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Cell Cycle and Senescence FoxM1 is Overexpressed in Helicobacter pyloriInduced Gastric Carcinogenesis and Is Negatively Regulated by miR-370 Yimin Feng 1 , Lixiang Wang 1,2 , Jiping Zeng 1,3 , Li Shen 1 , Xiuming Liang 1 , Han Yu 1 , Shili Liu 1 , Zhifang Liu 1,3 , Yundong Sun 1 , Wenjuan Li 1 , Chunyan Chen 1 , and Jihui Jia 1 Abstract Helicobacter pylori (H. pylori) infections are strongly implicated in human gastric mucosaassociated diseases. Forkhead box M1 (FoxM1), a key positive regulator of cell proliferation, is overexpressed in gastric cancer. MicroRNAs are important post-transcriptional regulators of gene expression. In this study, the effects of H. pylori infection on FoxM1 expression and possible mechanisms of carcinogenesis were explored. The expression of FoxM1 was gradually increased in human gastric specimens from inammation to cancer. FoxM1 upregulation was time- and concentration-dependent in gastric epithelial-derived cell lines infected with H. pylori. CagA, a key virulence factor of H. pylori, was associated with increased FoxM1 expression. H. pylori and CagA inhibited the expression of p27 Kip1 (CDKN1B) and promoted cell proliferation by upregulating FoxM1. The expression of miR-370 was decreased in human gastritis and gastric cancer. FoxM1 was directly downregulated by miR-370 in gastric cell lines. H. pylori and CagA inhibited miR-370 expression, which led to overexpression of FoxM1 and cell proliferation. Furthermore, the overexpression of FoxM1 and reduced expression of miR-370 was conrmed in H. pyloriinfected C57BL/6J mice. H. pylori infection and CagA upregulated FoxM1 expression, dependent on miR-370, altered the expression of p27 Kip1 , and promoted proliferation in gastric cells. Implications: These ndings delineate the mechanisms governing FoxM1 regulation and the role of H. pylori in the process of gastric carcinogenesis. Mol Cancer Res; 11(8); 83444. Ó2013 AACR. Introduction Gastric cancer is the fourth diagnosed cancer and the second most common cause of cancer-related death world- wide, especially in developing countries (1, 2). Most patients are diagnosed at an advantaged stage because of lack of early specic symptoms, so prognosis remains poor even after extensive surgery and adjuvant therapy. Gastric carcinogenesis is a complex, multistep, and mul- tifactorial event. Helicobacter pylori (H. pylori) infection is involved in the early stage of gastric cancer pathogenesis by inducing chronic gastritis (36). This chronic gastritis can persist for decades and may result in nonresolving inam- mation, which is a major driver of gastric cancer (6), and the interaction of hostpathogen contributes to this carcino- genesis (7). The protein CagA of H. pylori is closely asso- ciated with gastric cancer and can intervene in signal path- ways in cells (8). To date, however, the underlying molecular mechanisms of carcinogenesis induced by H. pylori remain to be elucidated. Forkhead box M1 (FoxM1), a member of the Fox tran- scription factor family (9), is a key positive cell-cycle regu- lator in cell proliferation. Aberrant expression of FoxM1 is involved in several tumor types, including hepatocellular carcinoma, basal cell carcinoma, breast cancer, lung cancer, prostate cancer, glioblastomas, and gastric cancer (1018), which implies an oncogene role in carcinogenesis. We previously reported that FoxM1 is upregulated in gastric cancer, and its inhibition leads to cellular senescence (18), but the relevance of H. pylori infection and FoxM1 expres- sion associated with the pathogenesis of gastric cancer remains undened. miRNAs, a family of small noncoding RNAs, are impor- tant negative regulators of posttranscriptional gene expres- sion by directly targeting the 3 0 untranslated regions (3 0 UTR) of target mRNAs, eventually promoting the degradation or translation suppression of target mRNAs Authors' Afliations: 1 Department of Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education; and Depart- ments of 2 Pharmacology and 3 Biochemistry, School of Medicine, Shan- dong University, Jinan, PR China Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Y. Feng and L. Wang contributed equally to this work. Corresponding Author: Jihui Jia, Department of Microbiology/Key Lab- oratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, Jinan 250012, PR China. Phone: 0086-531-88382672; Fax: 0086-531-88382502; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-13-0007 Ó2013 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 11(8) August 2013 834 on December 12, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst April 10, 2013; DOI: 10.1158/1541-7786.MCR-13-0007

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Page 1: FoxM1 is Overexpressed in Helicobacter pylori Induced Gastric … · Cell Cycle and Senescence FoxM1 is Overexpressed in Helicobacter pylori–Induced Gastric Carcinogenesis and Is

Cell Cycle and Senescence

FoxM1 is Overexpressed in Helicobacter pylori–InducedGastric Carcinogenesis and Is Negatively Regulated bymiR-370

Yimin Feng1, Lixiang Wang1,2, Jiping Zeng1,3, Li Shen1, Xiuming Liang1, Han Yu1, Shili Liu1, Zhifang Liu1,3,Yundong Sun1, Wenjuan Li1, Chunyan Chen1, and Jihui Jia1

AbstractHelicobacter pylori (H. pylori) infections are strongly implicated in human gastric mucosa–associated diseases.

