Overexpression of IFITM1 Has Clinicopathologic Effects on Gastric Cancer and Is Regulated by an Epigenetic Mechanism
2012; Elsevier BV; Volume: 181; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2012.03.027
ISSN1525-2191
AutoresJieun Lee, Sung‐Ho Goh, Naaleum Song, Jung-Ah Hwang, Seungyoon Nam, Il Ju Choi, Aesun Shin, In-Hoo Kim, Mi-Ha Ju, Jin Sook Jeong, Yeon-Su Lee,
Tópico(s)Signaling Pathways in Disease
ResumoIn an effort to identify novel genes related to the prognosis of gastric cancer, we performed gene expression profiling and found overexpressed levels of human interferon-induced transmembrane protein 1 (IFITM1). We validated the gastric cancer–specific up-regulation of IFITM1 and its association with cancer progression. We also studied its epigenetic regulation and tumorigenesis-related functions. Expression of IFITM1 was evaluated in various human gastric cancer cells and in 35 patient tumor tissues by quantitative RT-PCR and Western blot analyses. The results showed highly up-regulated IFITM1 in cancer cell lines and tissues. Furthermore, IHC studies were performed on 151 patient tissues, and a significant correlation was revealed between higher IFITM1 expression and Lauren's intestinal type (P = 0.007) and differentiated adenocarcinoma (P = 0.025). Quantitative studies of DNA methylation for 27 CpG sites in the regulatory region showed hypermethylation in cells expressing low levels of IFITM1. Methylation-dependent IFITM1 expression was confirmed further by in vitro demethylation using 5-aza-2′-deoxycytidine and luciferase assays. The functional analysis of IFITM1 by silencing of its expression with small-interfering RNA showed decreased migration and invasiveness of cancer cells, whereas its overexpression exhibited the opposite results. In this study, we demonstrated gastric cancer–specific overexpression of IFITM1 regulated by promoter methylation and the role of IFITM1 in cancer prognosis. In an effort to identify novel genes related to the prognosis of gastric cancer, we performed gene expression profiling and found overexpressed levels of human interferon-induced transmembrane protein 1 (IFITM1). We validated the gastric cancer–specific up-regulation of IFITM1 and its association with cancer progression. We also studied its epigenetic regulation and tumorigenesis-related functions. Expression of IFITM1 was evaluated in various human gastric cancer cells and in 35 patient tumor tissues by quantitative RT-PCR and Western blot analyses. The results showed highly up-regulated IFITM1 in cancer cell lines and tissues. Furthermore, IHC studies were performed on 151 patient tissues, and a significant correlation was revealed between higher IFITM1 expression and Lauren's intestinal type (P = 0.007) and differentiated adenocarcinoma (P = 0.025). Quantitative studies of DNA methylation for 27 CpG sites in the regulatory region showed hypermethylation in cells expressing low levels of IFITM1. Methylation-dependent IFITM1 expression was confirmed further by in vitro demethylation using 5-aza-2′-deoxycytidine and luciferase assays. The functional analysis of IFITM1 by silencing of its expression with small-interfering RNA showed decreased migration and invasiveness of cancer cells, whereas its overexpression exhibited the opposite results. In this study, we demonstrated gastric cancer–specific overexpression of IFITM1 regulated by promoter methylation and the role of IFITM1 in cancer prognosis. Gastric cancer is one of the most common human cancers. In 2008, there were approximately 989,600 new cases and 738,000 deaths worldwide.1Jemal A. Bray F. Center M.M. Ferlay J. Ward E. Forman D. Global cancer statistics.CA Cancer J Clin. 2011; 61: 69-90Crossref PubMed Scopus (30314) Google Scholar The most important prognostic indicator for patients with gastric cancer is the observation of metastasis to lymph nodes or distant organs since this occurs in 80% to 90% of patients in the United States and Europe.2Crew K.D. Neugut A.I. 