Am J Physiol Gastrointest Liver Physiol 305: G786 –G796, 2013. First published October 17, 2013; doi:10.1152/ajpgi.00279.2013.

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Physiology and GI Cancer

Strain-specific suppression of microRNA-320 by carcinogenic Helicobacter pylori promotes expression of the antiapoptotic protein Mcl-1 Jennifer M. Noto,1 M. Blanca Piazuelo,1 Rupesh Chaturvedi,1 Courtney A. Bartel,2 Elizabeth J. Thatcher,2 Alberto Delgado,1 Judith Romero-Gallo,1 Keith T. Wilson,1,3,4,5 Pelayo Correa,1 James G. Patton,2 and Richard M. Peek, Jr.1,3 1

Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee; 2Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee; 3Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee; 4Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee; and 5Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee Submitted 22 August 2013; accepted in final form 8 October 2013

Noto JM, Piazuelo MB, Chaturvedi R, Bartel CA, Thatcher EJ, Delgado A, Romero-Gallo J, Wilson KT, Correa P, Patton JG, Peek RM Jr. Strain-specific suppression of microRNA-320 by carcinogenic Helicobacter pylori promotes expression of the antiapoptotic protein Mcl-1. Am J Physiol Gastrointest Liver Physiol 305: G786 –G796, 2013. First published October 17, 2013; doi:10.1152/ajpgi.00279.2013.— Helicobacter pylori is the strongest risk factor for gastric cancer, and strains harboring the cag pathogenicity island, which translocates the oncoprotein CagA into host cells, further augment cancer risk. We previously reported that in vivo adaptation of a noncarcinogenic H. pylori strain (B128) generated a derivative strain (7.13) with the ability to induce adenocarcinoma, providing a unique opportunity to define mechanisms that mediate gastric carcinogenesis. MicroRNAs (miRNAs) are small noncoding RNAs that regulate expression of oncogenes or tumor suppressors and are frequently dysregulated in carcinogenesis. To identify miRNAs and their targets involved in H. pylori-mediated carcinogenesis, miRNA microarrays were performed on RNA isolated from gastric epithelial cells cocultured with H. pylori strains B128, 7.13, or a 7.13 cagA⫺ isogenic mutant. Among 61 miRNAs differentially expressed in a cagA-dependent manner, the tumor suppressor miR-320 was significantly downregulated by strain 7.13. Since miR-320 negatively regulates the antiapoptotic protein Mcl-1, we demonstrated that H. pylori significantly induced Mcl-1 expression in a cagA-dependent manner and that suppression of Mcl-1 results in increased apoptosis. To extend these results, mice were challenged with H. pylori strain 7.13 or its cagA⫺ mutant; consistent with cell culture data, H. pylori induced Mcl-1 expression in a cagA-dependent manner. In human subjects, cag⫹ strains induced significantly higher levels of Mcl-1 than cag⫺ strains, and Mcl-1 expression levels paralleled the severity of neoplastic lesions. Collectively, these results indicate that H. pylori suppresses miR320, upregulates Mcl-1, and decreases apoptosis in a cagAdependent manner, which likely confers an increased risk for gastric carcinogenesis. Helicobacter pylori; gastric cancer; microRNA; Mcl-1; apoptosis MICROBIAL INFECTIONS ARE SIGNIFICANT causes of cancer, and ⬃20% of all malignancies worldwide are due to infectious agents (27). Gastric cancer is a major global health concern (9) and remains

Address for reprint requests and other correspondence: R. M. Peek, Jr., 2215 Garland Ave., Medical Research Bldg. IV 1030C, Nashville, TN 37232 (e-mail: [email protected]). G786

the second leading cause of cancer-related death, with ⬃700,000 deaths/year attributable to this malignancy (28). The primary initiating factor in the development of gastric cancer is chronic gastritis induced by the bacterial pathogen, Helicobacter pylori. H. pylori selectively colonizes the gastric epithelium of over 50% of the world’s population and typically persists for the lifetime of its host. Chronic gastric inflammation induced by H. pylori persists for decades and significantly increases the risk of gastric adenocarcinoma (30). Although H. pylori-induced gastritis is the strongest known risk factor for gastric cancer, only a fraction of colonized individuals ever develop neoplasia. Strain-specific bacterial virulence factors, host responses, and environmental constituents, either alone or in combination, contribute to enhanced cancer risk. One bacterial factor that increases gastric cancer risk is the cag pathogenicity island (PAI). H. pylori strains that harbor the cag PAI induce more severe gastric injury and further augment the risk for developing gastric cancer compared with strains that lack this virulence constituent (30). The cag island encodes a bacterial type IV secretion system (T4SS), which translocates CagA, the product of the terminal gene within the island, into host cells. Intracellular CagA can become phosphorylated by Src kinases (23, 38, 39) or remain unphosphorylated. In either form, CagA affects multiple pathways that alter host cell morphology, signaling, and inflammatory responses (2, 21, 26, 32, 35). However, most persons infected by cag⫹ H. pylori strains never develop cancer. These observations underscore the importance of defining factors that may alone, or in tandem with known virulence determinants, increase risk for this malignancy. Host factors that may contribute to gastric cancer risk include oncogenic or tumor suppressor microRNAs (miRNAs). miRNAs are small, noncoding RNAs ⬃20 –25 nucleotides in length that function as posttranscriptional regulators of gene expression (3). miRNAs function by binding to the 3= untranslated region (3= UTR) of messenger RNAs (mRNAs), resulting in mRNA degradation and gene silencing or translational repression (3). It is estimated that the human genome encodes thousands of miRNAs, targeting up to 60% of all proteincoding genes (14). miRNAs are involved in many biological processes, including development, differentiation, angiogenesis, cell cycle progression, proliferation, apoptosis, and activahttp://www.ajpgi.org