Forkhead box M1 (FoxM1), a key positive regulator of cell proliferation, is overexpressed in gastric cancer.MicroRNAs are important post-transcriptional regulators of gene expression. In this study, the effects ofH. pylori infection on FoxM1 expression and possible mechanisms of carcinogenesis were explored. Theexpression of FoxM1 was gradually increased in human gastric specimens from inflammation to cancer.FoxM1 upregulation was time- and concentration-dependent in gastric epithelial-derived cell lines infectedwith H. pylori. CagA, a key virulence factor of H. pylori, was associated with increased FoxM1 expression.H. pylori and CagA inhibited the expression of p27Kip1 (CDKN1B) and promoted cell proliferation byupregulating FoxM1. The expression of miR-370 was decreased in human gastritis and gastric cancer. FoxM1was directly downregulated by miR-370 in gastric cell lines.H. pylori and CagA inhibited miR-370 expression,which led to overexpression of FoxM1 and cell proliferation. Furthermore, the overexpression of FoxM1 andreduced expression of miR-370 was confirmed in H. pylori–infected C57BL/6J mice. H. pylori infectionand CagA upregulated FoxM1 expression, dependent on miR-370, altered the expression of p27Kip1, andpromoted proliferation in gastric cells.Implications: These findings delineate the mechanisms governing FoxM1 regulation and the role of H. pylori

in the process of gastric carcinogenesis. Mol Cancer Res; 11(8); 834–44. �2013 AACR.

IntroductionGastric cancer is the fourth diagnosed cancer and the

second most common cause of cancer-related death world-wide, especially in developing countries (1, 2).Most patientsare diagnosed at an advantaged stage because of lack of earlyspecific symptoms, so prognosis remains poor even afterextensive surgery and adjuvant therapy.Gastric carcinogenesis is a complex, multistep, and mul-

tifactorial event. Helicobacter pylori (H. pylori) infection isinvolved in the early stage of gastric cancer pathogenesis by

inducing chronic gastritis (3–6). This chronic gastritis canpersist for decades and may result in nonresolving inflam-mation, which is a major driver of gastric cancer (6), and theinteraction of host–pathogen contributes to this carcino-genesis (7). The protein CagA of H. pylori is closely asso-ciated with gastric cancer and can intervene in signal path-ways in cells (8). To date, however, the underlyingmolecularmechanisms of carcinogenesis induced byH. pylori remain tobe elucidated.Forkhead box M1 (FoxM1), a member of the Fox tran-

scription factor family (9), is a key positive cell-cycle regu-lator in cell proliferation. Aberrant expression of FoxM1 isinvolved in several tumor types, including hepatocellularcarcinoma, basal cell carcinoma, breast cancer, lung cancer,prostate cancer, glioblastomas, and gastric cancer (10–18),which implies an oncogene role in carcinogenesis. Wepreviously reported that FoxM1 is upregulated in gastriccancer, and its inhibition leads to cellular senescence (18),but the relevance of H. pylori infection and FoxM1 expres-sion associated with the pathogenesis of gastric cancerremains undefined.miRNAs, a family of small noncoding RNAs, are impor-

tant negative regulators of posttranscriptional gene expres-sion by directly targeting the 30 untranslated regions(30UTR) of target mRNAs, eventually promoting thedegradation or translation suppression of target mRNAs

Authors' Affiliations: 1Department of Microbiology/Key Laboratory forExperimental Teratology of Chinese Ministry of Education; and Depart-ments of 2Pharmacology and 3Biochemistry, School of Medicine, Shan-dong University, Jinan, PR China

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Y. Feng and L. Wang contributed equally to this work.

Corresponding Author: Jihui Jia, Department of Microbiology/Key Lab-oratory for Experimental Teratology of Chinese Ministry of Education,School ofMedicine, ShandongUniversity, Jinan 250012, PRChina. Phone:0086-531-88382672; Fax: 0086-531-88382502; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-13-0007

�2013 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 11(8) August 2013834

on December 12, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst April 10, 2013; DOI: 10.1158/1541-7786.MCR-13-0007

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(19, 20). miRNAs are frequently deregulated in many typesof human cancers and play critical roles in tumorigenesis,serving as tumor suppressors or oncogenes (21, 22). Recentstudies have shown that several miRNAs, such as miR-146a,miR-155, and miR-21, are involved in H. pylori-inducedinfection (23–25). MiR-370 has been verified as a tumorsuppressor in bladder cancer and cholangiocarcinoma celllines (26, 27). Furthermore, FoxM1 is predicted as a putativetarget of miR-370 by bioinformatics.Here, we aimed to identify the role of FoxM1 inH. pylori

infection and the pathogenesis of gastric cancer and regu-lation by miR-370 in vitro and in vivo.