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Association of a leukemic stem cell gene expression signature with clinical outcomes in acute myeloid leukemia.JAMA. 2010; 304: 2706-2715Crossref Scopus (284) Google Scholar, 8Germano S. Kennedy S. Rani S. Gleeson G. Clynes M. Doolan P. McDonnell S. Hughes L. Crown J. O'Driscoll L. MAGE-D4B is a novel marker of poor prognosis and potential therapeutic target involved in breast cancer tumorigenesis.Int J Cancer. 2012; 130: 1991-2002Crossref PubMed Scopus (28) Google Scholar Not only genetic alternations but also epigenetic modifications are involved in tumorigenesis, and many researchers have reported the cancer-specific epigenetic regulation in human cancer.9Gaudet F. Hodgson J.G. Eden A. Jackson-Grusby L. Dausman J. Gray J.W. Leonhardt H. Jaenisch R. Induction of tumors in mice by genomic hypomethylation.Science. 2003; 300: 489-492Crossref PubMed Scopus (1231) Google Scholar, 10Luczak M.W. Jagodzinski P.P. 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Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing.Gastroenterology. 2011; 140: 879-891Abstract Full Text Full Text PDF Scopus (94) Google Scholar From a previous gene expression profiling study of human gastric tumor and normal tissues (GSE30727), we identified interferon-induced transmembrane protein 1 (IFITM1) as an up-regulated gene in tumors. Additional surveys of public databases [ArrayExpress (http://www.ebi.ac.uk/arrayexpress), European Bioinformatics Institute (http://www.ebi.ac.uk), and Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo)] also showed its up-regulated expression in diverse cancer cell lines. IFITM1 (alias Leu13 or 9-27) is a cell surface 17-kDa membrane protein that is encoded on chromosome arm 11p15.5. It is a member of the interferon-induced transmembrane protein family and was initially known to be a leukocyte antigen that is part of a membrane complex involved in the transduction of antiproliferative and homotypic adhesion signals in lymphocytes.14Evans S.S. Lee D.B. Han T. Tomasi T.B. Evans R.L. Monoclonal antibody to the interferon-inducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells.Blood. 1990; 76: 2583-2593Crossref Google Scholar, 15Evans S.S. Collea R.P. Leasure J.A. Lee D.B. IFN-alpha induces homotypic adhesion and Leu-13 expression in human B lymphoid cells.J Immunol. 1993; 150: 736-747Google Scholar, 16Deblandre G.A. Marinx O.P. Evans S.S. Majjaj S. Leo O. Caput D. Huez G.A. Wathelet M.G. Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth.J Biol Chem. 1995; 270: 23860-23866Crossref PubMed Scopus (143) Google Scholar In humans, the IFITM family consists of at least three other functional genes (IFITM1, IFITM2, and IFITM3), and IFITM1 has been studied for its involvement in the inhibition of viral replication,17Brass A.L. Huang I.C. Benita Y. John S.P. Krishnan M.N. Feeley E.M. Ryan B.J. Weyer J.L. van der Weyden L. Fikrig E. Adams D.J. Xavier R.J. Farzan M. Elledge S.J. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.Cell. 2009; 139: 1243-1254Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar promotion of cell invasion,18Hatano H. Kudo Y. Ogawa I. Tsunematsu T. Kikuchi A. Abiko Y. Takata T. IFN-induced transmembrane protein 1 promotes invasion at early stage of head and neck cancer progression.Clin Cancer Res. 2008; 14: 6097-6105Crossref PubMed Scopus (90) Google Scholar and drug response.19Fumoto S. Shimokuni T. Tanimoto K. Hiyama K. Otani K. Ohtaki M. Hihara J. Yoshida K. Hiyama E. Noguchi T. Nishiyama M. Selection of a novel drug-response predictor in esophageal cancer: a novel screening method using microarray and identification of IFITM1 as a potent marker gene of CDDP response.Int J Oncol. 