulation of the downstream

& 2014 Nature America, Inc. Cancer Gene Therapy (2014), 305 – 316 MiRNA and signaling pathways in gastric cancer Z Zhang et al 310 Table 4. Conflicting results

Name Study materials Methods Target gene Mechanism of action Reference

miRNA-148a Tissue and cell line Western blot CCKBR Cell proliferation, invasion 38,57,58 miRNA-148a Cell line Luciferase reporter p27 Cell proliferation 123 miRNA-126 Tissue and cell line Luciferase reporter Crk Cell proliferation, cell adhesion, migration 124 miRNA-126 Cell line Luciferase reporter SOX2 Cell proliferation, apoptosis 125 miRNA-107 Cell line Luciferase reporter CDK6 Cell proliferation, cell cycle 126 miRNA-107 Tissue and cell line Luciferase reporter DICER1 Invasion, migration 127 miRNA-146a Tissue and cell line Luciferase reporter SMAD4 Cell proliferation, apoptosis 128 miRNA-146a Tissue and cell line Luciferase reporter CARD10,COPS8 CARD10 and COPS8 are involved in 129 GPCR-mediated activation of NF-kB. NF-kB regulates expression of cytokines and growth factors involved in several aspects of tumor progression. miRNA-146a Tissue and cell line Luciferase reporter L1CAM Invasion, migration 130 miRNA-146a Tissue and cell line Luciferase reporter EGFR and IRAK1 Invasion, migration 131 miRNA-146a Tissue and cell line Luciferase reporter WASF2 Invasion, migration 132