Materials and MethodsPatients and tissue specimensResected tissues from 25 patients with gastric cancer were

harvested at surgery. Gastritis species were collected from 34patients undergoing gastroscopy. All these patients under-went surgery at Qilu Hospital, Shandong University (Jinan,PR China) from 2009 through 2011. None of the patientshad received adjuvant chemotherapy before surgery. Thediagnosis of all gastric cancer and gastritis was histopatho-logically confirmed. General information about patients isgiven in Supplementary Table S1. The study was approvedby the Ethics Committee of Shandong University School ofMedicine (Jinan, PR China).

Experimental animalsC57BL/6J mice were purchased from the Experimental

Animal Center of Shandong University and maintained inthe Animal Center in Shandong University School of Med-icine. Male mice aged 6 weeks were used in all experiments.We randomly divided 40 mice into 2 groups for treatment:infection (n¼ 24) and control (n¼ 16). The infection groupreceived by intragastric gavage 1� 109 colony-forming unitsofH. pylori (SS1, a standard strain ofH. pylori adapted to themurine stomach.) after 12-hour fasting every other day for 3times. The control group received 1mLPBS each time in thesame way. At 8months, mice were killed, and stomach tissuesamples underwent hematoxylin and eosin staining andimmunohistochemical (IHC) assay. All animal care andexperimental protocols were approved by the Ethics Com-mittee of Shandong University School of Medicine.

H. pylori and bacterial cultureThe standard strains of H. pylori (NCTC 11637,

26695, and SS1) were kindly provided by Dr. JianzhongZhang (Chinese Disease Control and Prevention Center;Beijing, China). All strains were cultured in Brucella brothwith 5% FBS under microaerobic conditions (5% O2,10% CO2, and 85% N2) at 37�C. The strains wereharvested at exponential amplification phases by centri-fugation and added to gastric cell lines at different bac-teria-to-cell ratios and times.

Cell lines and cell cultureThe gastric epithelial-derived cell lines, AGS and BGC-

823, were obtained from China Academia Sinica Cell

Repository (Shanghai, China). AGS cells were cultured inF12 medium supplemented with 10% FBS. BGC-823cells were cultured in RPMI-1640 medium supplementedwith 10% FBS. Both cell lines were incubated in a humid-ified atmosphere containing 5% CO2 at 37�C withoutantibiotics.

Plasmid construction and transfectionThe sequence fragment of 212 bp incorporating the

whole length of pre-miR-370 was amplified from normalhuman genomic DNA with the primers sense, 50-AAGG-GATCCTACTTGAGGGATGGGCGATA-30, and anti-sense, 50-TCAAAGCTTCCCGAGCTCTGGTGTTAG-AC-30. The amplified fragment was cloned into thespecific vector pSilencer3.1-H1 (Ambion) to constructthe pSilencer-miR-370 expression plasmid. Likewise, forconstructing the anti-miR-370 interfering plasmid, themiR-370 complementary sequence was synthesized asDNA oligonucleotides and cloned into the pSuper vector(Oligoengine) after annealing. Firefly luciferase reportervectors with the intact putative miR-370 recognitionsequence of the wild-type (pGL3-FoxM1-wild-30UTR)and mutant (pGL3-FoxM1-mutant-30UTR) were clonedfrom the FoxM1 30UTR. The authenticity of DNAsequences was confirmed by sequencing. All plasmidswere tranfected into cell lines by the Lipofectamine2000 method (Invitrogen). The si-FoxM1 oligonucletideswere also transected with Lipofectamine 2000, with finalconcentration 100 nmol/L.

RNA extraction and qRT-PCRTotal RNA was extracted by use of Trizol reagent (Invi-

trogen). For detection of miR-370, RNAwas converted intocDNA with use of the TaqMan MicroRNA Reverse Tran-scription Kit, then real-time PCR (RT-PCR) was conductedwith specific primers (TaqManMicroRNA Assay) and Taq-Man Universal PCR Master Mix (Applied Biosystems). Fordetecting FoxM1 and P27Kip1, 1mgRNAwas converted intocDNA by the MMLV Reverse Transcription System (Pro-mega) and RT-PCR involved use of the SYBR Premix ExTaq system (TaKaRa). All PCRs were run on an ABI 7500sequence detector (Applied Biosystems). U6 snRNA orb2-microglobulin was used as an endogenous control. AllRT-PCR reactions were conducted in triplicate, andrelative quantification was calculated by the DDCt method(95% confidence interval) with calibration to the corre-sponding endogenous control. Primers for RT-PCRwere forFoxM1, sense, 50-TGCAGCTAGGGATGTGAATCTTC-30, and antisense, 50-GGAGCCCAGTCCATCAGAACT-30; P27Kip1, sense, 50-ATGTCAAACGTGCGAGTGTC-TAA-30, and antisense, 50-TTACGTTTGACGTCTTCT-GAGG-30.