H. PYLORI SUPPRESSES miR-320 AND INDUCES MCL-1 EXPRESSION

tion of signal transduction pathways (1). Dysregulation of miRNA expression with subsequent disruption of these processes can result in immune and inflammatory disorders (37, 43) as well as malignancy (16, 41). Recent studies have demonstrated that H. pylori can modulate expression of miRNAs, which may contribute to disease (25). Animal models provide important insights into mechanisms that regulate gastric carcinogenesis. We previously identified a strain of H. pylori, 7.13, that reproducibly induces gastric cancer in two rodent models of gastritis, Mongolian gerbils and hypergastrinemic INS-GAS mice (11). This strain was derived via in vivo adaptation of a clinical H. pylori strain, B128, which induces inflammation, but not cancer, in rodent gastric mucosa. H. pylori strains B128 and 7.13 are closely related genetically (10) but differ in oncogenic potential; therefore, we capitalized on this unique resource to identify specific microRNAs altered in gastric epithelial cells by a carcinogenic H. pylori strain. MATERIALS AND METHODS

H. pylori strains and growth conditions. The H. pylori cag⫹ strains B128 (12), 7.13 (11), and a 7.13 cagA⫺ isogenic mutant strain were grown on trypticase soy agar-5% sheep blood plates (BD Biosciences, Franklin Lakes, NJ) at 37°C with 5% CO2. The cagA⫺ isogenic mutant was maintained under selection on Brucella agar (BD Biosciences) plates containing 20 ␮g/ml kanamycin (Sigma-Aldrich, St. Louis, MO). H. pylori strains were then grown in Brucella broth with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) for 18 h at 37°C with 5% CO2 prior to experimentation. Gastric epithelial cells and coculture conditions. MKN28 (human gastric epithelial cells isolated from a patient with gastric adenocarcinoma) and AGS (human gastric epithelial cells isolated from a 54-yr-old Caucasian female with gastric adenocarcinoma, ATCC, Manassas, VA) were grown in RPMI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals), L-glutamine (2 mM, BD Biosciences, Franklin Lakes, NJ), and HEPES buffer (1 mM, Cellgro, Manassas, VA) at 37°C with 5% CO2. H. pylori strains were cocultured with gastric epithelial cells at a multiplicity of infection (MOI) of 100:1 for indicated time points. RNA isolation and microRNA microarray analysis. Total RNA was isolated from MKN28 cells cocultured with or without H. pylori by use of the miRNeasy Mini Kit (Qiagen, Venlo, Limburg, the Netherlands) and small RNAs were isolated by use of the mirVana miRNA Isolation Kit (Ambion Life Technologies, Carlsbad, CA). Small RNAs were labeled with Cy5 at a 1:1 ratio. Unincorporated Cy5 was removed by use of a Nucleotide Removal Kit (Qiagen) and diluted to 2 mg per array. Synthetic lycopene synthase RNA was labeled and purified by the same protocol and was diluted to 50 pg per array. Hybridizations were performed in 25% deionized formamide, 5⫻ SCC, and 0.1% SDS, for 16 h in ArrayIt hybridization chambers followed by three washes in 2⫻ SCC 0.1% SDS, 1⫻ SCC, and 0.1⫻ SCC, as previously described (40). All arrays were scanned with GenePix4000B scanner (Molecular Devices, Sunnyvale, CA) and data were analyzed using GeneSpring GX 10.0.2 software (Aligent Technologies, Santa Clara, CA). All experiments were performed in triplicate. For normalization, hybridizations were spiked with identical amounts of lycopene synthase RNA. Predicted mRNA targets were obtained using the prediction algorithms TargetScan and miRanda. Northern blot analysis. Thirty micrograms of total RNA was separated on 12% urea-polyacrylamide gels and then transferred to positively charged membranes at 80 V for 1 h at 4°C. A miR-320 probe was radioactively labeled with ␣-32P-ATP by use of a StarFire