2008; 32: 413-423Google Scholar Furthermore, recent studies suggest an association between IFITM1 and several cancers, such as head and neck cancer,18Hatano H. Kudo Y. Ogawa I. Tsunematsu T. Kikuchi A. Abiko Y. Takata T. IFN-induced transmembrane protein 1 promotes invasion at early stage of head and neck cancer progression.Clin Cancer Res. 2008; 14: 6097-6105Crossref PubMed Scopus (90) Google Scholar serous ovarian cancer,20Johnatty S.E. Beesley J. Chen X. Macgregor S. 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Differential gene expression identified in Uigur women cervical squamous cell carcinoma by suppression subtractive hybridization.Neoplasma. 2010; 57: 123-128Crossref PubMed Scopus (32) Google Scholar Although one study indicated that IFITM1 may modulate the invasiveness of gastric cancer cells,23Yang Y. Lee J.H. Kim K.Y. Song H.K. Kim J.K. Yoon S.R. Cho D. Song K.S. Lee Y.H. Choi I. The interferon-inducible 9–27 gene modulates the susceptibility to natural killer cells and the invasiveness of gastric cancer cells.Cancer Lett. 2005; 221: 191-200Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar no further results were reported, and so the role of IFITM1 and its regulation in gastric cancer is not fully clarified. In this study, we report the up-regulation of IFITM1 expression in gastric cancer and the correlation of IFITM1 expression with clinicopathologic features of patients with gastric cancer. In addition, the promoter CpG methylation analysis revealed that IFITM1 expression is epigenetically regulated. The in vitro functional study showed the effect that IFITM1 exerts on cell migration and invasion of gastric cancer cells. Here, we propose that IFITM1 could be a good candidate as a potential target in the therapy of gastric cancer. All the cell lines were obtained from ATCC (Manassas, VA) or the Korean Cell Line Bank (Seoul, South Korea) and were cultured in the designated media (Thermo Scientific HyClone, Logan, UT) supplemented with 10% fetal bovine serum (Thermo Scientific HyClone) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). HMECs were obtained from Lonza Inc. (Walkersville, MD) and were cultured in MEGM basal medium (Lonza Inc.) supplemented with supplement reagent and growth factors. All human cell lines were grown in a 5% CO2–humidified incubator at 37°C. Thirty-five pairs of normal and gastric carcinoma tissues used in quantitative RT-PCR (RT-qPCR), RT-PCR, and Western blot studies were obtained by endoscopic biopsy from patients after obtaining their informed consent; the protocols were approved by the Institutional Review Board of the National Cancer Center, Goyang Gyeonggi-do, South Korea (see Supplemental Table S1 at http://ajp.amjpathol.org). For immunohistochemical studies, tissues from 151 patients who had undergone gastrectomy and were diagnosed as having tubular adenocarcinoma at the Dong-A University Hospital, Busan, South Korea, between January 1, 2005, and October 31, 2005, were investigated (Table 1). All the patients signed an Institutional Review Board–approved written informed consent form.Table 1Characteristics of the 151 Patients with Gastric Cancer Who Contributed Tissue for These StudiesIFITM1 expression⁎For IFITM1 expression, negative, mild, moderate, and marked were determined according to the immunostaining intensity, and positive cases were categorized.CharacteristicNegative (n = 18)Mild (n = 66)Moderate (n = 50)Marked (n = 17)P valueSex0.5848 Male11463710 Female720137Age (years)0.3026 <601036255 ≥608302512Differentiation0.0250†P < 0.05. Well016116 Moderately423217 Poorly1427184Lauren's classification0.0068†P < 0.05. Intestinal2372911 Mixed59104 Diffuse1120112LVI0.2080 Negative11472611 Positive719246T classification0.1264 T1629164 T231675 T3610111 T4311167LN metastasis0.1141 Negative839225 Positive10272812TNM stage‡Sixth edition of the American Joint Cancer Committee/Union Internationale Contre le Cancer.0.7857 I636207 II512104 III511135 IV2771Data are given as number of patients.LN, lymph node; LVI, lymphovascular invasion. For IFITM1 expression, negative, mild, moderate, and marked were determined according to the immunostaining intensity, and positive cases were categorized.