Luciferase assayThe P27Kip1 reporter plasmid, constructed to harbor the

P27Kip1 promoter sequence (�1358/þ132), and controlTK plasmid were added into cells by use of Lipofectamine2000. Cells were infected with H. pylori by the ratio 1:100

FoxM1 and hsa-miR-370 in H. pylori-Induced Gastric Carcinogenesis

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for 6 hours. Luciferase activity was determined with theDual Luciferase Reporter Assay system (Promega), andthe P27Kip1 promoter-driven firefly luciferase activity wasnormalized to that of TK Renilla. For luciferase assays ofmiRNA target validation, pSilencer-miR-370, wild-type ormutant 30UTR of FoxM1, and TK plasmids were cotrans-fected into AGS cells. After 48 hours, cells were lysed inpassive lysis buffer and luciferase activity was determined.

Western blot analysis and IHC assayAccording to standard Western blot procedures, proteins

were separated by 10% SDS-PAGE and then transferred toPVDF membrane (Bio-Rad), which were blocked in 5%nonfat milk, then incubated with the primary antibodiesFoxM1 (1:500), P27Kip1 (1:200), and b-actin (1:500) for 1to 2 hours under room temperature, then horseradishperoxidase-labeled goat-anti-mouse or goat-anti-rabbitimmunoglobulin G antibody (1:5,000, all Santa Cruz Bio-technology). Protein levels were detected by use of enhancedchemiluminescence (Pierce) and visualized on X-ray film.FoxM1 levels in tissue specimens were determined by IHCstaining with primary antibody (FoxM1, 1:250) incubationat 4�Covernight. Negative controls were treated without theprimary antibody.

Colony formation assayAGS cells were infected with H. pylori standard strain

26695 or transfected with FoxM1 siRNA and pSilencer-miR-370 for corresponding time, and then seeded into6-well plates (300 cells/well) for 10 to 14 days. Plates werestained with Giemsa, and the number of colonies with morethan 50 cells was counted.

Statistical analysisQuantitative data are expressed as mean � SEM. Statis-

tical analysis involved SPSS 13.0 (SPSS Inc.,) with 2-tailedStudent's t test or 1-way ANOVA for analysis of more than2 subgroups. Statistical significance was set at P < 0.05.

ResultsExpression of FoxM1 is upregulated in human gastritisand primary gastric cancerWe investigated the expression of FoxM1 in gastric speci-

mens at different disease stages, including 20 normal, 12superficial gastritis, 12 atrophic gastritis associated withintestinal metaplasia (AG/IM), and 20 primary gastric can-cer tissues (Supplementary Table S1). The mRNA expres-sion of FoxM1 gradually increased from gastritis to cancer ascompared with noncancerous gastric tissues (P < 0.01; Fig.1A), so upregulation of FoxM1 was an early event in humangastric carcinoma. This overexpression was confirmed byIHC assay, which showed FoxM1 expressed in superficialgastritis tissue (21.7% positive cells), AG/IM tissue (36.4%positive cells), and gastric cancer tissue (89.2% positive cells)as compared with noncancerous gastric tissues (6.7% pos-itive cells; P < 0.01; Fig. 1B and C). FoxM1 expressionwas not associated with patient age or sex (SupplementaryTable S1).

H. pylori and its key virulence factor CagA promoteFoxM1 expression in gastric epithelial cell linesNext, we determined whether infection with H. pylori

(standard strain 26695) could affect FoxM1 expression in 2gastric epithelial cell lines: poorly differentiated AGS andundifferentiated BGC-823 cells. Both the mRNA andprotein levels of FoxM1 were significantly upregulated withH. pylori infection time and dose dependently (Fig. 2A–D).Furthermore, to evaluate whether overexpression of FoxM1byH. pylori infection was strain specific, AGS and BGC-823cell lines were infected with H. pylori strains 26695, 11637,and SS1 (1:100) for 12 hours. FoxM1 expression wassubstantially elevated after infection by all of the 3 strainsas compared with the control (Fig. 2E and G). To determinewhich virulence factor was responsible for the increasedFoxM1 expression, we treated cells with CagA (CagA full-length plasmid), VacA (centrifugation of H. pylori liquidmedium), and lipopolysaccharide (LPS; hot inactivation ofH. pylori; ref. 28) and examined FoxM1 expression. In AGS

Figure 1. FoxM1 was overexpressed in human specimens of gastritis andgastric cancer. A, quantitative RT-PCR (qRT-PCR) analysis of relativemRNA level of FoxM1 in gastric biopsy specimens. �,P < 0.01. Each pointrepresented one sample. B, quantitative IHC results of percentage ofFoxM1-positive cells in gastric tissues. �, P < 0.01. C, IHC staining ofFoxM1 expression in normal (top left), superficial gastritis (SG; top right),atrophic gastritis (AG; bottom left), and cancerous (bottom right) gastrictissues.