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Labeling Kit (Integrated DNA Technologies IDT, Coralville, IA) and added to the membrane. A U6 probe was radioactively labeled with ␥-32P-ATP and used as a loading control. This reaction was left overnight at 37°C and unhybridized probe was removed with 2⫻ SSPE 0.1% SDS, 1⫻ SSPE 0.1% SDS, and 0.5⫻ SSPE 1% SDS, as previously described (40). Membranes were developed overnight by using a Phosphor screen and viewed on a Typhoon phosphorimager (GE Healthcare, Pittsburgh, PA). Quantification of Northern blots was performed with ImageJ software (National Institutes of Health, Bethesda, MD). Real-time RT-PCR analysis. MKN28 or AGS cells were cocultured with H. pylori at an MOI of 100:1 for 2, 4, 8, or 24 h. RNA was isolated using the RNeasy RNA isolation kit (Qiagen), according to the manufacturer’s instructions. Reverse transcriptase PCR and quantitative real-time PCR (7300 Real-Time PCR System, Applied Biosystems Life Technologies, Carlsbad, CA) were performed, according to the manufacturer’s instructions. Levels of human mcl-1 mRNA expression (TaqMan, Applied Biosystems Life Technologies) were standardized to levels of human gapdh mRNA expression (TaqMan, Applied Biosystems Life Technologies). Western blot analysis. MKN28 or AGS cells were cocultured with H. pylori at an MOI of 100:1 for 8, 24, and 48 h. Protein lysates were harvested using RIPA buffer (50 mM Tris, pH 7.2; 150 mM NaCl; 1% Triton X-100; and 0.1% SDS) containing protease (1:100, Roche, Indianapolis, IN) and phosphatase (1:100, Sigma-Aldrich) inhibitors. Protein concentrations were determined by a bicinchoninic acid assay (Pierce, Thermo Scientific, Waltham, MA). Forty micrograms of proteins were separated by SDS-PAGE and transferred (Bio-Rad, Hercules, CA) to polyvinylidene difluoride membranes (Millipore, Billerica, MA). Human Mcl-1 protein expression was quantified by use of a rabbit polyclonal anti-Mcl-1 antibody (1:500, Santa Cruz Biotechnologies, Dallas, TX). Human Mcl-1 expression was standardized to human GAPDH using a mouse polyclonal anti-GAPDH antibody (1:5,000, Millipore). Primary antibodies were detected using goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)conjugated secondary antibodies (1:5,000, Santa Cruz Biotechnology). Immunoreactive bands were visualized by Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Waltham, MA) according to the manufacturer’s instructions and then quantified by densitometry by using the ChemiGenius Gel Bio Imaging System (Syngene, Frederick, MD). Transient transfection of siRNA. MKN28 or AGS cells were transiently transfected with Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA), in accordance with the manufacturer’s instructions. Briefly, transfection reagent was mixed with 40 pmol of nontargeting (NT) small interfering RNA (siRNA) (Thermo Scientific, Waltham, MA) or mcl-1-targeting siRNA (Thermo Scientific) in Opti-MEM cell culture medium (Life Technologies). Cells were incubated with transfection mixtures for 48 h, during which time cells were cocultured with or without H. pylori at an MOI of 100:1 for 24 h. Flow cytometry. Untransfected or transfected MKN28 or AGS cells were cocultured with H. pylori at an MOI of 100:1 for 24 h. Cocultures were washed with phosphate-buffered saline (Cellgro) and harvested by using 0.25% trypsin/EDTA (GIBCO Life Technologies, Carlsbad, CA). Cells were collected by centrifugation and resuspended in binding buffer [10⫻; 0.1 mol/l HEPES (pH 7.4), 1.4 mol/l NaCl, and 25 mmol/l CaCl2] at a concentration of 5 ⫻ 105 cells/ml. Cells were then stained with a rabbit polyclonal anti-Mcl-1 antibody (1:200, Santa Cruz Biotechnologies) to assess Mcl-1 expression or a polyclonal anti-active caspase-3 antibody (1:100, Cell Signaling Technology, Danvers, MA) to assess apoptosis and analyzed by quantitative flow cytometry, as previously described (24). Murine model of H. pylori infection. All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Vanderbilt University Medical Center’s Institutional Animal Care and Use Committee approved all protocols and all

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efforts were made to minimize animal suffering. Male hypergastrinemic FVB/N INS-GAS mice were bred and housed in the Vanderbilt University Animal Care Facilities in a room with a 12-h light-dark cycle at 21–22°C. Mice were orogastrically challenged with Brucella broth, as an uninfected control, with the rodent-adapted H. pylori strain 7.13, or a 7.13 cagA⫺ isogenic mutant. Mice were euthanized at 12–24 wk postchallenge and gastric tissue was harvested. To assess H. pylori-induced inflammation, linear strips of gastric tissue, extending from the squamocolumnar junction through the proximal duodenum, were fixed in 10% neutral-buffered formalin