† P < 0.05.‡ Sixth edition of the American Joint Cancer Committee/Union Internationale Contre le Cancer. Open table in a new tab Data are given as number of patients. LN, lymph node; LVI, lymphovascular invasion. Total RNA purified using TRIzol reagent (Invitrogen) was reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen) and was subjected to relative quantification of IFITM1 mRNA by quantitative reverse transcription normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-2-microglobulin.24Rho H.W. Lee B.C. Choi E.S. Choi I.J. Lee Y.S. Goh S.H. Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR.BMC Cancer. 2010; 10: 240Crossref Scopus (68) Google Scholar The primers used are listed in Table 2. QuantiFast SYBR green PCR master mix (Qiagen Inc., Valencia, CA) was used in the LightCycler 480 system (Roche Applied Science, Mannheim, Germany). RT-PCR was performed using Taq DNA polymerase (SolGent Co. Ltd., Daejeon, South Korea).Table 2Primers Used in this StudyExperimentPrimer IDSequenceRT-qPCRIFITM1-Q-F5′-ACTAGTAGCCGCCCATAGCC-3′IFITM1-Q-R5′-GCACGTGCACTTTATTGAATG-3′GAPDH-F5′-TGCACCACCAACTGCTTA-3′GAPDH-R5′-GGATGCAGGGATGATGTTC-3′B2M-F5′-ACTGAATTCACCCCCACTGA-3′B2M-R5′-CCTCCATGATGCTGCTTACA-3′RT-PCRTNF-α-F5′-AAGAATTCAAACTGGGGCCT-3′TNF-α-R5′-GGCTACATGGGAACAGCCTA-3′IL-6-F5′-GAACTCCTTCTCCACAAGCG-3′IL-6-R5′-TTTTCTGCCAGTGCCTCTTT-3′IL-8-F5′-TAGCAAAATTGAGGCCAAGG-3′IL-8-R5′-GGACTTGTGGATCCTGGCTA−3′EpiTYPER analysisIFITM1-01-10-F5′-AGGAAGAGAGTATGATTTTTGTGTTTGAGGGTTTT-3′IFITM1-01-10-R5′-CAGTAATACGACTCACTATAGGGAGAAGGCTCTAATAACCAAAATTTCCTACCCCA−3′IFITM1-02-10-F5′-AGGAAGAGAGTTTTTTGTGTATTTTTTGGTTTTGG-3′IFITM1-02-10-R5′-CAGTAATACGACTCACTATAGGGAGAAGGCTAAAAAACCTCTCTCCTTAACCTTCA-3′IFITM1-03-10-F5′-AGGAAGAGAGTTTGGGTTAATGGATAGTTAGGGAT−3′IFITM1-03-10-R5′-CAGTAATACGACTCACTATAGGGAGAAGGCTCCAAAACCAAAAAATACACAAAAAA-3′IFITM1-04-10-F5′-AGGAAGAGAGGGTTATGAGGATGTTTAGAATTAGGG-3′IFITM1-04-10-R5′-CAGTAATACGACTCACTATAGGGAGAAGGCTAACTCACAAATAACTTCACCCCATA-3′MSP analysisIFITM1-Meth-F5′-GAGATTTTCGTGTTCGATTATGTC-3′IFITM1-Meth-R5′-ATAAAACCCCAAACTCACCG−3′IFITM1-UnMeth-F5′-AGATTTTTGTGTTTGATTATGTTGT-3′IFITM1-UnMeth-R5′-ATAAAACCCCAAACTCACCAAC-3′Promoter assayIFITM1-C1-F(−750)5′-GGGGTACCGTGGCACATGTGTGCCCTG-3′IFITM1-C2-F(−200)5′-GGGGTACCTGGCTAATTCACCAATTTACAAACAGC-3′IFITM1-C1C2-R(−1)5′-CCAAGCTTCTTCTGGCTTTGGGGAAGGAAG-3′B2M, β-2-microglobulin. Open table in a new tab B2M, β-2-microglobulin. Immunoblotting was performed using anti-IFITM1 (sc-66827; Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG antibody (F7425; Sigma-Aldrich, St. Louis, MO), and anti-β-actin (Abcam Inc., Cambridge, MA). The signal intensity was measured after subtracting the background by using Multi Gauge V3.0 software (Fujifilm, Tokyo, Japan). Immunohistochemical detection of IFITM1 was performed on three core cancer tissues and nonneoplastic gastric mucosa specimens (2 mm in diameter) for each individual. Tissues were arranged as arrays of 4.5-μm sections mounted on silicon-coated glass slides with anti-IFITM1 antibody (sc-66827; Santa Cruz Biotechnology) in the BenchMark XT automated system (Ventana Medical Systems Inc., Tucson, AZ) as described previously.25Goh S.H. Hong S.H. Lee B.C. Ju M.H. Jeong J.S. Cho Y.R. Kim I.H. Lee Y.S. eIF3m expression influences the regulation of tumorigenesis-related genes in human colon cancer.Oncogene. 