Feng et al.

Mol Cancer Res; 11(8) August 2013 Molecular Cancer Research836

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cells, CagA promoted the mRNA and protein levels ofFoxM1, with almost no impact with other treatments (Fig.2F and H). These results were confirmed in cells with CagA

overexpression (Fig. 2I and J). Thus, upregulation of FoxM1may have been induced by H. pylori infection through itscomponent CagA.

Figure 2. The mRNA and proteinlevels of FoxM1were upregulated byHelicobacater pylori and its keyvirulent factor CagA in gastricepithelial cell lines. A andC, AGSandBGC-823 cells were infected with H.pylori [multiplicity of infection (MOI)1:100 (cell:H. pylori)] for 6 and12 hours. qRT-PCR and Westernblot analysis of mRNA and proteinlevels of FoxM1 over time. �,P < 0.01and ��, P < 0.001 versus con. B andD, cells were infected by H. pyloriwith MOI at 1:25, 1:50, and 1:100 for12 hours. qRT-PCR and Westernblot analysis of mRNA andprotein levels of FoxM1 expression.�,P < 0.01 versus con. E andG, qRT-PCR and Western blot analysis ofmRNA and protein levels of FoxM1by infection with all 3 H. pylori stains26695, 11637, and SS1 atMOI 1:100for 12 hours. �,P < 0.01 versus con. Fand H, qRT-PCR and Western blotanalysis of mRNA and protein levelsof FoxM1 with CagA, LPS, and VacAtreatment in AGS cells. �, P < 0.01versus con. I and J, CagA full-lengthplasmid was transfected into AGSand BGC-823 cells for 48 hours.qRT-PCR and Western blot analysisofmRNAandprotein levels of FoxM1with CagA transfection. �, P < 0.01versus pcDNA3.1 con. Data aremean � SEM of 3 independentexperiments.

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FoxM1 and hsa-miR-370 in H. pylori-Induced Gastric Carcinogenesis

www.aacrjournals.org Mol Cancer Res; 11(8) August 2013 837

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H. pylori and CagA were involved in cell proliferation bysuppressing P27Kip1 expression mediated by FoxM1We previously reported that P27Kip1 expression was

suppressed by FoxM1 in gastric cancer cell lines (18).Hence,we examined whether H. pylori infection could inhibitP27Kip1 expression by upregulating FoxM1. Both themRNA and protein levels of P27Kip1 were significantlyinhibited in AGS and BGC-823 cell lines after H. pyloriinfection and CagA transfection, separately (Figs. 3A and Band 4A and B). Furthermore, both H. pylori and CagAtreatment reduced the luciferase activity of P27Kip1 promot-er and expression of P27Kip1, which could be partiallyreversed by knockdown of FoxM1 with effective siRNA(Figs. 3C–E and 4C and D), which suggests that inhibitionof P27Kip1 induced by H. pylori or CagA was partially

mediated by FoxM1 through p27Kip1 promoter activity.Finally, we examined the function ofH. pylori infection andCagA transfection on cell proliferation by colony formationassay in AGS cells. H. pylori promoted colony formationcompared with control, and this promotion was reversed bysilencing of FoxM1 (Figs. 3F and G and 4E and F).

FoxM1 is a direct target of hsa-miR-370We used computer-aided algorithmic programs

[TargetScanHuman (http://www.targetscan.org), PicTar(http://pictar.mdc-berlin.de), and miRBase (http://www.mirbase.org)] to predict FoxM1 as a putative target ofhsa-miR-370 (Fig. 5A). The mRNA level of miR-370 wasgradually decreased from superficial gastritis, AG/IM togastric cancer samples, which indicates the importance of

Figure 3. H. pylori was involved inthe proliferation of gastric epithelialcells by inhibiting p27Kip1 promoteractivity through FoxM1. A and B,mRNA and protein levels of p27Kip1

in AGS and BGC-823 cells infectedwith H. pylori 26695 [multiplicity ofinfection (MOI) 1:100] for 12 hours,�, P < 0.01 versus con. C–E,luciferase activity of P27Kip1

promoter and P27Kip1 expressionin AGS cells with H. pylori (1:100)infection (Hp) for 12 hours, controlsiRNA transfection for 48 hours(Cs), and FoxM1 silencing withspecific siRNA for 48 hours (Fs).Luciferase activity was normalizedto that of Renilla of the TK plasmid.�, P < 0.01 versus con and#, P < 0.05 versus H. pyloriinfection. F and G, the foci numberof AGS cells after H. pylori (MOI1:100) infection for 12 hours andFoxM1 silencing. �, P < 0.01 versuscon and #, P < 0.01 versus H. pyloriinfection. Data are mean � SEM of3 independent experiments.

Feng et al.

