Skip to main content

Advertisement

SpringerLink
Log in

Helicobacter pylori infection induces abnormal expression of pro-angiogenic gene ANGPT2 and miR-203a in AGS gastric cell line

  • Clinical Microbiology - Research Paper
  • Published:
Brazilian Journal of Microbiology Aims and scope Submit manuscript

Abstract

Helicobacter pylori colonizes the stomach and induces an inflammatory response that can develop into gastric pathologies including cancer. The infection can alter the gastric vasculature by the deregulation of angiogenic factors and microRNAs. In this study, we investigate the expression level of pro-angiogenic genes (ANGPT2, ANGPT1, receptor TEK), and microRNAs (miR-135a, miR-200a, miR-203a) predicted to regulate those genes, using H. pylori co-cultures with gastric cancer cell lines. In vitro infections of different gastric cancer cell lines with H. pylori strains were performed, and the expression of ANGPT1, ANGPT2, and TEK genes, and miR-135a, miR-200a, and miR-203a, was quantified after 24 h of infection (h.p.i.). We performed a time course experiment of H. pylori 26695 infections in AGS cells at 6 different time points (3, 6, 12, 28, 24, and 36 h.p.i.). The angiogenic response induced by supernatants of non-infected and infected cells at 24 h.p.i. was evaluated in vivo, using the chicken chorioallantoic membrane (CAM) assay. In response to infection, ANGPT2 mRNA was upregulated at 24 h.p.i, and miR-203a was downregulated in AGS cells co-cultured with different H. pylori strains. The time course of H. pylori 26695 infection in AGS cells showed a gradual decrease of miR-203a expression concomitant with an increase of ANGPT2 mRNA and protein expression. Expression of ANGPT1 and TEK mRNA or protein could not be detected in any of the infected or non-infected cells. CAM assays showed that the supernatants of AGS-infected cells with 26695 strain induced a significantly higher angiogenic and inflammatory response. Our results suggest that H. pylori could contribute to the process of carcinogenesis by downregulating miR-203a, which further promotes angiogenesis in gastric mucosa by increasing ANGPT2 expression. Further investigation is needed to elucidate the underlying molecular mechanisms.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data Availability

Datasets are available on request.

References

  1. Atherton JC (2006) The pathogenesis of Helicobacter pylori-induced gastro-duodenal diseases. Annu Rev Pathol 1:63–96. https://doi.org/10.1146/annurev.pathol.1.110304.100125

    Article  CAS  PubMed  Google Scholar 

  2. Basso D, Zambon CF, Letley DP, Stranges A, Marchet A, Rhead JL et al (2008) Clinical relevance of Helicobacter pylori cagA and vacA gene polymorphisms. Gastroenterology 135(1):91–99. https://doi.org/10.1053/j.gastro.2008.03.041

    Article  CAS  PubMed  Google Scholar 

  3. Covacci A, Telford JL, Del Giudice G, Parsonnet J, Rappuoli R (1999) Helicobacter pylori virulence and genetic geography. Science 284(5418):1328–1333

    Article  CAS  PubMed  Google Scholar 

  4. Peng C, Ouyang Y, Lu N, Li N (2020) The NF-κB signaling pathway, the microbiota, and gastrointestinal tumorigenesis: recent advances. Front Immunol 11:1387

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kitadai Y (2010) Angiogenesis and lymphangiogenesis of gastric cancer. J Oncol 2010:468725. https://doi.org/10.1155/2010/468725

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Pousa ID, Gisbert JP (2006) Gastric angiogenesis and Helicobacter pylori infection. Rev Esp Enferm Dig 98(7):527–541

    Article  CAS  PubMed  Google Scholar 

  7. Strowski MZ, Cramer T, Schäfer G, Jüttner S, Walduck A, Schipani E et al (2004) Helicobacter pylori stimulates host vascular endothelial growth factor-A (vegf-A) gene expression via MEK/ERK-dependent activation of Sp1 and Sp3. FASEB J 18(1):218–220. https://doi.org/10.1096/fj.03-0055fje

    Article  CAS  PubMed  Google Scholar 

  8. Kitadai Y, Sasaki A, Ito M, Tanaka S, Oue N, Yasui W et al (2003) Helicobacter pylori infection influences expression of genes related to angiogenesis and invasion in human gastric carcinoma cells. Biochem Biophys Res Commun 311(4):809–814. https://doi.org/10.1016/j.bbrc.2003.10.077