2011; 30: 398-409Crossref Scopus (43) Google Scholar Negative or positive controls were determined by immunostaining intensity of gastric carcinoma cells, and positive cases were categorized into mild, moderate, and marked expression. None of the patients had received any preoperative treatments. Outcomes were determined from the date of surgery until death or December 31, 2009, which resulted in follow-up of 0.8 to 59.8 months (mean, 48.9 months). We determined postoperative pathologic stage using the sixth edition of the American Joint Cancer Committee/Union Internationale Contre le Cancer classification system.26Sobin L.H. Wittekind C. TNM Classification of Malignant Tumors.ed 6. Wiley-Liss Inc., New York2002Google Scholar Genomic DNA extracted from the cell lines using the DNeasy kit (Qiagen Inc.) was sodium bisulfite treated using the EZ DNA Methylation-Gold kit (Zymo Research Corp., Orange, CA). Primers for EpiTYPER and methylation-specific PCR (MSP) analysis were designed using EpiDesigner software (Sequenom Inc., San Diego, CA) and are presented in Table 2. The methylation level was analyzed using EpiTYPER software (Sequenom Inc.). For MSP, genomic DNA was bisulfite modified and amplified using HotStarTaq DNA polymerase (Qiagen Inc.) using MSP primers. For demethylation by 5-azacytidine treatment, cells were seeded 24 hours before treatment and were incubated in media containing 20 μmol/L 5-aza-2′-deoxycytidine (5-aza-dC; Sigma-Aldrich) for 3 days; the medium was changed every 24 hours and harvested for further analysis. For methylation analysis by cloning and sequencing, the promoter CpG site 10–15–contained region was amplified by PCR and was cloned into the pGEM-T easy vector system (Promega Corp., Madison, WI). The sequences of the PCR products were analyzed using a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA). The amplified fragments of the IFITM1 promoter region (construct 1: −200 to −1, and construct 2: −750 to −1 from the start ATG codon; the primer sequences are presented in Table 2) were cloned into the pGL3-basic vector containing the firefly luciferase gene (Promega Corp.). For in vitro methylation, the promoter was treated with insert fragments with SssI CpG methylase (New England Biolabs, Ipswich, MA) and were religated. H23 human lung cancer cells (2 × 105) and each construct (1 μg) were co-transfected with 10 ng of pRL-CMV vector and Renilla luciferase as internal control for the transfection efficiency assessment, and 24 hours later, the luciferase activities were measured using the Dual-Luciferase reporter assay system (Promega Corp.). For the silencing of IFITM1 expression, 1.5 × 105 AGS human gastric cancer cells were transfected using small-interfering RNA (siRNA) specific for human IFITM1 (SI00054117; 5′-ACAGTCTACCATATTATGTTA-3′) or nontargeting siRNA (NC) (Qiagen Inc.) in a 6-well plate and were checked every 24 hours by Western blot analysis. Human IFITM1 open reading frame isolated by PCR using forward primer 5′-GAATTCCACCCTTCCCCAAAGCCAGAAGATG-3′ and reverse primer 5′-GGATCCAGTGCAAAGGTTGCAGGCTA-3′ (Bioneer Corp., Daejeon, South Korea) was cloned into pFLAG-CMV-2 vector (Sigma-Aldrich) to make pFLAG-CMV2-IFITM1. This construct was transfected into 293 cells using Lipofectamine 2000 (Invitrogen). Transfilter migration and invasion assays were performed using 8.0-μm pore inserts in a 24-well Transwell (Corning Inc., Corning, NY). For this assay, AGS cells were transfected with IFITM1 siRNA or NC siRNA for 2 days, or IFITM1-overexpressing 293 cells were seeded after 1 day of incubation. They were isolated and added to the upper chamber of a Transwell with a noncoated filter in the migration assay. The invasion assay was performed using 10% Matrigel (BD Bioscience, San Jose, CA)-coated filters at 5 × 104 cells per well, and the