Mol Cancer Res; 11(8) August 2013 Molecular Cancer Research838

on December 12, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

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miR-370 during gastric cancer progression (Fig. 5B). Theeffect of miR-370 on FoxM1 expression was evaluatedwith effective overexpressing and interfering plasmids ofmiR-370 (Fig. 5C). Both AGS and BGC cell lines trans-fected with pSilencer-miR-370 expression plasmid showedreduced mRNA and protein levels of FoxM1 (Fig. 5Dand E). Meanwhile, FoxM1 was upregulated after plasmidknockdown of miR-370 (Fig. 5D and E). Next, we valid-ated FoxM1 as a target of miR-370 by dual luciferase

report assay. AGS cells were cotransfected with pSilencer-miR-370 and FoxM1 wild-type or mutant-type 30UTRplasmids. Cotransfection of hsa-miR-370 and wild-type30UTR plasmid reduced the luciferase activity by approxi-mately 80% relative to the control, whereas mutant 30UTRcotransfection almost restored the luciferase activity (Fig.5F). Thus, miR-370 directly targeted the binding sitelocated at FoxM1 30UTR and FoxM1may be a direct targetof miR-370.

Figure 4. CagA induced theproliferation of gastric epithelial cellsby inhibiting P27Kip1 promoteractivity through FoxM1. A and B,qRT-PCR and Western blot analysisof mRNA and protein levels of theexpression of P27Kip1 in AGS andBGC-823 cells with CagA full-lengthplasmid transfection for 48 hours.�, P < 0.01 versus pcDNA3.1 con.C and D, luciferase activity of P27Kip1

promoter and P27Kip1 mRNAexpression with CagA transfectionfor 48 hours (CagA), control siRNAtransfection for 48 hours (Cs) andFoxM1 silencing with specific siRNAfor 48 hours (Fs). Luciferase activitywas normalized to that of Renilla ofthe TK plasmid. �, P < 0.01 versuspcDNA3.1 con and #, P < 0.05 versusCagAplasmid treatment. E and F, thefoci number of AGS cells with H.pylori (1:100) infection (Hp), CagAtransfection (Cs), and FoxM1silencing with specific siRNA (Fs).�, P < 0.01 versus pcDNA3.1 and#, P < 0.01 versus CagA plasmidtreatment. Data aremean�SEMof 3independent experiments.

FoxM1 and hsa-miR-370 in H. pylori-Induced Gastric Carcinogenesis

www.aacrjournals.org Mol Cancer Res; 11(8) August 2013 839

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MiR-370–FoxM1 pathway mediates H. pylori-inducedcell proliferationIn AGS and BGC-823 cells, the expression of hsa-miR-

370 was inhibited by H. pylori infection and transfection ofCagA full-length plasmid, respectively, which suggests thatsuppression of hsa-miR-370 by H. pylori may play animportant role in gastric carcinogenesis (Fig. 6A and B).Next, we examined whether the miR-370–FoxM1 pathwaywas involved in H. pylori-induced gastric cancer cell prolif-eration. miR-370 could inhibit FoxM1 expression andsuppress H. pylori and CagA-induced upregulation ofFoxM1 in AGS cells (Fig. 6C and D), which implies theexistence of a miR-370–FoxM1 pathway in H. pylori-induced cell proliferation.

Furthermore, the results were confirmed by colony-formation assay, which showed that miR-370 significantlyinhibited H. pylori and CagA-induced cell proliferation(P < 0.01; Fig. 6E and F).

FoxM1 is involved in H. pylori-induced gastritis in vivoThe validation of our mouse model of gastritis induced by

H. pylori infection was confirmed by hematoxylin and eosinstaining (Fig. 7A). After administration of H. pylori for 8months, 90% (18/20) of mice were validated as gastritismodels in the H. pylori infection group as compared with13% (2/15) in the control group (Supplementary Table S2).FoxM1 protein level was more in the model than the controlon IHC assay (Fig. 7A and B; P < 0.01). In addition, the

Figure 5. Hsa-miR-370 directlytargeted to FoxM1 in vitro. A, thesequence with hsa-miR-370directly binding to the 30UTR ofFoxM1 (from TargetscanHuman).B, qRT-PCR analysis of theexpression of hsa-miR-370 ingastric biopsy specimens. �, P <0.01 versus normal tissues. Eachpoint represented one sample. C,in AGS and BGC-823 cells, hsa-miR-370 was overexpressed bypSilencer-miR-370 and inhibitedby pSuper-anti-miR-370,respectively. �, P < 0.01 and #, P <0.05 versus con. D and E, mRNAand protein levels of FoxM1 in AGSand BGC-823 cells withoverexpression (370) and inhibition(inhibitor) of miR-370, respectively.�, P < 0.01 and #, P < 0.05 versuscon. F, relative luciferase activitywith reporter plasmids (pGL3-FoxM1-wild-30UTR and pGL3-FoxM1-mutant-30UTR)cotransfected with pSilencer-miR-370 into AGS cells. Luciferaseactivity was normalized to that ofRenilla of the TK plasmid.�,P<0.01 versuspSilencerþ30UTRcon, #, P < 0.01 versus miR-370þ30UTR. Data aremean�SEMof 3 independent experiments.