    Article  CAS  PubMed  Google Scholar 

  9. Wu CY, Wang CJ, Tseng CC, Chen HP, Wu MS, Lin JT et al (2005) Helicobacter pylori promote gastric cancer cells invasion through a NF-kappaB and COX-2-mediated pathway. World J Gastroenterol 11(21):3197–3203. https://doi.org/10.3748/wjg.v11.i21.3197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lazaraki G, Kountouras J, Metallidis S, Vrettou E, Tzioufa V, Germanidis G et al (2008) Helicobacter pylori infection upregulates endothelial nitric oxide synthase expression and induces angiogenesis in gastric mucosa of dyspeptic patients. Eur J Gastroenterol Hepatol 20(5):441–449. https://doi.org/10.1097/MEG.0b013e3282f4c35a

    Article  CAS  PubMed  Google Scholar 

  11. Tuccillo C, Cuomo A, Rocco A, Martinelli E, Staibano S, Mascolo M et al (2005) Vascular endothelial growth factor and neo-angiogenesis in H. pylori gastritis in humans. J Pathol 207(3):277–84. https://doi.org/10.1002/path.1844

    Article  CAS  PubMed  Google Scholar 

  12. Yeo M, Kim DK, Han SU, Lee JE, Kim YB, Cho YK et al (2006) Novel action of gastric proton pump inhibitor on suppression of Helicobacter pylori induced angiogenesis. Gut 55(1):26–33. https://doi.org/10.1136/gut.2005.067454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liu N, Zhou N, Chai N, Liu X, Jiang H, Wu Q et al (2016) Helicobacter pylori promotes angiogenesis depending on Wnt/beta-catenin-mediated vascular endothelial growth factor via the cyclooxygenase-2 pathway in gastric cancer. BMC Cancer 16:321. https://doi.org/10.1186/s12885-016-2351-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. https://doi.org/10.1016/j.cell.2011.02.013

    Article  CAS  PubMed  Google Scholar 

  15. Macedo F, Ladeira K, Longatto-Filho A, Martins SF (2017) Gastric cancer and angiogenesis: is VEGF a useful biomarker to assess progression and remission? J Gastric Cancer 17(1):1–10. https://doi.org/10.5230/jgc.2017.17.e1

    Article  PubMed  PubMed Central  Google Scholar 

  16. Huang H, Bhat A, Woodnutt G, Lappe R (2010) Targeting the ANGPT-TIE2 pathway in malignancy. Nat Rev Cancer 10(8):575–585. https://doi.org/10.1038/nrc2894

    Article  CAS  PubMed  Google Scholar 

  17. O’Brien J, Hayder H, Zayed Y, Peng C (2018) Overview of MicroRNA biogenesis, mechanisms of actions, and circulation. Front Endocrinol (Lausanne) 9:402. https://doi.org/10.3389/fendo.2018.00402

    Article  CAS  PubMed  Google Scholar 

  18. Kent OA, Mendell JT (2006) A small piece in the cancer puzzle: microRNAs as tumor suppressors and oncogenes. Oncogene 25(46):6188–6196. https://doi.org/10.1038/sj.onc.1209913

    Article  CAS  PubMed  Google Scholar 

  19. Otmani K, Lewalle P (2021) Tumor suppressor miRNA in cancer cells and the tumor microenvironment: mechanism of deregulation and clinical implications. Front Oncol 11:708765. https://doi.org/10.3389/fonc.2021.708765

    Article  PubMed  PubMed Central  Google Scholar 

  20. Funamizu N, Lacy CR, Kamada M, Yanaga K, Manome Y (2015) MicroRNA-203 induces apoptosis by upregulating Puma expression in colon and lung cancer cells. Int J Oncol 47(5):1981–1988. https://doi.org/10.3892/ijo.2015.3178

    Article  CAS  PubMed  Google Scholar 

  21. Tang R, Zhong T, Dang Y, Zhang X, Li P, Chen G (2016) Association between downexpression of MiR-203 and poor prognosis in non-small cell lung cancer patients. Clin Transl Oncol 18(4):360–368. https://doi.org/10.1007/s12094-015-1377-9

    Article  CAS  PubMed  Google Scholar 

  22. Zhao G, Guo Y, Chen Z, Wang Y, Yang C, Dudas A et al (2015) miR-203 functions as a tumor suppressor by inhibiting epithelial to mesenchymal transition in ovarian cancer. J Cancer Sci Ther 7(2):34–43. https://doi.org/10.4172/1948-5956.1000322

