Helicobacter pylori promotes VEGF expression via the p38 MAPK‑mediated COX‑2‑PGE2 pathway in MKN45 cells

  • Authors:
    • Ningning Liu
    • Qiong Wu
    • Yan Wang
    • Hua Sui
    • Xuan Liu
    • Ning Zhou
    • Lihong Zhou
    • Yifei Wang
    • Naijing Ye
    • Xiaoling Fu
    • Nikitin Alexander Yu
    • Qi Li
  • View Affiliations

  • Published online on: August 6, 2014     https://doi.org/10.3892/mmr.2014.2458
  • Pages: 2123-2129
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Abstract

Helicobacter pylori has been suggested to be the major cause of gastric malignancy. However, the pathogenesis and molecular mechanisms of gastric tumorigenesis induced by H. pylori infection are yet to be elucidated. In the present study, the expression levels of vascular endothelial growth factor (VEGF), which has been suggested to promote angiogenesis in gastric cancer, were found to be elevated in H. pylori‑infected MKN45 cells. Furthermore, it was demonstrated that the expression of VEGF was modulated by the p38 mitogen‑activated protein kinases (MAPK) pathway via regulation of the cyclooxygenase (COX)‑2 pathway. It was also found that prostaglandin E2 (PGE2) and its receptor EP2/EP4 may mediate the upregulation of VEGF in gastric cells exposed to H. pylori. In combination, these results suggest that VEGF expression is regulated by the p38 MAPK COX‑2-PGE2‑EP2/EP4 pathway in gastric cancer cells induced by H. pylori. This provides a theoretical basis for the investigation of the pathogenesis of H. pylori‑induced gastric cancer.

Introduction

Helicobacter pylori, a Gram-negative, spiral-shaped, microaerophilic bacterium found in the human stomach, is recognized as a major risk factor for chronic gastritis, peptic ulcers and gastric cancer (1). H. pylori infects approximately half of the world’s population and has been classified as a carcinogen by the World Health Organization and the International Agency for Research on Cancer (2,3). Gastric cancer is the second leading cause of cancer-associated mortality worldwide (4). Although considerable research has shown that H. pylori infection is closely associated with gastric cancer, the molecular mechanisms of gastric tumor initiation and development induced by H. pylori infection remain poorly understood (5).

Cyclooxygenase (COX), also known as prostaglandin-endoperoxide synthase, is a key rate-limiting enzyme responsible for the formation of prostanoids and thromboxanes (6,7). There are three COX isoenzymes, COX-1, COX-2 and COX-3 (a splice variant of COX-1). COX-1 is considered a constitutive enzyme that is expressed in nearly all mammalian cells, whilst COX-2 is an inducible enzyme that is undetectable in the majority of normal tissues (8,9). However, COX-2 expression is elevated in many types of cancer, including gastric cancer, and is closely correlated with the clinical outcome (1015). Furthermore, a previous study demonstrated that COX-2 expression is associated with H. pylori infection in human gastric cancer, and the elevation of COX-2 expression may be mediated by the p38 mitogen-activated protein kinases (MAPK) pathway (16).

Prostaglandin E2 (PGE2) is the main product generated from arachidonic acid catalyzed by COX-2, and it exerts a wide range of pathological effects via receptors on the cell and nuclear membranes (1719). Enhanced expression of PGE2 is found in several types of cancer and is associated with tumor growth and angiogenesis (20,21). PGE2 receptors (EPs) are types of G protein-coupled receptor (GPCR), and exist in at least four isoforms: EP1, EP2, EP3 and EP4 (2225). PGE2 binds to EPs to promote the expression of vascular endothelial growth factor (VEGF) in human prostate PC3 and gastric MKN28 cancer cells (20,26). However, whether EPs mediate VEGF expression in gastric cancer cells following H. pylori infection, and the type of EP involved in such regulation, have yet to be elucidated.

The MAPK family of proteins are involved in cell differentiation, migration, apoptosis and autophagy (27). p38 MAPK is a key member of this family and it is part of a signaling cascade that modulates cellular responses to cytokines and stresses, including inflammatory cytokines, osmotic shock, lipopolysaccharide, ultraviolet light and growth factors (26). Previous studies have demonstrated that p38 MAPK is also activated following H. pylori infection, and promotes COX-2 expression in MKN45 gastric cancer cells, which has been found to be involved in mediating H. pylori-induced gastric tumorigenesis (16,2830). Several studies have investigated the oncogenic potential of the p38 MAPK pathway, since p38 MAPK also has a critical role in regulating VEGF expression leading to angiogenesis (27,31). However, whether p38 MAPK is involved in regulating VEGF expression in H. pylori-infected gastric cells is yet to be elucidated.