target Cyclin D2.74 MiRNA-106b and miRNA-93, both of which are The Ras/Raf/MEK/ERK pathway and Raf by itself also have upregulated in gastric cancer and are downstream targets of the diverse effects on key molecules involved in the prevention of oncogenic transcription factor E2F1, directly target p21 and thus apoptosis. It has been revealed that the Ras/Raf/MEK/ERK pathway impair the tumor-suppressive activity of transforming growth can phosphorylate Bad on S112 that contributes to its inactivation factor-b.39 E2F1 was verified to be a promising target gene of both and subsequent sequestration by 14-3-3 proteins.151 This allows miRNA-331-3p and miRNA-106a, thereby modulating the G1/S B-cell lymphoma 2 (Bcl-2) to form homodimers and generate an transition.42,99 The former, miRNA-331-3p, exhibits action as a tumor antiapoptotic response. Activation of the Raf/MEK/ERK cascade suppressor whereas the latter does the opposite. Cho et al.94 have can also result in phosphorylation of the antiapoptotic Mcl-1 confirmed that miRNA-372 is expressed in human gastric cancer (myeloid cell leukemia 1) protein and the proapoptotic Bim AGS cells and its expression supports the growth of these cells protein. Phosphorylation of Bim results in its disassociation from through downregulation of the tumor suppressor LATS2. However, Bcl-2, Bcl-XL and Mcl-1, after which Bim becomes ubiquitionated previous reports suggest that overexpression of LATS2 results in cell and targeted to the proteasome. This allows Bcl-2, Bcl-XL and Mcl- cycle arrest at the G2/M phase via inhibition of Cdc2-Cyclin B kinase 1 to bind Bax and prevent Bax activation as well as the formation activity.146 The p42.3 gene is the target gene of miRNA-29a and one of Bax: Bax homodimers, thus inhibiting apoptosis.152–154 Depend- result indicated that p42.3 silencing could alter the expression of ing on the cell type as well as the stimuli used to induce apoptosis, two key proteins that are involved in cell cycle regulation: CHK2 and Raf can function as either an inhibitor155 or a promoter156 of cyclin B1.52 The activities of CDK (cyclin-dependent kinase) are apoptosis. regulated by a class of molecules known as CDK inhibitors. Currently, The execution of mitochondria-mediated apoptosis is mainly CDK inhibitors are composed of the p16 family members (p15, p16, governed by opposing activities of proapoptotic (Bax, Bak, Bik, p18 and p19)147 and the p21 family members (p21, p27, p28 and Bim, Bad, Bid, HRK, NOXA, PUMA, BNIP3 and BNIP3L) and p57).148,149 Kim et al.19 reported that miRNA-106b-93-25 and miRNA- antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, A1 and Mcl-1) members of the 222-221 clusters suppress the p21 family of CDK inhibitors, and Bcl-2 family. MiRNA dysregulation has been shown to regulate specifically that miRNA-25 targets p57 through the 30-untranslated apoptosis by altering the expression of Bcl-2 family members in region. In addition, miRNA-106b and miRNA-93 control p21, whereas gastric cancer. The miRNA-106b-25 cluster protects gastric cancer miRNA-222 and miRNA-221 downregulate both p27 and p57. P27 cells from apoptosis. Bim, the farthest downstream apoptotic has been verified to be a direct target of miRNA-148a, thereby effector of the transforming growth factor-b pathway,157 is a key suggesting that miRNA-148a might promote gastric cell proliferation target of miRNA-25.27 Overexpression of miRNA-130b has also by suppressing p27 expression.123 MiRNA-196a is upregulated in been reported to suppress transforming growth factor-b-induced gastric cancer tissues and cell lines and high expression of miRNA- Bim expression and apoptosis by targeting RUNX3 in gastric 196a is significantly associated with tumor size, poor pT stage, pN cancer cells.101 Several miRNAs, including miRNA-15b, miRNA-16, stage and patients’ overall survival times. Moreover, downregulation miRNA-34, miRNA-181b and miRNA-497, have been shown to of miRNA-196a suppresses gastric cancer cell proliferation both directly target the antiapoptotic protein Bcl-2 and regulate in vitro and in vivo by targeting p27kip1.114 apoptosis.28,30,67,100 One finding suggests that the miRNA-200bc/ Nuclear factor (NF)-kB is an important transcription factor 429 cluster might play a role in the development of multidrug involved in a variety of tumorigenic mechanisms.150 Experimental resistance in both gastric and lung cancer cell lines, at least in part evidence indicates that miRNA-9 targets NF-kB1 and regulates the by modulating apoptosis via targeting BCL2 and XIAP.110 growth of gastric cancer cells.32 Overexpression of miRNA-218 also Despite the mounting evidence for miRNA interaction with inhibits NF-kB transcriptional activation as well as transcription of members of the mitochondrial apoptosis pathway, whether or not cyclooxygenase-2, a proliferative gene regulated by NF-kB.40 In they directly interact with members of the Ras/Raf/MEK/ERK another study, the biological relevance of OLFM4 as a miRNA-486 pathway has not yet been reported. Through further study of this target was supported by evidence that OLFM4 silencing can pathway, there may be more meaningful discoveries regarding reduce proliferation of gastric cancer cells, and that OLFM4 this unanswered question. overexpression can rescue the antioncogenic effects of miRNA- 486.26 MiRNA-429 plays a role in the pathogenesis of gastric carcinoma and may function as a recessive cancer gene. C-Myc is a Slit/Robo signaling pathway transcription factor that is an important miRNA-429 target gene.56 The Slit family of guidance cues binds to Roundabout (Robo) According to work published by Xu et al.,46 it may be one of the receptors to modulate neuronal, leukocytic and endothelial targets of miRNA-212. migration. Mammals have three Slit proteins (Slit1–3) and four