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zeng X, Qu X, Zhao C, Xu L, Hou K, Liu Y et al (2019) FEN1 mediates miR-200a methylation and promotes breast cancer cell growth. FASEB J 33(10):10717–30. https://doi.org/10.1096/fj.201900273R

    Article  CAS  PubMed  Google Scholar 

  24. Zhang B, Li Y, Zhao H, An R (2022) High expression of microRNA-200a/b indicates potential diagnostic and prognostic biomarkers in epithelial ovarian cancer. Dis Markers 2022:2751696. https://doi.org/10.1155/2022/2751696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Xu LM, Zhang J, Ma Y, Yuan YJ, Yu H, Wang J et al (2022) MicroRNA-135 inhibits initiation of epithelial-mesenchymal transition in breast cancer by targeting ZNF217 and promoting m6A modification of NANOG. Oncogene 41(12):1742–1751. https://doi.org/10.1038/s41388-022-02211-2

    Article  CAS  PubMed  Google Scholar 

  26. Argent RH, Hale JL, El-Omar EM, Atherton JC (2008) Differences in Helicobacter pylori CagA tyrosine phosphorylation motif patterns between western and East Asian strains, and influences on interleukin-8 secretion. J Med Microbiol 57(Pt 9):1062–1067. https://doi.org/10.1099/jmm.0.2008/001818-0

    Article  PubMed  Google Scholar 

  27. Costa AM, Ferreira RM, Pinto-Ribeiro I, Sougleri IS, Oliveira MJ, Carreto L et al (2016) Helicobacter pylori activates matrix metalloproteinase 10 in gastric epithelial cells via EGFR and ERK-mediated pathways. J Infect Dis 213(11):1767–1776. https://doi.org/10.1093/infdis/jiw031

    Article  CAS  PubMed  Google Scholar 

  28. Matsushima K, Isomoto H, Inoue N, Nakayama T, Hayashi T, Nakayama M et al (2011) MicroRNA signatures in Helicobacter pylori-infected gastric mucosa. Int J Cancer 128(2):361–370. https://doi.org/10.1002/ijc.25348

    Article  CAS  PubMed  Google Scholar 

  29. Agarwal V, Bell GW, Nam JW, Bartel DP (2015) Predicting effective microRNA target sites in mammalian mRNAs. Elife 4. https://doi.org/10.7554/eLife.05005

  30. Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42(Database issue):D68-73. https://doi.org/10.1093/nar/gkt1181

    Article  CAS  PubMed  Google Scholar 

  31. Leite M, Marques MS, Melo J, Pinto MT, Cavadas B, Aroso M et al (2020) Targets the EPHA2 receptor tyrosine kinase in gastric cells modulating key cellular functions. Cells 9(2). https://doi.org/10.3390/cells9020513

  32. Correa P, Houghton J (2007) Carcinogenesis of Helicobacter pylori. Gastroenterology 133(2):659–672. https://doi.org/10.1053/j.gastro.2007.06.026

    Article  CAS  PubMed  Google Scholar 

  33. Kuipers EJ (1999) Review article: exploring the link between Helicobacter pylori and gastric cancer. Aliment Pharmacol Ther 13(Suppl 1):3–11

    Article  PubMed  Google Scholar 

  34. Polk DB, Peek RM (2010) Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer 10(6):403–414. https://doi.org/10.1038/nrc2857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Olivera-Severo D, Uberti AF, Marques MS, Pinto MT, Gomez-Lazaro M, Figueiredo C et al (2017) A new role for Helicobacter pylori urease: contributions to angiogenesis. Front Microbiol 8:1883. https://doi.org/10.3389/fmicb.2017.01883

    Article  PubMed  PubMed Central  Google Scholar 

  36. Otrock ZK, Mahfouz RA, Makarem JA, Shamseddine AI (2007) Understanding the biology of angiogenesis: review of the most important molecular mechanisms. Blood Cells Mol Dis 39(2):212–220. https://doi.org/10.1016/j.bcmd.2007.04.001

    Article  CAS  PubMed  Google Scholar 

  37. Ucuzian AA, Gassman AA, East AT, Greisler HP (2010) Molecular mediators of angiogenesis. J Burn Care Res 31(1):158–175. https://doi.org/10.1097/BCR.0b013e3181c7ed82