To investigate the mechanism of H. pylori-induced gastric cancer, VEGF expression was analyzed in MKN45 gastric cells following H. pylori infection. It was found that p38 MAPK has a critical role in regulating VEGF expression in H. pylori-infected MKN45 cells, and this effect may be mediated via the COX-2-EP2/EP4 pathway.

Materials and methods

Cell culture and reagents

MKN45 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 (Invitrogen™ Life Technologies, Carlsbad, CA, USA) containing 10% (v/v) bovine serum albumin (BSA; Invitrogen Life Technologies) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen Life Technologies). Cells were cultured in a humidified incubator containing 5% CO2 at 37°C. The p38 MAPK inhibitor SB203580, the COX-2 inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulfonamide (NS-398), the EP2 inhibitor AH6089 and the EP4 inhibitor AH23848 were all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). For the inhibition treatment, confluent cells were treated with 20 μM SB203580, 50 μM NS-398, 50 μM AH6089 or 50 μM AH23848 for the indicated times.

H. pylori culture

The cagA- and vacA-positive H. pylori strain (NCTC11637) was acquired and cultured as previously described (15). In brief, H. pylori were cultured on Columbia Agar plates (Oxoid, Thermo Fisher Scientific, Basingstoke, UK) containing 10% sheep blood and incubated at 37°C for 48–72 h, with 5% O2, 10% CO2 and 85% N2. Prior to use, H. pylori were identified using Gram staining, colony morphology and positive oxidase, catalase and urease reactions. To prepare H. pylori for infection, the cells were suspended in phosphate-buffered saline (PBS) and cell density was determined using spectrophotometry (Eppendorf BioSpectrometer; Eppendorf, Hamburg, Germany). Confluent MKN45 cells were then incubated with H. pylori at a quantity of 100 bacteria per cell for the indicated times.

RNA isolation and quantitative polymerase chain reaction (qPCR)

Total RNA was isolated from MKN45 cells using RNAisol Reagent kit (Takara Bio, Inc., Shiga, Japan) in accordance with the manufacturer’s instructions. RNA quality was verified using spectrophotometry at an absorbance ratio of A260/280. Total RNA (1 μg) was used for reverse transcription into cDNA using Prime-Script™ RT-PCR kit (Takara Bio, Inc.) under the following conditions: 37°C for 15 min and 85°C for 5 sec. A total of 1 μl cDNA was then used for qPCR amplification using the ABI 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95°C for 10 sec, 95°C for 5 sec, and 60°C for 31 sec, run for 40 cycles. The forward and reverse primers for VEGF, COX-2 and GAPDH were used at a final concentration of 200 nM and the sequences were as follows: VEGF, 5′-GGCCTCCGAAACCATGAACT-3′ (forward) and 5′-CACTTGGCATGGTGGAGGTA-3′ (reverse); COX-2, 5′-AATGAGTACCGCAAACGCTTCT-3′ (forward) and 5′-TTCTGCAGCCATTTCCTTCTC-3′ (reverse); GAPDH, 5′-CCACTCCTCCACCTTTGAC-3′ (forward) and 5′-ACCCTGTTGCTGTAGCCA-3′ (reverse). The TaqMan® probes (Invitrogen Life Technologies) used were as follows: 5′-TGTCTTGGGTGCATTGGAGC-3′ for VEGF, 5′-CCTGAAGCCGTACACATCATTTG-3′ for COX-2 and 5′-TTGCCCTCAACGACCACTTTGTC-3′ for GAPDH.