Cancer Gene Therapy (2014), 305 – 316 & 2014 Nature America, Inc. MiRNA and signaling pathways in gastric cancer Z Zhang et al 311 Robos (Robo 1–4).158 Slit2 interacts with Robo1 to mediate Peng et al.175 reported an association between miRNA-196a-2 repulsive cues in axon guidance,159 neuronal migration160 and gene polymorphism and gastric cancer risk in a Chinese popula- leukocyte chemotaxis.161 Slit/Robo1 signaling is now recognized tion. Polymorphisms in miRNA-27a act as an important factor in as having a paradoxical role in carcinogenesis.162,163 gastric cancer susceptibility.96,176 Tie et al.41 have demonstrated that miRNA-218 coding genes Polymorphisms falling in miRNA binding sites can alter the are located in and transcribed together with Slit genes, which are strength of miRNA binding and disturb miRNA-mediated post- Robo1 ligands, thus creating a negative feedback loop that transcriptional regulation. Undoubtedly, single-nucleotide poly- regulates Slit/Robo1 signaling. They also found that Slit2, but not morphisms in some target genes may be of as much importance Slit3, interacts with Robo1 to promote gastric cancer invasion. in gastric cancer as those located within the miRNAs themselves. Several results177,178 have been demonstrated to be associated JAK/STAT signaling pathway with gastric cancer. For example, a polymorphism in the puta- Cellular responses to dozens of cytokines and growth factors are tive binding site for miRNA-181a within MTMR3 (rs12537CT) is associated with the susceptibility and prognosis of gastric cancer mediated by the evolutionarily conserved Janus kinase/signal 115 transducers and activators of its transcription signaling pathway in southern Han Chinese. (JAK/STAT). These responses include proliferation, differentiation, Despite work on the polymorphic status of miRNAs and their migration, apoptosis and cell survival, depending on signal, tissue binding sites being in its infancy, significant associations between and cellular context. JAK/STAT signaling is essential for numerous numerous single-nucleotide polymorphisms and gastric cancer developmental and homeostatic processes including hematopoi- risk and onset are already coming to light. These single-nucleotide esis, immune cell development, stem cell maintenance, organis- polymorphisms will be of use for the identification of a subgroup mal growth and mammary gland development.164 In mammals, of high-risk patients, with subsequent monitoring yielding earlier there are four members of the JAK family and seven STATs. JAK2 is tumor detection. a member of the Janus family of cytoplasmic nonreceptor tyrosine 165 kinases that also includes JAK1, JAK3 and TYK2. Previous The miRNAs as diagnostic and prognostic markers research has shown that miRNA-375 may function as a tumor sup- pressor that potentially regulates gastric cancer cell proliferation Noninvasive tests are seeing increasing use as tools for disease 39 diagnostics and serum/plasma miRNAs have been identified as by targeting the JAK2 oncogene. Another study showed that 179 miRNA-135a may function as a tumor suppressor by targeting stable blood-based markers for cancer detection. Circulating JAK2 to repress p-STAT3 activation, reduce cyclin D1 and Bcl-xL methods for miRNA profiling have been suggested as a useful tool expression and inhibit gastric cancer cell proliferation.71 for the early diagnosis of gastric cancer. It has been demonstrated that the plasma concentrations of various miRNAs, such as miRNA- 17-5p, miRNA-21, miRNA-106a and miRNA-106b, are higher Wnt/b-Catenin/Tcf signaling pathway whereas let-7a is lower in gastric cancer patients. The values of The Wnt pathway plays key roles in development and cancer.166 the area under the receiver-operating characteristic curve were The canonically known pathway involves extracellular Wnt 0.721 for the miRNA-106b assay and 0.879 for the miRNA-106a/let- proteins binding to and activating membrane-bound Frizzled 7a ratio assay in gastric cancer.180 Three plasma miRNAs (miRNA- receptors that in turn mediate phosphorylation of Dishevelled. 106b, miRNA-20a and miRNA-221) were significantly elevated Through binding to Axin, Dishevelled inhibits phosphorylation of in gastric cancer patients as compared with healthy controls. b-catenin by disrupting a complex consisting of the adenomatous Furthermore, in another study, areas under the receiver-operat- polyposis coli, Axin and glycogen synthase kinase-3b proteins. ing characteristic curves using miRNA-106b, miRNA-20a and Unphosphorylated b-catenin then binds to transcription factors of miRNA-221 for gastric cancer diagnosis were 0.7733, 0.8593, and the T-cell factor/lymphoid-enhancer factor (Tcf/Lef) family and 0.7960, respectively.181 Quantitative reverse transcriptase-PCR activates transcription of specific target genes including c-Myc, analysis further identified a profile of five serum miRNAs (miRNA-1, cyclin D1, gastrin and ITF-2.167–170 miRNA-20a, miRNA-27a, miRNA-34 and miRNA-423-5p) as a bio- Scott et al.171 revealed that miRNA-27a suppresses expression of marker for gastric cancer detection.182 In another study, the ZBTB10 mRNA that could potentially inhibit gastrin expression by biomarker for detection of early gastric cancer is another five- interfering with Sp1 activation. Expression and activation of the miRNA panel present in plasma.183 However, despite numerous Sp1 transcription factor could contribute to gastric cancer cell studies, the determination of biomarkers still lacks consistency. survival, growth and angiogenesis.172 Another research group Significant work will be required to integrate the various obtained similar conclusions in gastric miRNA research.96 One studies184,185 and enhance the sensitivity and specificity of non- result suggests that MAPRE1 may be a direct and functional target invasive diagnostic tests. of miRNA-10b in gastric cancers,66 and another study showed that Aside from the traditionary prognostic factors, miRNAs may MAPRE1 may also act as an oncogene in gastric cancer by serve as biological prognostic markers for outcome prediction as activating the b-catenin/TCF pathway to promote cellular growth they have recently been used to predict the outcomes of patients and inhibit apoptosis.173 Recent studies have revealed that many with gastric cancer. The probability of survival was significantly proteins, such as adenomatous polyposis coli and Axin, are lower in patients with high expression levels of miRNA-20b or involved in the regulation of the Wnt signaling pathway. miRNA-150. A correlation was found between miRNA-27a and Therefore, miR-27 may modulate Wnt signaling by interacting lymph node metastasis.18 Downregulation of miRNA-451,31 with adenomatous polyposis coli.98 miRNA-218,41 miRNA-145,75 miRNA-409,63 miRNA-125a-3p,186 miRNA-206,187 or miRNA-22,188 are also associated with poor survival outcomes. Ueda et al.24 reported that low expression of CLINICAL IMPLICATIONS OF miRNA IN GASTRIC CANCER let-7g and miRNA-433 and high expression of miRNA-214 was The miRNA polymorphisms associated with gastric cancer associated with unfavorable outcomes in overall survival A miRNA mutation can be defined as a mutation that interferes independent of clinical covariates including depth of invasion, with miRNA function. The miR polymorphisms/mutations can lymph node metastasis and stage. A seven-miRNA signature cause a gain or loss of miRNA function. Functional miR poly- is closely associated with relapse-free and overall survival morphisms or mutations can create or destroy a miRNA binding among patients with gastric cancer. A patient’s prognostic site within a target mRNA and affect gene expression by signature could be applicable to future decisions concerning interfering with the function of a miRNA.174 treatment.189