    Article  PubMed  Google Scholar 

  38. Etoh T, Inoue H, Tanaka S, Barnard GF, Kitano S, Mori M (2001) Angiopoietin-2 is related to tumor angiogenesis in gastric carcinoma: possible in vivo regulation via induction of proteases. Cancer Res 61(5):2145–2153

    CAS  PubMed  Google Scholar 

  39. Tang S, Wang D, Zhang Q, Li L (2016) miR-218 suppresses gastric cancer cell proliferation and invasion via regulation of angiopoietin-2. Exp Ther Med 12(6):3837–3842. https://doi.org/10.3892/etm.2016.3893

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wang J, Wu K, Zhang D, Tang H, Xie H, Hong L et al (2005) Expressions and clinical significances of angiopoietin-1, -2 and Tie2 in human gastric cancer. Biochem Biophys Res Commun 337(1):386–393. https://doi.org/10.1016/j.bbrc.2005.09.051

    Article  CAS  PubMed  Google Scholar 

  41. Kim JS, Kim JM, Jung HC, Song IS (2004) Helicobacter pylori down-regulates the receptors of vascular endothelial growth factor and angiopoietin in vascular endothelial cells: implications in the impairment of gastric ulcer healing. Dig Dis Sci 49(5):778–786. https://doi.org/10.1023/b:ddas.0000030089.76514.e4

    Article  CAS  PubMed  Google Scholar 

  42. Baj J, Forma A, Sitarz M, Portincasa P, Garruti G, Krasowska D et al (2020) Virulence factors-mechanisms of bacterial pathogenicity in the gastric microenvironment. Cells 10(1). https://doi.org/10.3390/cells10010027

  43. Fiedler U, Augustin HG (2006) Angiopoietins: a link between angiogenesis and inflammation. Trends Immunol 27(12):552–558. https://doi.org/10.1016/j.it.2006.10.004

    Article  CAS  PubMed  Google Scholar 

  44. Potente M, Gerhardt H, Carmeliet P (2011) Basic and therapeutic aspects of angiogenesis. Cell 146(6):873–887. https://doi.org/10.1016/j.cell.2011.08.039

    Article  CAS  PubMed  Google Scholar 

  45. Hayes AJ, Huang WQ, Yu J, Maisonpierre PC, Liu A, Kern FG et al (2000) Expression and function of angiopoietin-1 in breast cancer. Br J Cancer 83(9):1154–1160. https://doi.org/10.1054/bjoc.2000.1437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Stoeltzing O, Ahmad SA, Liu W, McCarty MF, Wey JS, Parikh AA et al (2003) Angiopoietin-1 inhibits vascular permeability, angiogenesis, and growth of hepatic colon cancer tumors. Cancer Res 63(12):3370–3377

    CAS  PubMed  Google Scholar 

  47. Yu X, Ye F (2020) Role of angiopoietins in development of cancer and neoplasia associated with viral infection. Cells 9(2). https://doi.org/10.3390/cells9020457

  48. Fawzy A, Gaafar R, Kasem F, Ali SS, Elshafei M, Eldeib M (2012) Importance of serum levels of angiopoietin-2 and survivin biomarkers in non-small cell lung cancer. J Egypt Natl Canc Inst 24(1):41–45. https://doi.org/10.1016/j.jnci.2011.12.006

    Article  PubMed  Google Scholar 

  49. Yu H, Lu J, Zuo L, Yan Q, Yu Z, Li X et al (2012) Epstein-Barr virus downregulates microRNA 203 through the oncoprotein latent membrane protein 1: a contribution to increased tumor incidence in epithelial cells. J Virol 86(6):3088–3099. https://doi.org/10.1128/JVI.05901-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Liu W, Dong Z, Liang J, Guo X, Guo Y, Shen S et al (2016) Downregulation of potential tumor suppressor miR-203a by promoter methylation contributes to the invasiveness of gastric cardia adenocarcinoma. Cancer Invest 34(10):506–516. https://doi.org/10.1080/07357907.2016.1242010

    Article  CAS  PubMed  Google Scholar 

  51. Zhou X, Xu G, Yin C, Jin W, Zhang G (2014) Down-regulation of miR-203 induced by Helicobacter pylori infection promotes the proliferation and invasion of gastric cancer by targeting CASK. Oncotarget 5(22):11631–40. https://doi.org/10.18632/oncotarget.2600

