Published online Jun 28, 2022. doi: 10.3748/wjg.v28.i24.2689
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
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.
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.
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.
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.
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.
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.
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.