Lenti-virus RNA interference (RNAi) plasmid construction and virus infection

The plasmids of lenti-virus RNAi system were obtained from Shanghai Genechem Co., Ltd. (Shanghai, China). Four small interfering RNAs (siRNAs) against human COX-2 mRNA [National Center for Biotechnology Information (NCBI) GenBank, NM-000963.2] were designed using the siRNA Target Finder from GenScript (Piscataway, NJ, USA). The target sequences used were as follows: Clone 1, 5′-GCT GAATTTAACACCCTCTAT-3′ (1230–1250 bp); clone 2, 5′-CCATTCTCCTTGAAAGGACTT-3′ (1677–1697 bp); clone 3, 5′-GCAGATGAAATACCAGTCTTT-3′ (1463–1483 bp); and clone 4, 5′-CATTCCCTTCCTTCGAAAT-3′ (407–425 bp). The clones were then inserted into a green fluorescent protein (GFP)-expressing pFU-GW-RNAi vector in accordance with the manufacturer’s instructions. The pFU-GW-RNAi vector was co-transfected with pHelper 1.0 and pHelper 2.0 vectors into 293T cells using Lipofectamine® 2000 Transfection Reagent (Invitrogen Life Technologies). The virus was subsequently analyzed and amplified in 293T cells. The appropriate amount of virus was then used to infect MKN45 cells for 72 h in order to suppress the endogenous COX-2 expression.

Western blot analysis

Cells were lysed using lysis buffer solution (50 mM Tris-HCl, pH 7.5; 150 mM NaCl) containing 1% nonyl phenoxypolyethoxylethanol-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethanesulfonylfluoride, 10 nM microcystin, 1 μg/ml aprotinin and 1 μg/ml leupeptin (all constituents of the lysis buffer were purchased from Sangon Biotech Shanghai Co., Ltd., Shanghai, China). Following centrifugation at 14,000 × g for 20 min, the protein in the supernatant was quantified using the BCA Protein Assay Reagent (Merck Millipore, Billerica, MA, USA) and equal amounts of protein were separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology, Shanghai, China), prior to being transferred onto a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). Following blocking with 5% fat-free milk in Tris-buffered saline in 0.05% Tween 20 (TBST; Sangon Biotech Shanghai Co., Ltd.) for 1 h at room temperature, the membranes were then separately incubated overnight at 4°C with the following antibodies: Rabbit anti-human COX-2 monoclonal antibody and rabbit anti-human β-actin monoclonal antibody (1:1,000; Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit anti-human EP-2 polyclonal antibody and rabbit anti-human EP-4 polyclonal antibody (1:1,000; Abcam, Cambridge, UK). Membranes were rinsed three times with TBST (5 min each time) and then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature prior to visualization using the Pierce enhanced chemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Results were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

ELISA

The cell culture medium was centrifuged at 3,000 × g for 5 min, and the supernatant was then used for further analysis. ELISA was performed in accordance with the manufacturer’s instructions (Invitrogen™ Life Technologies). Briefly, the microtiter plates were incubated with 100-μl samples at 37°C for 120 min. The plates were washed five times with 10 mM PBS, prior to incubation with 100 μl VEGF and PGE2 primary antibodies labeled with biotin at 37°C for 60 min. The plates were then rinsed five times with 10 mM PBS and 100 μl avidin-biotin-peroxidase complex was added, prior to the plates being cultured at 37°C for 30 min. Following extensive rinsing, the plates were then filled with 100 μl TMB Microwell substrate and incubated in darkness at 37°C for 15 min. The reaction was terminated using 100 μl TMB stop solution and the optical density (OD) values were analyzed within 30 min using a microplate reader (Multiskan Spectrum; Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. The OD values were subsequently converted into concentrations deduced from a calibration curve.

Statistical analysis

Statistical analysis was performed using the SPSS 19.0 software package (SPSS, Inc., Chicago, IL, USA). Statistical significance was determined using a one-way analysis of variance (ANOVA) followed by a Newman-Keuls test. All results are presented as the mean ± standard error (SE) for three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

Results

H. pylori infection enhances expression levels of VEGF in MKN45 cells

To determine whether H. pylori induced VEGF expression, confluent MKN45 cells were co-cultured with H. pylori for 0, 6, 12 and 24 h. Cells were then harvested to examine the relative expression levels of VEGF mRNA compared with GAPDH mRNA expression levels using qPCR. It was found that VEGF mRNA levels in cells treated with H. pylori for 6 h (P<0.05), 12 h (P<0.01) and 24 h (P<0.01) were significantly elevated compared with cells not exposed to H. pylori (Fig. 1A). The VEGF mRNA expression was highest in cells treated with H. pylori for 12 h. VEGF mRNA expression in cells treated with H. pylori for 12 h was significantly higher than that in cells treated for 6 h (P<0.01); however, expression appeared to decline after 24 h (Fig. 1A). The VEGF protein expression levels were analyzed using ELISA. MKN45 cells were incubated with H. pylori for 0, 12, 24, 36 and 48 h, and the supernatant was harvested and analyzed. It was found that VEGF protein levels increased with the length of incubation, and were significantly elevated in cells cultured with H. pylori for 36 h (P<0.05) and 48 h (P<0.01) (Fig. 1B).