cells were allowed to incubate for 48 hours. The migrating and invading cells were stained with Diff-Quik (using a staining kit from Sysmex Corp., Kobe, Japan). For wound measuring, a scratch on complete confluence was made, and the percentage of cell-free area at 24 hours was measured relative to the distance at 0 hour (100%) using photographed images. Each experiment was performed in triplicate, and mean values are presented. The t-test was used to evaluate the significance of the difference between two groups. The χ2 test was conducted to determine the significance of the difference between the covariates. Survival durations were calculated via the Kaplan-Meier method. The log-rank test was used to compare cumulative survival in the patient groups. We applied P < 0.05 for the consideration of statistical significance throughout the study. The MedCalc software program (version 11.4; MedCalc Software, Mariakerke, Belgium) was used for the analyses. IFITM1 mRNA and protein expressions in six human gastric cancer cell lines (SNU-216, SNU-638, SNU-719, AGS, KATO III, and MKN-28) and two human noncancer cell lines (293 and HMEC) were analyzed by RT-qPCR and Western blot analysis. We found that the expressions of IFITM1 mRNA (Figure 1A) and IFITM1 protein (Figure 1B) were significantly higher in AGS and SNU-638 cell lines than in normal cell lines. The IFITM1 expressions in terms of protein level in gastric cancer cell lines were correlative to the mRNA level (Figure 1B). In parallel studies of IFITM1 mRNA and protein expressions in 27 pairs of gastric cancer tissues and matched normal tissues, IFITM1 was highly up-regulated in 25 of 27 cancer tissues (92.6%) compared with in their normal counterparts (paired t-test P < 0.001) (Figure 1C). The mRNA expression levels of IFITM1 in 10 intestinal-type (Lauren's classification) tumor tissues showed a much higher elevation (5.7-fold increase in average) than in its normal counterpart. In accordance with the mRNA level difference, the protein levels in the paired patient tissues also showed an elevation in most cancer tissues (five of eight patients; 62.5%) compared with in its normal counterpart (P = 0.008, 3.4-fold increase in average) (Figure 1D). Since IFITM1 is an inflammation-inducible gene, the higher level of IFITM1 in some cancer tissues may reflect increased inflammation in these tissues. We, therefore, compared the expression of IFITM1 with that of other inflammation marker genes, including tumor necrosis factor-α (TNF-α), IL-6, and IL-8. In the 10 patients showing increased IFITM1expression (fold change >3 times), IL-6 and IL-8 levels also increased, although TNF-α level was not correlated with IFITM1 expression (Figure 1E). These results suggest that up-regulation of IFITM1 expression levels in cancer reflect increased inflammatory responses. One hundred thirty-three of 151 patients (88.1%) showed positive IFITM1 expression in gastric adenocarcinomas. Mostly, IFITM1 was expressed in cytoplasms and occasionally was co-expressed at luminal borders, especially in well-differentiated adenocarcinomas (Figure 2; see also Supplemental Figure S1 at http://ajp.amjpathol.org). Although detected in infiltrating mononuclear leukocytes, nonneoplastic gastric epithelial cells did not express IFITM1. The characteristics of the 151 patients examined are presented in Table 1. Increased IFITM1 expression was more frequent in histologically differentiated gastric carcinomas (P = 0.025). In particular, the intestinal type (by Lauren's classification) showed a higher proportion of moderate or marked IFITM1 expression (P = 0.007), which is in accordance with mRNA expression analyzed by RT-qPCR. Patients with gastric carcinoma with moderate and marked expression of IFITM1 were found to have poorer survival than those with its