(Azer Scientific), paraffin embedded, and stained with hematoxylin and eosin. A single pathologist (M. B. Piazuelo), blinded to treatment groups, scored indices of inflammation. Severity of acute or chronic inflammation was graded on a scale of 0 to 3 in both the gastric antrum and corpus, leading to a maximum combined score of 12. Immunohistochemistry. To assess Mcl-1 expression in murine gastric tissue, immunohistochemical (IHC) analysis was performed on deparaffinized gastric tissue sections by using a murine polyclonal anti-Mcl-1 antibody (1:3,000, Santa Cruz Biotechnologies). A single pathologist (M. B. Piazuelo), blinded to treatment groups, scored

Fig. 1. A carcinogenic Helicobacter pylori strain induces distinct microRNA expression profiles compared with its noncarcinogenic progenitor strain or an isogenic cagA⫺ mutant and specifically downregulates miR-320 in a cagA-dependent manner. A: total RNA was isolated from uninfected (UI) MKN28 gastric epithelial cells, or MKN28 cells infected with H. pylori strains B128, 7.13, or a 7.13 cagA⫺ isogenic mutant for 24 h. Small RNAs were isolated, fluorescently labeled, and hybridized to an array containing 346 unique miRNAs. Each line within a row indicates a unique miRNA. Blue color indicates background levels of expression, and red color indicates high levels of expression. B: Northern blot analyses were used to assess miR-320 expression levels relative to an endogenously expressed small RNA, U6. Error bars indicate standard error of the mean (SE) from experiments performed on at least 3 independent occasions. ANOVA tests were used to determine statistical significance among groups. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00279.2013 • www.ajpgi.org

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Mcl-1 epithelial staining in the entire length of the antral mucosa. The percentage of Mcl-1⫹ staining epithelial cells was assessed semiquantitatively and the intensity of epithelial Mcl-1 staining was graded on a scale of 1–3 (weak, moderate, or strong), as previously reported (24). The Mcl-1 epithelial score was determined by multiplying the Mcl-1 staining intensity by the percentage of positively stained cells. Gastric biopsies from human subjects from the Andean mountain Nariño region of Colombia (18) were previously obtained under Vanderbilt University Institutional Review Board-approved protocols. Biopsies from patients who were uninfected or infected with H. pylori cagA⫹ or cagA⫺ strains were used for Mcl-1 IHC by using a human polyclonal anti-Mcl-1 antibody (1:100, Santa Cruz Biotechnology) followed by a MACH 2 Universal HRP-conjugated secondary antibody (Biocare Medical, Concord, CA). Statistical analysis. All experiments were performed on at least three independent occasions. Statistical analysis was performed by Mann-Whitney, ANOVA with Bonferroni’s multiple comparison, or linear regression tests in GraphPad PRISM. A P value of ⬍0.05 was considered statistically significant. RESULTS

A carcinogenic H. pylori strain induces distinct microRNA expression profiles compared with its noncarcinogenic progenitor strain or an isogenic cagA⫺ mutant. To define microRNA profiles specific to carcinogenic H. pylori, MKN28 gastric epithelial cells were left uninfected or cocultured with wildtype noncarcinogenic H. pylori parental strain B128, wild-type carcinogenic H. pylori strain 7.13, or a 7.13 cagA⫺ isogenic mutant. Small RNAs were isolated, fluorescently labeled, and hybridized to an array containing 346 unique miRNAs (Fig. 1A) (39). Sixty-four miRNAs were significantly altered (⬎5-fold) between cells cocultured with strain B128 and strain 7.13 (data not shown). Similarly, 61 miRNAs were significantly altered (⬎5-fold) between cells cocultured with strain 7.13 vs. the cagA⫺ isogenic mutant (Table 1). Of interest, miR-320, which functions to regulate the expression of specific oncogenes, was downregulated in gastric epithelial cells following infection with carcinogenic strain 7.13 compared with both the parental strain B128 and the 7.13 cagA⫺ mutant (Table 1). To verify the microarray results, Northern blot analyses were performed to assess miR-320 expression levels in gastric epithelial cells. Consistent with the array data, strain 7.13 significantly decreased miR-320 expression compared with either strain B128 or the cagA⫺ mutant (Fig. 1B). These data indicate that a subset of miRNAs can be selectively altered by a carcinogenic H. pylori strain in a cagA-dependent manner. H. pylori upregulates mcl-1 in a cagA-dependent manner, consistent with downregulation of miR-320. To identify potential miR-320 mRNA targets linked to oncogenesis, a variety of prediction algorithms were utilized. Myeloid cell leukemia-1 (Mcl-1), a member of the antiapoptotic Bcl-2 protein family, was the most highly predicted target of miR-320, as defined by the conservation and number of binding sites (N ⫽ 5) within the 3= untranslated region (UTR) of mcl-1 mRNA (Fig. 2A). Prior data have also demonstrated that miR-320 negatively regulates mcl-1 expression and facilitates the induction of apoptosis (6). To assess mcl-1 expression in our microbial-host epithelial interaction model system, gastric epithelial cells were cocultured with H. pylori strain 7.13 or its cagA⫺ isogenic mutant and levels of mcl-1 mRNA were determined by quantitative real-time RT-PCR (Fig. 2B). Consistent with the downregula-