Inhibition of p38 MAPK attenuates the effects of H. pylori on VEGF expression

To examine whether p38 MAPK modulated VEGF expression, confluent MKN45 cells were pretreated with p38 MAPK inhibitor SB203580 for 2 h prior to incubation with or without H. pylori for 12 or 48 h. Cells were then harvested and the levels of VEGF mRNA expression relative to GAPDH mRNA expression were analyzed using qPCR (cells treated with H. pylori for 12 h), while VEGF protein expression levels were analyzed using ELISA (cells treated with H. pylori for 48 h). In cells that were not exposed to H. pylori, treatment with SB203580 did not affect the VEGF mRNA or protein expression levels (Fig. 2A and B). Without SB203580 pretreatment, VEGF mRNA and protein levels in H. pylori-treated cells were significantly increased compared with cells not treated with H. pylori (P<0.01) (Fig. 2A and B). However, when pretreated with SB203580, VEGF mRNA and protein expression levels in H. pylori-treated cells significantly decreased compared with H. pylori-treated cells without incubation with SB203580 (P<0.01) (Fig. 2A and B).

Blocking COX-2 with the inhibitor NS-398 attenuates the effects of H. pylori on VEGF expression

It was demonstrated in our previous study that p38 MAPK activity was essential for increased expression of COX-2 in MKN45 cells following H. pylori infection (15); therefore, in the present study, it was investigated whether COX-2 was involved in the p38 MAPK-mediated upregulation of VEGF expression. Confluent MKN45 cells were pretreated with the COX-2 inhibitor NS-398 for 2 h prior to incubation with or without H. pylori for 12 h. The cells were then harvested to measure the expression levels of VEGF mRNA compared with GAPDH mRNA using qPCR. In cells not exposed to H. pylori, treatment with NS-398 did not affect the VEGF mRNA expression levels (Fig. 3A). Consistent with our previous results, VEGF mRNA levels in H. pylori-treated cells increased significantly (P<0.01) (Fig. 3A). However, in H. pylori-infected cells pretreated with NS-398, VEGF mRNA expression levels were downregulated compared with H. pylori-infected cells not incubated with NS-398 (P<0.01) (Fig. 3A). Our previous studies demonstrated that H. pylori increased COX-2 expression in MKN45 cells (16); therefore, it was hypothesized that the downstream products of COX-2 may have been elevated. The present study analyzed the protein expression levels of PGE2, one such downstream product of COX-2, in MKN45 cells incubated with H. pylori for 0, 6, 12, 24 and 48 h using ELISA. PGE2 protein levels in cells treated with H. pylori for 12, 24 and 48 h were significantly higher compared with cells not exposed to H. pylori (P <0.01), with the highest expression observed in in cells treated with H. pylori for 24 h (Fig. 3B).

RNAi-mediated suppression of COX-2 attenuates the effects of H. pylori on VEGF expression

In order to specifically suppress endogenous COX-2 expression, four lentiviral based RNAi clones targeted to different parts of the COX-2 gene were designed, and the inhibitory effect was analyzed by infecting MKN45 cells for 72 h with each clone separately. The cells were then harvested and the expression levels of COX-2 mRNA relative to GAPDH mRNA expression were analyzed using qPCR, while COX-2 protein expression was measured using western blot analysis. All four of the clones of lenti-viral RNAi targeted to COX-2 efficiently suppressed mRNA and protein expression levels of COX-2; however, clone 4 suppressed COX-2 mRNA and protein expression levels by ~95 and ~81%, respectively and, therefore, was used in the following experiments (Fig. 4A and B). To determine the effects of COX-2 RNAi on VEGF expression, confluent MKN45 cells were infected with control RNAi (GFP control), or COX-2 RNAi for 72 h, and then cells were incubated with or without H. pylori for 48 h. The supernatant was harvested to measure the protein expression of VEGF using ELISA. The results showed that background expression of VEGF in MKN45 cells was significantly reduced in cells infected with COX-2 siRNA (P<0.01), and the same effect was observed in cells also treated with H. pylori. This indicates that RNAi-mediated suppression of COX-2 inhibits the upregulation of VEGF expression in MKN45 cells following H. pylori treatment (Fig. 4C).