& 2014 Nature America, Inc. Cancer Gene Therapy (2014), 305 – 316 MiRNA and signaling pathways in gastric cancer Z Zhang et al 312 The miRNAs as therapeutic molecules complex 1) pathways are commonly activated during oncogen- The ability of miRNAs to regulate cellular processes and the aber- esis. Owing to cross-activation and pathway convergence, the rant dysregulation of their expression levels in cancer illustrate the resulting activation of Ras/ERK and PI3K/mTOR signaling could potential of miRNA modulation as a viable therapeutic strategy theoretically facilitate the development of resistance to therapeu- and a powerful intervention tool. By virtue of their ability to target tics targeting only one pathway. Indeed, concurrent KRAS/BRAF multiple genes and their aberrant perturbations in widespread and PI3K/PTEN mutations reduce the cytostatic response of cancer 197,198 cancers, miRNAs have emerged to be promising, novel therapeutic cell lines to AKT and mTOR inhibitors. Furthermore, chronic targets and intervention tools. treatment of melanoma cells harboring activating BRAFV600E There are two main strategies for targeting miRNA expression in mutations can induce resistance via Raf isoform switching and cancer. Direct strategies involve the use of oligonucleotides or upregulation of the MAPKKK COT (also called TPL2) and IGF1R/ 199–201 virus-based constructs to either block the expression of an PI3K/AKT signaling pathways. Tumor samples from phase I oncogenic miRNA or to reintroduce a tumor-suppressor miRNA clinical trials with Raf inhibitors also exhibit NRAS mutations and 202 lost in cancer. Indirect strategies involve the use of drugs to platelet-derived growth factor receptor upregulation. Encourag- modulate miRNA expression by targeting their transcription and ingly, treatment with inhibitors to both pathways has been found their processing. For example, reintroduction of miRNA-15a/16-1 to kill resistant melanoma lines and effectively inhibit tumor 199,203,204 induced apoptosis in leukemic MEG01 cells and inhibited tumor growth in prostate and lung cancer mouse models. growth in vivo in a xenograft model.190 The overall review of miRNAs in gastric cancer is an exciting one The miRNA is believed to be relatively safe and has proven to be as significant progress has been made in this field over the past more effective in cancer treatment in earlier studies.191,192 few years. There is no doubt that the relationship between Nevertheless, up till now, no preclinical study has been carried miRNAs and signaling pathways in gastric cancer is complicated out in gastric cancer research. At present, there are still several but new pieces of the puzzle are rapidly being uncovered. Further obstacles to overcome before clinical testing of miRNA treatment investigation into the correlation between the expression of can be carried out, such as delivery of miRNA to a specific tissue or miRNAs and outcomes in gastric cancer patients may clarify the disease site, avoidance of off-target effects, optimization of dosing regulation network and make big steps forward in the treatment and minimization of the likelihood of immune activation. of cancer.

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