Table 1. microRNAs altered in a cagA-dependent manner microRNA

Fold Change (7.13/7.13 cagA⫺)

miR-291-as miR-17 miR-278 miR-87 miR-198 miR-301 miR-351 miR-19 miR-218 miR-128 miR-115 miR-411 miR-29c miR-117 miR-185 miR-305 miR-69 miR-61 miR-40 miR-126 miR-196 miR-139 miR-310 miR-62 miR-46 miR-307 miR-186 miR-90 miR-148 miR-64 miR-36 miR-409 miR-44 miR-338 miR-309 miR-34b miR-137 miR-73 miR-57 miR-68 miR-154 miR-52 miR-337 miR-190 miR-123 miR-145 miR-99a miR-285 miR-6 miR-34c miR-10a miR-146 miR-30-as miR-226a miR-320 miR-86 miR-343 miR-306 miR-304 miR-39 miR-308

20.98 19.43 12.06 11.37 11.04 9.69 9.21 7.53 7.32 7.30 7.08 7.06 6.06 5.80 5.37 5.35 5.08 ⫺5.03 ⫺5.69 ⫺5.89 ⫺5.90 ⫺5.94 ⫺6.04 ⫺6.06 ⫺6.56 ⫺6.58 ⫺6.69 ⫺6.72 ⫺6.97 ⫺7.12 ⫺7.81 ⫺8.07 ⫺8.22 ⫺8.96 ⫺9.80 ⫺10.11 ⫺10.49 ⫺10.67 ⫺11.15 ⫺11.74 ⫺14.78 ⫺15.30 ⫺15.40 ⫺16.12 ⫺16.17 ⫺16.40 ⫺18.94 ⫺19.08 ⫺22.73 ⫺24.12 ⫺26.70 ⫺31.21 ⫺32.16 ⫺39.92 ⫺46.06 ⫺50.68 ⫺51.26 ⫺59.09 ⫺64.73 ⫺177.12 ⫺700.10

tion of miR-320, H. pylori strain 7.13 induced significantly increased levels of mcl-1 expression at 2, 4, 8, and 24 h postinfection compared with either uninfected control cells or cells infected with the cagA⫺ isogenic mutant. To assess levels of Mcl-1 protein expression, Western blot analyses (Fig. 2, C and D) were performed. Consistent with the mRNA expression

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Fig. 2. miR-320 negatively regulates Mcl-1 and H. pylori upregulates Mcl-1 in a cagA-dependent manner. A: schematic representation of 5 predicted miR-320 binding sites within the 3= untranslated region (UTR) of mcl-1 mRNA. The expanded region depicts 1 of the 5 predicted miR-320 binding sites, which is complementary to the 3= UTR target. The box indicates the 8-nucleotide sequence that is exactly complementary to the 3= UTR target and regulates expression. B–F: human gastric epithelial cells were cocultured with strain 7.13 or its cagA⫺ isogenic mutant at an multiplicity of infection (MOI) of 100:1 for 2, 4, 8, or 24 h. B: quantitative real-time RT-PCR was used to assess mcl-1 mRNA expression relative to gapdh mRNA expression. C: Western blot analysis was used to assess Mcl-1 protein expression relative to GAPDH protein expression. A representative blot is shown. D: Western blot analysis replicates were quantified by densitometry. E and F: flow cytometry was used to assess Mcl-1 protein expression. A representative histogram showing relative fluorescence intensity of APC-Mcl-1 is shown (E) as well as the average mean fluorescent units (MFU) from 3 independent experiments (F). max, Maximum. Error bars indicate SE from experiments performed on at least 3 independent occasions. ANOVA tests were used to determine statistical significance among groups.

data, strain 7.13 induced Mcl-1 expression to significantly higher levels compared with uninfected cells or cells cocultured with the cagA⫺ isogenic mutant. To confirm these findings, flow cytometry (Fig. 2, E and F) was performed on uninfected control cells or cells infected with wild-type H. pylori strain 7.13 or the 7.13 cagA⫺ isogenic mutant strain to

assess Mcl-1 expression levels. Consistent with the Western blot data, strain 7.13 induced significantly higher levels of Mcl-1 protein compared with both uninfected cells or cells cocultured with the cagA⫺ isogenic mutant. Collectively, these results indicate that H. pylori downregulates miR-320 and upregulates Mcl-1 in a cagA-dependent manner in vitro.

AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00279.2013 • www.ajpgi.org

H. PYLORI SUPPRESSES miR-320 AND INDUCES MCL-1 EXPRESSION

Mcl-1 attenuates apoptosis during H. pylori infection. To define the functional role of Mcl-1 within the context of H. pylori infection, siRNA was used to silence mcl-1 expression. Gastric epithelial cells were transfected with NT or Mcl-1-targeting (Mcl-1) siRNA. Mcl-1 mRNA and protein levels were quantified by quantitative real-time RT-PCR (Fig. 3A), Western blot (Fig. 3, B and C), and flow cytometry (Fig. 3D) analyses, respectively. These data demonstrated that Mcl-1-targeting siRNA results in an approximate 75% decrease in mcl-1 mRNA levels (Fig. 3A) and an 80% decrease in Mcl-1 protein levels (Fig. 3, B–D). Since Mcl-1 can function as a suppressor of apoptosis, we next evaluated this cellular response by flow cytometry. Consistent with previous reports (5, 17, 22), infection of gastric epithelial cells with H. pylori resulted in a significant induction of apoptosis, as assessed by increased active caspase-3 expression levels, compared with uninfected, Lipofectamine controls. However, H. pylori-induced apoptosis was significantly increased in cells treated with Mcl-1-targeting siRNA, compared with either Lipofectamine and NT siRNA controls (Fig. 3E). These data indicate that downregulation of miR-320 and subsequent upregulation of Mcl-1 by H. pylori may be one mechanism to suppress cellular apoptosis.

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H. pylori induces inflammation and Mcl-1 expression in a cagA-dependent manner within murine gastric mucosa. To extend our in vitro results into an in vivo model of carcinogenesis, male INS-GAS mice, which are hypergastrinemic and at heightened risk for developing gastric cancer (15), were challenged with Brucella broth as a negative control, wild-type carcinogenic H. pylori strain 7.13, or a 7.13 cagA⫺ isogenic mutant for 12–24 wk (Fig. 4A). Colonization efficiency was 100% following challenge with wild-type strain 7.13 or its cagA⫺ isogenic mutant. Because of the persistent hypergastrinemia in this model, uninfected mice are susceptible to the development of gastritis. Despite low levels of gastric inflammation in uninfected INS-GAS mice, infection with wild-type strain 7.13 resulted in a significant and robust inflammatory response compared with uninfected controls or infection with the cagA⫺ isogenic mutant, indicating that H. pylori-induced inflammation occurs in a cagA-dependent manner in this model (Fig. 4B). To assess expression of Mcl-1 in this model, immunohistochemistry was performed. Consistent with the in vitro results (Fig. 2), infection with wild-type strain 7.13 resulted in a

Fig. 3. Mcl-1 attenuates apoptosis during H. pylori infection. Gastric epithelial cells were transiently transfected by using Lipofectamine 2000 (Lipo) with nontargeting siRNA (NT) or Mcl-1-targeting siRNA (Mcl-1). A: quantitative real-time RT-PCR was used to assess mcl-1 mRNA expression relative to gapdh mRNA expression. B: Western blot analysis was used to assess Mcl-1 protein expression relative to GAPDH protein expression. A representative blot is shown. C: Western blot analysis replicates were quantified by densitometry. D and E: flow cytometry was used to assess Mcl-1 expression in mean fluorescent units (D) and apoptosis, as assessed by quantification of active caspase-3 (E), in uninfected transfected cells or transfected cells infected with H. pylori strain 7.13 at an MOI of 100:1 for 24 h. Error bars indicate SE from experiments performed on at least 3 independent occasions. ANOVA tests were used to determine statistical significance among groups.

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Fig. 4. H. pylori induces inflammation and Mcl-1 expression in a cagA-dependent manner within murine gastric mucosa. A: experimental design. Male hypergastrinemic INSGAS mice were challenged with Brucella broth as an uninfected (UI) control, H. pylori strain 7.13, or its cagA⫺ mutant for 12–24 wk. B: the severity of inflammation was assessed in gastric tissue from uninfected mice, 7.13-infected mice, or mice infected with a cagA⫺ mutant. Severity of acute and chronic inflammation was graded on a scale of 0 to 3 in both the gastric antrum and corpus, leading to a maximum combined score of 12. C: Mcl-1 expression was evaluated by immunohistochemistry on gastric tissue from uninfected mice, 7.13-infected mice, or mice infected with a cagA⫺ mutant. Representative sections are shown at ⫻100 magnification. D: a single pathologist determined a Mcl-1 epithelial staining score by assessing the percentage of Mcl-1⫹ epithelial cells as well as the intensity of Mcl-1 staining (on a scale of 1–3). E: linear regression analysis was used to determine the relationship between the severity of gastric inflammation and Mcl-1 epithelial expression in H. pyloriinfected mice. Each data point represents mean scores from an individual animal. ANOVA or linear regression tests were used to determine statistical significance among groups.