EP2/EP4-mediated regulation of VEGF expression upon H. pylori infection

Since it was found that COX-2 promotes VEGF expression in H. pylori-treated MKN45 cells and that the downstream product of COX-2, PGE2, was upregulated, further studies were performed to determine whether the PGE2 receptors EP2/EP4 were also associated with COX-2-mediated upregulation of VEGF. Confluent MKN45 cells were incubated with H. pylori for 0, 2, 6, 12, 24 and 48 h and then harvested. Western blot analysis was performed to determine the protein expression levels of EP2 and EP4, using β-actin as the internal control. As shown in Fig. 5A, no significant difference was observed in the protein expression levels of EP2 or EP4 during the continual induction of H. pylori (Fig. 5A). To examine whether EP2/EP4 expression was associated with VEGF expression in MKN45 cells, confluent cells were pretreated with specific inhibitors against EP2 and EP4, AH6089 and AH23848, respectively, for 2 h prior to incubation with or without H. pylori for 12 or 48 h. The cells were then harvested and the expression levels of VEGF mRNA relative to GAPDH mRNA were analyzed using qPCR (cells treated with H. pylori for 12 h), while expression levels of VEGF protein were analyzed using ELISA (cells treated with H. pylori for 48 h). In cells not exposed to H. pylori, treatment with AH6089 or AH23848 did not affect VEGF mRNA or protein expression levels (P>0.05) (Fig. 5B and C). In cells not treated with AH0689 or AH2348, VEGF mRNA and protein levels in H. pylori-infected cells increased significantly compared with cells not infected with H. pylori (P<0.01) (Fig. 5B and C). However, when cells were pretreated with AH6089, VEGF mRNA (P<0.05) and protein (P<0.01) expression levels in H. pylori-infected cells significantly decreased compared with H. pylori-infected cells without incubation of AH6089 (Fig. 5B and C). A similar result was observed in cells pretreated with the inhibitor AH23848: VEGF mRNA and protein expression levels in H. pylori-infected cells significantly decreased compared with H. pylori-infected cells without incubation of AH23848 (P<0.01) (Fig. 5B and C).

Discussion

VEGF, an oncogenic marker in cancer diagnosis, has potent angiogenic activity on endothelial cells and promotes tumor growth (32). Enhanced expression of VEGF is often observed in malignant tumors and is frequently used as a therapeutic target (32). A previous study demonstrated that H. pylori infection promoted gastric cancer cell invasion via upregulation of VEGF expression (33). However, the mechanism by which H. pylori induces VEGF expression in gastric cancer has yet to be elucidated. The p38 MAPK pathway has been found to be involved in tumor growth and metastasis through regulation of the production of VEGF in cancer. p38 MAPK activity also has a key role in increasing H. pylori-induced COX-2 expression in gastric cancer cells (16,27,31). Therefore, in the present study, the role of p38 MAPK in the regulation of VEGF expression in gastric cancer cells exposed to H. pylori was investigated. It was found that mRNA and protein expression levels of VEGF were significantly increased following H. pylori infection; however, the p38 MAPK-specific inhibitor, SB203580, significantly attenuated this effect, indicating that p38 MAPK was involved in promoting VEGF expression in H. pylori-infected MKN45 cells (Figs. 1 and 2).

COX-2 activity is associated with H. pylori infection in gastric cells, and may promote the production of PGE2 (16,26). In a previous study, we demonstrated that H. pylori infection upregulates the expression of COX-2 via the p38 MAPK/activating transcription factor 2 pathway (16). In this present study, the expression of PGE2 in H. pylori-infected cells was analyzed and it was found that PGE2 levels were significantly increased, suggesting that COX-2 activity was upregulated following H. pylori infection (Fig. 3B). To investigate whether the p38 MAPK-associated upregulation of VEGF expression was mediated by COX-2, the COX-2 specific inhibitor, NS-398, was used in cells exposed to H. pylori. The results show that VEGF expression significantly decreased in NS-398-treated cells (Fig. 3A). Endogenous COX-2 expression was also downregulated using RNAi and it was found that background expression levels of VEGF in MKN45 cells were significantly reduced, and the same effect was observed in H. pylori-treated cells, indicating that RNAi-mediated inhibition of COX-2 suppressed the upregulation of VEGF expression in MKN45 cells following H. pylori infection (Fig. 4C).