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mRNA level of mmu-miR-370 was reduced and that ofFoxM1 increased in mucosal epithelial samples ofH. pylori-induced gastritis (Fig. 7C and D; P < 0.01). The results invivo revealed the involvement of FoxM1 and miR-370 inH.pylori-induced gastritis.

DiscussionIn this study, we found overexpression of FoxM1 and

reduced expression of hsa-miR-370 in human gastritis andgastric cancer. H. pylori and its key virulent factor CagAinduced the expression of FoxM1 by inhibiting that ofhsa-miR-370, which directly targeted FoxM1, resulting incell proliferation for gastric carcinogenesis by reducingP27Kip1 promoter activity and its expression (SupplementaryFig. S1).Gastric carcinomas may occur on the background of

chronic gastric inflammation caused by persistent infection

of H. pylori (29). Accumulating studies indicate that H.pylori may be involved in superficial gastritis formation,which might later progress into AG/IM, or even gastriccancer. The chronic inflammatory status over decadesincreases the risk of gastric cancer with H. pylori considereda Group 1 human carcinogen (30). Studies have focused onthe mechanism by which H. pylori induces carcinogenesis,such as bacterium products that cause gastric mucosaldamage (31), generation of oxidative stress (32), and favor-ing the formation of mutagenic substances (33). Moreover,H. pylori may inhibit apoptosis and promote proliferation,invasion, and angiogenesis of cancer cells via CagA, the keyvirulence factor of H. pylori (34).Despite the importance of H. pylori, a susceptible host is

also required in gastric tumorigenesis. Some typical path-ways have been verified in H. pylori infection-related gastriccancer and include NF-kB and Wnt/b-catenin signaling

Figure 6. H. pylori and CagAinhibited hsa-miR-370 expressionand thus induced FoxM1 expressionfor cell proliferation. A, Hsa-miR-370expression after H. pylori infection(MOI 1:100) for 12 hours. �, P < 0.01versus con. B, Hsa-miR-370expression with CagA transfectionfor 48 hours. �, P < 0.01 versuspcDNA3.1 con. C and D, qRT-PCRand Western blot analysis of mRNAand protein levels of FoxM1 withoverexpressionof hsa-miR-370 (370)with H. pylori infection and CagAtransfection. �, P < 0.01 vs pSilencer(Psil), #, P < 0.01 versus con. E and F,the foci number of AGS cells after H.pylori infection and CagAtransfection, with pSilencer-miR-370(Psil) and has-miR-370 (370)treatment. �, P < 0.01 versuspSilencer (Psil), #, P < 0.01 versuscon. Data are mean � SEM of 3independent experiments.

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pathways (35). However, the involvement of the transcrip-tion factor FoxM1, shown upregulated in gastric cancersamples in our previous studies (18), remained unknown. Asa typical cell-cycle–related transcription factor, one of thekey roles of FoxM1 in carcinogenesis is to promote tumorproliferation (9). In addition, upregulation of FoxM1 is afrequent event in different tumor types, and FoxM1 defi-ciency triggers reduced cellular proliferation or even growtharrest (10–14), which suggests that FoxM1 may play animportant role in tumorigenesis and progression. Our resultsrevealed that overexpression of FoxM1 occurred from gas-tritis to gastric cancer progression and H. pylori-inducedFoxM1 expression in vivo and in vitro, so FoxM1might takepart in the early stage of gastric cancer. Because of the geneticheterogeneity present within H. pylori genomes, infectionoutcomes vary for different H. pylori strains and theirvirulence factors, such as CagA, VacA, and LPS.In this study, we found that all 3H. pylori strains, 26695,

11637, and SS1, increased the expression of FoxM1 indifferent gastric cells, further indicating the key role ofFoxM1 in H. pylori-induced pathologic process. However,

only CagA was able to promote FoxM1 upregulation.Microdissection and microarray were used to determine thedifferentially expressed mRNA levels with progression ofsuperficial gastritis, AG/IM, and gastric cancer; data analysisrevealed the involvement of FoxM1 in this process (data notshown). In short, we found that upregulation of FoxM1existed in the early stage of gastric cancer associated withH. pylori infection, which suggests an important role ofFoxM1 in this progress.Cell-cycle checkpoint genes play a pivotal role in gastric

carcinogenesis (36). P27Kip1, the negative regulator of cellcycle, is commonly downregulated in gastric cancer. It hasbeen considered a tumor suppressor and could serve as acandidate molecular marker for early gastric carcinoma (37).In the diffuse gastric cancer subtype, P27Kip1-negative–C-MYC–positive was the most frequent combination and wasassociated with more pathogenic H. pylori strains (38).Furthermore, transcription of P27Kip1 was verified to beinhibited by CagA via a PI3K/Akt1 pathway (39). Inagreement with these observations, we found inhibitedP27Kip1 mRNA and protein levels with H. pylori CagA