    Article  PubMed  PubMed Central  Google Scholar 

  52. Wang L, Tong D, Guo Q, Wang X, Wu F, Li Q et al (2018) HOXD3 targeted by miR-203a suppresses cell metastasis and angiogenesis through VEGFR in human hepatocellular carcinoma cells. Sci Rep 8(1):2431. https://doi.org/10.1038/s41598-018-20859-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Gilmore TD (2021) NF-κB and human cancer: what have we learned over the past 35 years? Biomedicines 9(8). https://doi.org/10.3390/biomedicines9080889

Download references

Acknowledgements

This study was supported by Vicerrectoría de Investigación, Universidad de Costa Rica, Consejo Nacional para Investigaciones Científicas y Tecnológicas (CONICIT, Ministerio de Ciencia y Tecnología, Costa Rica) and European Regional Development Fund (COMPETE, ON.2 North Portugal Regional Operational Programme, NORTE-07-0124-FEDER-000022). Authors wish to thank Professor John Atherton (University of Nottingham) for the kind gift of H. pylori mutant strains, Dr. Cristine Varon (INSERM, University of Bordeaux, F33000 Bordeaux, France) for the kind gift of H. pylori 7.13 strain and MKN45 cell line, Dr. Marina Leite for support and instruction with cellular and molecular techniques, Dr. Silvia Molina-Castro for intellectual contributions and critical revision of the manuscript, and Elena Vásquez and Melany Calderón for excellent technical assistance. Rui M Ferreira is funded by the “FCT Scientific Employment Stimulus—Individual Call” program (CEECIND/01854/2017).

Author information

Authors and Affiliations

  1. Institute of Health Research (INISA), University of Costa Rica, 11501-2060, San José, Costa Rica

    Wendy Malespín-Bendaña, Clas Une & Vanessa Ramírez

  2. Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal

    Rui M. Ferreira, Marta T. Pinto & Ceu Figueiredo

  3. Instituto de Investigação E Inovação Em Saúde, Universidade Do Porto (i3S), Porto, Portugal

    Rui M. Ferreira, Marta T. Pinto & Ceu Figueiredo

  4. Faculty of Medicine of the University of Porto, Porto, Portugal

    Ceu Figueiredo

  5. Center for Research On Microscopic Structures (CIEMic), University of Costa Rica, San José, Costa Rica

    Warner Alpízar-Alpízar & Lucía Figueroa-Protti

  6. Department of Biochemistry, School of Medicine, University of Costa Rica, San José, Costa Rica

    Warner Alpízar-Alpízar

  7. Faculty of Microbiology, University of Costa Rica, San José, Costa Rica

    Lucía Figueroa-Protti

  8. Department Public Nutrition, School of Nutrition, University of Costa Rica, San José, Costa Rica

    Vanessa Ramírez

Authors
  1. Wendy Malespín-Bendaña
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rui M. Ferreira
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marta T. Pinto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ceu Figueiredo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Warner Alpízar-Alpízar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Clas Une
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lucía Figueroa-Protti
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Vanessa Ramírez
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Wendy Malespín-Bendaña: Conceptualization, Methodology, Investigation, Formal analysis, Writing-original draft. Rui M. Ferreira: Methodology, Writing-review and editing, Funding acquisition. Marta T. Pinto: Methodology, Formal analysis, Writing-review and editing. Ceu Figueiredo: Conceptualization, Funding acquisition, Project administration, Writing-original draft. Warner Alpízar-Alpízar: Conceptualization, Writing-review and editing. Clas Une: Conceptualization, Writing-review and editing. Lucía Figueroa Protti: Methodology, Writing-review and editing. Vanessa Ramírez: Conceptualization, Funding acquisition, Project administration, Formal analysis, Writing-original draft.

Corresponding author

Correspondence to Wendy Malespín-Bendaña.

Ethics declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Responsible Editor: Cristiano Gallina Moreira

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Malespín-Bendaña, W., Ferreira, R.M., Pinto, M.T. et al. Helicobacter pylori infection induces abnormal expression of pro-angiogenic gene ANGPT2 and miR-203a in AGS gastric cell line. Braz J Microbiol 54, 791–801 (2023). https://doi.org/10.1007/s42770-023-00940-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s42770-023-00940-4

Keywords

Search

Navigation