The results of the present study suggest that COX-2 is involved in the regulation of VEGF expression, downstream of p38 MAPK. EP2 and EP4 are PGE2 receptors, and have been shown to have an important role in modulating VEGF production in prostate cancer cells (20,26). In the present study, the roles of EP2/EP4 in VEGF production in gastric cancer cells following H. pylori infection were investigated. The results demonstrated that inhibition of EP2 and EP4 with the specific inhibitors AH6089 and AH23848 significantly decreased H. pylori-induced VEGF levels in cells, indicating that EP2/EP4 mediate the upregulation of VEGF expression in H. pylori-infected gastric cancer cells (Fig. 5B and C). The protein expression of EP2/EP4 was then analyzed, and it was found that H. pylori infection did not alter the EP2/EP4 protein levels. This suggests that the EP2/EP4-associated upregulation of VEGF is not mediated by EP2/EP4 protein levels, but by increased levels of PGE2 product, induced by enhanced COX-2 activity (Fig. 5A).

In combination, these results suggest a novel pathway of p38 MAPK-COX-2-PGE2-EP2/EP4 for the regulation of VEGF expression in H. pylori-infected gastric cells (Fig. 6). Following H. pylori infection, p38 MAPK is activated and COX-2 expression levels are upregulated, the level of PGE2 is therefore increased. PGE2 then binds to EP2/EP4 to promote VEGF expression. In a previous study we demonstrated that Jianpi Jiedu, a formulation used in traditional Chinese medicine, was demonstrated to downregulate COX-2 expression via inhibition of the H. pylori-induced p38 MAPK pathway (34). Therefore, further studies may be performed to examine whether the Jianpi Jiedu recipe regulates VEGF expression via modulation of this pathway, as shown in Fig. 6.

In conclusion, the present study elucidated a p38 MAPK-mediated signaling pathway that regulates VEGF expression in H. pylori-infected gastric cancer cells. This contributes to the investigation into the pathogenesis of H. pylori-induced gastric cancer. Furthermore, this study may provide novel therapeutic targets for H. pylori-induced gastric cancer.

Acknowledgements

This study was funded and supported by the National Natural Science Foundation of China (nos. 81072955, 81273958 and 81202663), the Program of Shanghai Municipal Education Commission (12ZZ118), the Science and Technology Commission of Shanghai Municipality (12ZR1449300, 1214090250), the Shanghai Municipal Health Bureau (2010019, XBR2011061, 2010044) and the Major Program of Technology Innovation, Putuo District, Shanghai (2009PTKW001).

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October 2014
Volume 10 Issue 4

Print ISSN: 1791-2997
Online ISSN:1791-3004

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APA
Liu, N., Wu, Q., Wang, Y., Sui, H., Liu, X., Zhou, N. ... Li, Q. (2014). Helicobacter pylori promotes VEGF expression via the p38 MAPK‑mediated COX‑2‑PGE2 pathway in MKN45 cells. Molecular Medicine Reports, 10, 2123-2129. https://doi.org/10.3892/mmr.2014.2458
MLA
Liu, N., Wu, Q., Wang, Y., Sui, H., Liu, X., Zhou, N., Zhou, L., Wang, Y., Ye, N., Fu, X., Yu, N. A., Li, Q."Helicobacter pylori promotes VEGF expression via the p38 MAPK‑mediated COX‑2‑PGE2 pathway in MKN45 cells". Molecular Medicine Reports 10.4 (2014): 2123-2129.
Chicago
Liu, N., Wu, Q., Wang, Y., Sui, H., Liu, X., Zhou, N., Zhou, L., Wang, Y., Ye, N., Fu, X., Yu, N. A., Li, Q."Helicobacter pylori promotes VEGF expression via the p38 MAPK‑mediated COX‑2‑PGE2 pathway in MKN45 cells". Molecular Medicine Reports 10, no. 4 (2014): 2123-2129. https://doi.org/10.3892/mmr.2014.2458