Basic Study
Copyright ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 28, 2022; 28(24): 2689-2704
Published online Jun 28, 2022. doi: 10.3748/wjg.v28.i24.2689
Downregulation of TNFR2 decreases survival gene expression, promotes apoptosis and affects the cell cycle of gastric cancer cells
Ana Flávia Teixeira Rossi, Fernanda da Silva Manoel-Caetano, Joice Matos Biselli, Ágata Silva Cabral, Marilia de Freitas Calmon Saiki, Marcelo Lima Ribeiro, Ana Elizabete Silva
Ana Flávia Teixeira Rossi, Fernanda da Silva Manoel-Caetano, Joice Matos Biselli, Ágata Silva Cabral, Marilia de Freitas Calmon Saiki, Ana Elizabete Silva, Department of Biological Sciences, Sao Paulo State University (UNESP), São José do Rio Preto 15054-000, São Paulo, Brazil
Marcelo Lima Ribeiro, Clinical Pharmacology and Gastroenterology Unit, São Francisco University (USF), Bragança Paulista 12916-900, São Paulo, Brazil
Author contributions: Rossi AFT analysed and interpreted the results; Rossi AFT, Manoel-Caetano FS and Biselli JM performed the experiments; Rossi AFT and Silva AE outlined the study, and drafted and revised the manuscript; Cabral AS performed the Western blot assays; Saiki MFC provided technical support for the shRNA transfection experiments; Ribeiro ML provided the HGC-27 cell line and produced the H. pylori extract; all authors approved the final version of the manuscript.
Supported by São Paulo Research Foundation (FAPESP), No. 2015/21464-0 and No. 2015/23392-7; and National Counsel of Technological and Scientific Development (CNPq), No. 310120/2015-2.
Institutional review board statement: Not applicable. This study was performed with cell line only.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Data sharing statement: This study was performed with cell line only. The datasets generated during the current study are available from the corresponding author on reasonable request.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Ana Elizabete Silva, PhD, Adjunct Associate Professor, Department of Biological Sciences, Sao Paulo State University (UNESP), Rua Cristóvão Colombo 2265, São José do Rio Preto 15054-000, São Paulo, Brazil. ae.silva@unesp.br
Received: January 15, 2022
Peer-review started: January 15, 2022
First decision: February 7, 2022
Revised: February 21, 2022
Accepted: May 7, 2022
Article in press: May 7, 2022
Published online: June 28, 2022
Abstract
BACKGROUND

Chronic inflammation due to Helicobacter pylori (H. pylori) infection promotes gastric carcinogenesis. Tumour necrosis factor-α (TNF-α), a key mediator of inflammation, induces cell survival or apoptosis by binding to two receptors (TNFR1 and TNFR2). TNFR1 can induce both survival and apoptosis, while TNFR2 results only in cell survival. The dysregulation of these processes may contribute to carcinogenesis.

AIM

To evaluate the effects of TNFR1 and TNFR2 downregulation in AGS cells treated with H. pylori extract on the TNF-α pathway.

METHODS

AGS cell lines containing TNFR1 and TNFR2 receptors downregulated by specific shRNAs and nonsilenced AGS cells were treated with H. pylori extract for 6 h. Subsequently, quantitative polymerase chain reaction with TaqMan® assays was used for the relative quantification of the mRNAs (TNFA, TNFR1, TNFR2, TRADD, TRAF2, CFLIP, NFKB1, NFKB2, CASP8, CASP3) and miRNAs (miR-19a, miR-34a, miR-103a, miR-130a, miR-181c) related to the TNF-α signalling pathway. Flow cytometry was employed for cell cycle analysis and apoptosis assays.

RESULTS

In nonsilenced AGS cells, H. pylori extract treatment increased the expression of genes involved in cell survival and inhibited both apoptosis (NFKB1, NFKB2 and CFLIP) and the TNFR1 receptor. TNFR1 downregulation significantly decreased the expression of the TRADD and CFLIP genes, although no change was observed in the cellular process or miRNA expression. In contrast, TNFR2 downregulation decreased the expression of the TRADD and TRAF2 genes, which are both important downstream mediators of the TNFR1-mediated pathway, as well as that of the NFKB1 and CFLIP genes, while upregulating the expression of miR-19a and miR-34a. Consequently, a reduction in the number of cells in the G0/G1 phase and an increase in the number of cells in the S phase were observed, as well as the promotion of early apoptosis.

CONCLUSION

Our findings mainly highlight the important role of TNFR2 in the TNF-α pathway in gastric cancer, indicating that silencing it can reduce the expression of survival and anti-apoptotic genes.

Keywords: Gastric cancer, Helicobacter pylori, Tumour necrosis factor-α signalling pathway, TNFR1, TNFR2, miRNAs

Core Tip: This study demonstrated for the first time the effect of TNFR1 and TNFR2 downregulation on an AGS cell line treated with Helicobacter pylori extract. Although TNFR1 downregulation did not promote significant changes in the expression of mRNA and miRNAs of the tumour necrosis factor-α (TNF-α) signalling pathway, TNFR2 downregulation promoted important changes in the signalling mediators evaluated. We observed a reduction in the expression of cell survival and anti-apoptotic genes and an increase in the expression of miR-19a and miR-34a, which affected cell cycle kinetics and contributed to early apoptosis. Thus, our findings highlight the important role of TNFR2 in the TNF-α signalling pathway in gastric carcinogenesis.