significant increase in Mcl-1 immunostaining throughout the gastric mucosa, whereas only minimal Mcl-1 immunostaining was observed in uninfected mice or mice infected with the cagA⫺ mutant, indicating that H. pylori-induced Mcl-1 expression occurs in a cagA-dependent manner in vivo (Fig. 4, C and D). To define the relationship between H. pyloriinduced inflammation and Mcl-1 expression, gastritis scores and Mcl-1 epithelial staining were compared via linear regression analysis. There was a significant correlation between the severity of gastric inflammation and expression of Mcl-1 (Fig. 4E), indicating that H. pylori-induced inflammation may contribute to increased expression of Mcl-1 within the gastric mucosa. H. pylori-induced mcl-1 expression within human gastric epithelium segregates with cagA status of infecting H. pylori isolates. To extend the cell culture and rodent findings into the natural niche of H. pylori infection, Mcl-1 expression was assessed by immunohistochemistry in gastric biopsies

from uninfected individuals with normal gastric mucosa or from H. pylori cag⫹- or cag⫺-infected individuals with gastritis from the Andean mountain Nariño region of Colombia. Consistent with the findings in vitro and in the INS-GAS mouse model, individuals infected with H. pylori cag⫹ strains demonstrated a significant increase in the intensity of Mcl-1 immunostaining compared with uninfected controls or individuals infected with H. pylori cag⫺ strains, indicating that upregulation of Mcl-1 occurs in a cagA-dependent manner in a human population at high risk for gastric cancer (Fig. 5, A–D). Mcl-1 expression increases in parallel to gastric neoplastic progression in humans. To further evaluate the role of Mcl-1 in neoplastic transformation of the stomach, Mcl-1 expression was assessed by immunohistochemistry in uninfected individuals with normal gastric mucosa and H. pyloriinfected subjects diagnosed with nonatrophic gastritis, intestinal metaplasia, or adenocarcinoma. Expression of Mcl-1

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Fig. 5. H. pylori-induced Mcl-1 expression within human gastric epithelium segregates with cagA status of infecting H. pylori isolates. Mcl-1 expression was evaluated by immunohistochemistry in human gastric biopsies from patients residing in a high-risk area for gastric cancer. Gastric biopsies from uninfected patients with normal gastric mucosa, gastritis patients harboring H. pylori cag⫹ strains, or gastritis patients harboring H. pylori cag⫺ strains were evaluated for Mcl-1 immunostaining. Representative sections (A–C) are shown at ⫻100 magnification. D: a single pathologist assessed the percentage of Mcl-1⫹-expressing epithelial cells. Error bars indicate SE. ANOVA tests were used to determine statistical significance among groups.

paralleled the severity of gastric preneoplastic lesions (Fig. 6, A–D). Following quantification of epithelial staining and intensity, Mcl-1 epithelial expression levels progressively increased at each stage of neoplastic progression, providing further evidence that altered expression of Mcl-1 by H. pylori cag⫹ strains may play a role in gastric carcinogenesis. DISCUSSION

Gastric adenocarcinoma is the second leading cause of cancerrelated death in the world, and H. pylori infection is the strongest known risk factor for this malignancy (30). We have now shown that carcinogenic strains of H. pylori significantly alter host miRNA expression profiles that may affect cellular responses involved in carcinogenesis. Previous studies have assessed miRNA expression profiles in response to H. pylori infection as well as in gastric cancer specimens, as reviewed in Ref. 25. Although the majority of miRNAs that were altered in response to H. pylori infection in this study were novel, alteration of miR-17, miR-34b, miR-34c, and miR-320 were consistent with previous reports. Furthermore, miR-17, miR-19, miR34b, miR-34c, miR-126, miR-128, miR-139, and miR-185 expression changes in response to H. pylori infection were consistent with expression patterns previously observed in gastric cancer specimens (25). We prioritized identified miRNAs based on the oncogenic potential of their corresponding targets and miR-320 was specifically investigated in greater depth based on its ability to regulate the antiapoptotic protein Mcl-1. miR-320

was significantly downregulated by carcinogenic H. pylori in a cagA-dependent manner. miR-320 has been previously shown to be decreased in specimens from breast cancer (45), intrahepatic cholangiocarcinoma (6), prostate cancer (13), colon cancer (34), and acute myelogenous leukemia (33), suggesting that miR-320 may act as a tumor suppressor during cancer progression. Although miR-320 likely exhibits a wide range of tumor suppressor activities, it has been demonstrated to negatively regulate Mcl-1 and attenuate apoptosis (6). Mcl-1 is a member of the Bcl-2 protein family. The rapid induction and degradation of Mcl-1 suggests it plays an important role in apoptotic control in response to rapidly changing environmental cues (7). Mcl-1 is one of the most highly amplified genes in human cancers, including hematopoietic, lymphoid, and solid tumors (4). Mcl-1 overexpression is often associated with chemotherapeutic resistance and relapse of tumors (42, 44), and its depletion is sufficient to promote apoptosis in cancer cells (8, 19, 36), suggesting that Mcl-1 represents a potential therapeutic target in the treatment of a variety of human malignancies. Apoptosis is a normal component of epithelial cell turnover in many tissues, including the gastrointestinal tract (31). Tissue integrity is maintained when the rate of cell loss by apoptosis is matched by the rate of new cell production through proliferation. Hyperproliferation not balanced by increased levels of cell death may contribute to malignant transformation of the stomach via enhanced exposure to mutagenic substances; thus dissociation between levels of proliferation and apoptosis may