negative and mild expression, even though this difference was not statistically significant (P = 0.086) (see Supplemental Figure S2 at http://ajp.amjpathol.org). DNA methylation levels of IFITM1 were determined in five gastric cancer cell lines and one normal cell line covering 27 CpG sites over the promoter, exon 1, and intron 1 regions (Figure 3A; also see the detailed CpG sites in Supplemental Figure S3 at http://ajp.amjpathol.org). In the vicinity of the transcriptional start site, high IFITM1-expressing gastric cancer cell lines (SNU-638 and AGS) showed low methylation compared with the normal cell line (293) and the low IFITM1-expressing gastric cancer cell lines (SNU-216, SNU-719, and MKN-28) (Figure 3B). Although CpG sites 1 to 5 seemed to be unrelated to gene expression, the methylation status of CpG sites 11 to 26 inversely correlated with IFITM1 gene expression, indicating promoter methylation-regulated gene expression. Based on the results of the quantitative methylation analysis, primers were designed to target four CpG sites (No. 12, 13, 19, and 20). Although there was significant methylation of the four CpG sites in cells expressing low levels of IFITM1 (SNU-216, SNU-719, MKN-28, and 293), there was little methylation of these CpG sites in cells highly expressing IFITM1 (SNU-638 and AGS) (Figure 3C). Therefore, these results suggest that the vicinity of the transcriptional start site region in the IFITM1 promoter, especially CpG sites 12 and 13, may play an important role in the regulation of methylation-related expression of IFITM1. When we searched the transcription factor binding sites of this region using MatInspector analysis software (Genomatix Software GmbH, Munich, Germany), the predicted binding site of the human proto-oncogene ETS factor and cyclin D binding myb-like transcription factor, were found situated in the exact position of CpG site 12, with core similarity of 1.0 for all and matrix similarity of 0.946 and 0.874, respectively (see Supplemental Figure S3 at http://ajp.amjpathol.org). We examined whether DNA demethylation by 5-aza-dC treatment affected IFITM1 mRNA expression. 5-Aza-dC–treated SNU-216, SNU-719, and MKN-28 gastric cancer cells showed a greater increase in IFITM1 mRNA expression compared with in controls, as judged by RT-qPCR analysis (2.5-, 2.5-, and 30.3-fold increases, respectively) (Figure 3D). These results suggest that DNA methylation suppresses IFITM1 gene expression and that demethylation increases it. Methylation-dependent IFITM1 expression was further confirmed by in vitro methylation studies in which two promoter constructs (1: −750 to −1, and 2: −200 to −1; Figure 3A) were methylated by methyltransferase and ligated to a luciferase reporter gene. Comparison of the luciferase activity of the methylated and unmethylated IFITM1 promoter showed an approximately 28% decrement in the case of construct 2 (Figure 3E), suggesting that the methylation status of the −200 to −1 promoter region (containing CpG 10 to 15) is important for the regulation of IFITM1 expression. To investigate the role of IFITM1 in the progression of malignancy, we examined the influence of IFITM1 on the migration and invasion of AGS cells. We verified that IFITM1 expression is reduced by siRNA in AGS cells 24 hours after siRNA transfection and remains reduced for 96 hours. Nontargeting siRNA (NC) had no effect on IFITM1 mRNA or protein levels (Figure 4A). At 96 hours, wound healing of IFITM1 siRNA-transfected cells was inhibited (Figure 4B). The cell migration or invasion counted from 10 randomly selected areas per well at 24 hours showed that IFITM1 siRNA also inhibited migration (Figure 4C) and invasion (Figure 4D) remarkably. We also confirmed that overexpression of IFITM1, c
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