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
Processing time: 159 Days and 18.2 Hours
ARTICLE HIGHLIGHTS
Research background

The tumour necrosis factor-α (TNF-α) signalling pathway triggered by TNFR1 and TNFR2 controls several biological processes, influencing cell fate. Thus, the deregulation of this pathway to cause an imbalance between the processes of cell survival and death may contribute to the tumorigenic process. This variety of functions is exercised by the ability of TNF-α to bind to TNFR1 or TNFR2, which results in different cellular processes. Both receptors are transmembrane proteins and are largely similar in extracellular structure, but their intracellular domains are different, and dictate the cellular fate for either survival or death. Since only TNFR1 has a death domain, the TNF-α signalling pathway triggered by TNFR1 is able to induce both cell survival and apoptosis, whereas TNFR2 results only in cell survival. The TNF-α signalling pathway also modulates the immune response and inflammation, so deregulation of this pathway has been implicated in inflammatory diseases and cancer. Therefore, studies are needed to better understand the relationships of this signalling network via TNFR1 and TNFR2 and its protumorigenic or antitumorigenic effects.

Research motivation

We proposed the present study based on our previous studies, which showed deregulation in the expression of genes and miRNAs of the TNF-α signalling pathway and its receptors TNFR1 and TNFR2 in fresh tissues of chronic gastritis and gastric cancer (GC) patients. Therefore, we decided to evaluate the effect of silencing TNFR1 and TNFR2 on GC cell lines.

Research objectives

According to the role of TNFR1 and TNFR2 in cellular responses triggered by TNF-α, and considering that studies addressing the role of these receptors in gastric neoplasm are limited and inconclusive, we proposed to couple the silencing of TNFR1 and TNFR2 receptors in an AGS gastric cell line and the treatment with Helicobacter pylori (H. pylori) extract to determine the effects on TNF-α mRNA expression and on downstream genes related to its signalling pathway. Moreover, we also investigated previously studied miRNAs that target genes in the TNF-α pathway to jointly determine their influence on the cell cycle and apoptosis.

Research methods

Stable AGS GC cells containing TNFR1 and TNFR2 receptors downregulated by specific shRNAs and nonsilenced cell lines were treated with 30% v/v H. pylori extract [H. pylori Tox+ strain (cagA+/vacA s1m1)] for 6 h. After silencing, TNFR1 and TNFR2 levels were assessed by quantitative polymerase chain reaction (qPCR) and Western blotting to confirm the knockdown effect. Subsequently, mRNA and miRNAs were quantified by qPCR using TaqMan gene and miRNA expression assays. The MTT assay was employed to evaluate the viability of silenced and nonsilenced AGS cells after different treatment conditions with H. pylori extract, and flow cytometry was used for cell cycle analysis and apoptosis.

Research results

Our results showed that H. pylori extract treatment increased the expression of genes involved in cell survival (NFKB1 and NFKB2) and inhibited apoptosis (CFLIP) and TNFR1 in nonsilenced AGS cells. TNFR1 downregulation significantly decreased the expression of the TRADD and CFLIP genes; however, no change in the cell cycle, apoptosis or miRNA levels was observed. In turn, 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 the NFKB1 and CFLIP genes, while upregulating the expression of miR-19a and miR-34a. Consequently, there was a decrease in the ratio of cells in the G0/G1 phase and an increase in cells in the S phase, as well as the promotion of early apoptosis.

Research conclusions

Our findings highlight that treatment with H. pylori extract increased the expression of pro-survival genes, mainly through TNFR1-mediated TNF-α signalling, emphasizing the role of bacterial infection in promoting GC progression. In the AGS cell line, TNFR1 and TNFR2 downregulation decreased the expression of prosurvival and antiapoptotic genes and affected miRNA expression and cellular processes, such as the cell cycle and apoptosis, emphasizing that shRNA-mediated downregulation of these receptors can have an antitumor effect.

Research perspectives

According to our results, we can mainly highlight the important role of TNFR2 in the TNF-α pathway in GC, indicating that silencing TNFR2 can reduce the expression of survival and anti-apoptotic genes. Thus, blocking this receptor may result in antitumor effects, suggesting possible targets for future in vivo studies into therapeutic strategies for treating GC.