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Fig. 6. Mcl-1 expression increases in parallel with gastric neoplastic progression in humans. Mcl-1 expression was evaluated by immunohistochemistry in a human population at high risk for gastric cancer. Gastric biopsies from uninfected patients with normal gastric mucosa and H. pylori-infected patients with nonatrophic gastritis, intestinal metaplasia, or adenocarcinoma were evaluated for Mcl-1 immunostaining. Representative sections (A–D) are shown at ⫻400 magnification. E: a single pathologist determined a Mcl-1 epithelial staining score by assessing the percentage of Mcl-1⫹ epithelial cells as well as the intensity of Mcl-1 staining (on a scale of 1–3). Each data point represents an individual biopsy and mean values are shown. ANOVA tests were used to determine statistical significance between groups.

be an important mechanism leading to gastric carcinogenesis within the context of H. pylori infection. We have now demonstrated that miR-320 is suppressed by a carcinogenic strain of H. pylori in a cagA-dependent manner, which, in turn, promotes increased Mcl-1 expression and decreased levels of apoptosis in vitro, as knockdown of Mcl-1 potentiated H. pylori-induced apoptosis. Extending these findings, we also demonstrated that Mcl-1 is upregulated in a rodent model of H. pylori infection and cancer, which is dependent on the oncoprotein CagA. Consistent with our in vitro and in vivo findings, Mimuro et al. (20) previously demonstrated that CagA can inhibit apoptosis in vitro as well as in Mongolian gerbil gastric epithelium via increasing levels of Mcl-1 following H. pylori infection. Furthermore, we have demonstrated that Mcl-1 is upregulated in gastric tissue harvested from human subjects infected with cagA⫹ strains of H. pylori and Mcl-1 expression increases in parallel with the stages of gastric neoplastic progression. We have also previously demonstrated that persons infected with cagA⫹ strains exhibit significantly lower levels of gastric epithelial cell apoptosis compared with persons infected with cagA⫺ strains, indicating that CagA may modulate levels of apoptosis in H. pylori-infected human populations (29). Collectively, these data demonstrate that downregulation of miR-320 by carcinogenic cag⫹ strains of H. pylori facilitates increased expression of the antiapoptotic protein Mcl-1 and suppresses apoptosis, which may promote H. pylori persistence within the gastric mucosa. Since Mcl-1 is highly upregulated in gastric cancer,

this could serve as a prognostic factor for this deadly malignancy. ACKNOWLEDGMENTS We acknowledge the following core laboratories at Vanderbilt University Medical Center for contributions to these studies: VANGARD Vanderbilt Technologies for Advanced Genomics Analysis and Research Design, Vanderbilt Division of Animal Care, Vanderbilt Tissue Acquisition and Pathology Core, Vanderbilt Flow Cytometry Core, and the Vanderbilt Digestive Disease Research Center. GRANTS We acknowledge the following funding sources for support of this work: F32CA153539 (J. M. Noto), K01AT007324 (R. Chaturvedi), R01DK053620 (K. T. Wilson), P01CA028842 (K. T. Wilson and P. Correa), R01GM075790 (J. G. Patton), R01CA077955 (R. M. Peek), R01DK058587 (R. M. Peek), P01CA116087 (K. T. Wilson and R. M. Peek), and P30DK058404 (R. M. Peek). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS J.M.N., J.G.P., and R.M.P.J. conception and design of research; J.M.N., M.B.P., R.C., C.A.B., E.J.T., A.D., and J.R.-G. performed experiments; J.M.N., M.B.P., R.C., C.A.B., E.J.T., and A.D. analyzed data; J.M.N., M.B.P., R.C., C.A.B., E.J.T., J.G.P., and R.M.P.J. interpreted results of experiments; J.M.N. prepared figures; J.M.N. drafted manuscript; J.M.N., M.B.P., R.C., C.A.B., E.J.T., A.D., J.R.-G., K.T.W., P.C., J.G.P., and R.M.P.J. edited and revised manuscript; J.M.N., M.B.P., R.C., C.A.B., E.J.T., A.D., J.R.-G., K.T.W., P.C., J.G.P., and R.M.P.J. approved final version of manuscript.

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