Figure 7. FoxM1 was upregulatedand mmu-miR-370 wasdownregulated in H. pylori-induced astritis in mice. A,hematoxylin and eosin (HE)staining and IHC assay of FoxM1in mucosal epithelial tissue withcontrol and H. pylori infection.B, proportion of FoxM1-positive cells as determinedimmunohistochemically. �,P <0.01versus con. qRT-PCR analysis ofrelative mRNA level of (C) FoxM1and (D) Mmu-miR-370 expressionin mucosal epithelial tissue cells.�, P < 0.01 versus con. Each pointrepresented one sample. Data aremean � SEM of 3 independentexperiments.

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treatment by upregulating FoxM1 expression, whichdecreased P27Kip1 promoter activity in AGS and BGC-823 cells. However, P27Kip1 inhibition and colony numberwere only partially reversed after knockdown of FoxM1 byits specific siRNA, which suggests other molecules involvedin this process. One study showed that H. pylori decreasedP27Kip1 expression through delta opioid receptor–mediatedinhibition of histone acetylation within the P27Kip1 pro-moter (40). Other research confirmed the involvement ofcytoplasmic mislocalization of P27Kip1 induced by H. pyloriin gastric cancer (41). These findings also link low gastricP27Kip1 expressionwith gastric carcinogenesis induced byH.pylori infection. Also, they could explain why P27Kip1

expression could not be completely reversed by FoxM1silencing: H. pylori-induced FoxM1 may be a critical butnot the only way for downregulation of p27Kip1 for cellproliferation. Two oncogenes, c-myc and human telomerasereverse transcriptase (hTERT), can be induced by H. pyloriand are the direct target genes of FoxM1 (9, 42). We alsofound substantial induction of the protein levels of c-mycand hTERT in vivo and in vitro afterH. pylori infection (datanot shown), which further indicates that FoxM1 plays a keyrole in gastric tumorigenesis induced by H. pylori infection.We failed to induce gastric cancer after 8 months ofH. pyloriinfection in animal models. However, we elucidate thatH. pylori and its key virulent factor CagA take part ininflammation-related gastric cancer through upregulationof FoxM1.miRNAs are important negative regulators of posttran-

scriptional gene expression and involved in several biologicprocesses, including tumorigenesis, by regulating tumorsuppressors or oncogenes. Numerous deregulated miRNAsare involved in gastric cancer and include miR-146a, miR-155, miR-21, miR-27a, miR-106-93-25, miR-221-222clusters, and the miR-200 family (23–25, 43–46). A grow-ing number of studies also suggest the involvement ofmiRNAs in various steps of gastric carcinogenesis: fromgastritis to metastatic disease (47). In this study, we found

reduced hsa-miR-370 expression in different diseasestages, and the expression could be downregulated byH. pylori infection and CagA treatment in gastric celllines. Moreover, FoxM1 was validated as a direct target ofmiR-370. Overexpression of hsa-miR-370 inhibited thecell proliferation induced by H. pylori infection or CagAtreatment by silencing FoxM1, which suggested a tumor-suppressive role of miR-370 in H. pylori-induced gastritistoward gastric cancer.In summary, our study shows that the miR-370–FoxM1

pathway is involved in the progress of gastritis toward gastriccancer induced by H. pylori infection by affecting theexpression of P27Kip1, which suggests potential applicationin early intervention and treatment of gastric cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Feng, L. Wang, J. Zeng, J. JiaDevelopment of methodology: L. Wang, J. JiaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): Y. Feng, L. Wang, J. Zeng, L. Shen, X. Liang, H. YuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): Y. Feng, J. Zeng, L. Shen, X. Liang, H. Yu, S. Liu, Z. Liu, Y. Sun,W. Li, C. ChenWriting, review, and/or revision of the manuscript: Y. Feng, L. Wang, J. Zeng,J. JiaAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): L. Wang, J. JiaStudy supervision: J. Jia

Grant SupportThis work was supported by National Natural Science Foundation of China (No:

81172354, 81001098, 81272654, 81171536 81071313, 81000868, and81170514), the National Basic Research Program of China (Grant No. 973 Program2012CB911202), the Science Foundation of Shandong Province (ZR2010HZ003),and Independent Innovation Foundation of Shandong University (No. 2012TS106).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received January 8, 2013; revised March 19, 2013; accepted March 20, 2013;published OnlineFirst May 10, 2013.

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2013;11:834-844. Published OnlineFirst April 10, 2013.Mol Cancer Res   Yimin Feng, Lixiang Wang, Jiping Zeng, et al.   Carcinogenesis and Is Negatively Regulated by miR-370

Induced Gastric−Helicobacter pyloriFoxM1 is Overexpressed in

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