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

  • Clinical Microbiology - Research Paper
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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.

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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).

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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

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  1. Wendy Malespín-Bendaña
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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.

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Correspondence to Wendy Malespín-Bendaña.

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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

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