Published online Apr 15, 2019. doi: 10.4251/wjgo.v11.i4.281
Peer-review started: January 11, 2019
First decision: January 26, 2019
Revised: February 16, 2019
Accepted: February 27, 2019
Article in press: February 28, 2019
Published online: April 15, 2019
Tumor necrosis factor (TNF)-α is a proinflammatory cytokine with opposite effects according activation of its TNFR1 and TNFR2 receptors, can provide signals for activation, differentiation, survival cell, invasion and propagation of cancer cells or induce apoptosis. This way, TNF-α signaling pathway perform several biological functions, as well as modulate the immune response and inflammation, so deregulation of this pathway has been implicated with inflammatory diseases and cancer. Therefore, studies are needed to better understand the relationships of this signaling network and the protumorigenic or antitumorigenic effects, as well as the tumor development and progression in different types of neoplasms.
We previously evaluated the effect of eradication therapy of Helicobacter pylori (H. pylori) on expression levels of cytokines genes and the expression of miRNAs involved with regulation of inflammatory process. We observed up-regulation of TNF-α mRNA and protein and other inflammatory mediators in samples of chronic gastritis patients infected by H. pylori. Moreover, we also observed deregulation of miRNAs that interact with genes encoding cytokines. Interestingly, after bacterial eradication treatment, the expression levels of TNF mRNA were reduced and that of several miRNAs, such as miR-103a and miR-181c, were increased. Considering that TNF-α can have both pro- and anti-tumoral effects activating processes as apoptosis and cell survival depending on the interaction with its TNFR1 and TNFR2 receptors, we proposed the present study. Therefore, we decided to quantify the transcript levels of the TNF-α signaling pathway genes and of TNFR1 and TNFR2 receptors, as well as the involvement of miRNAs that may participate in the regulation of this signaling pathway, in fresh tissues of gastric cancer (GC) patients.
Considering that while the TNF-α/TNFR1 binding promotes the activation of the apoptosis cascade and the TNF-α/TNFR2 leads to activation of the cell survival pathway, the main objective of this study was to investigate which of the two receptors are most expressed in GC, and downstream genes of TNF-α signaling pathway, thus favoring the cell survival or apoptosis processes. A secondary objective was to evaluate the relationship between miRNA and mRNA via construction of an interaction network. The results may highlight important genes that regulate cell proliferation and possible molecular targets that act on gastric carcinogenesis.
Sensitive and validated techniques were employed for RNA quantification of the genes and miRNAs in normal and tumor tissues. For this purpose, we used TaqMan gene and miRNA expression assays (Applied Biosystems, Foster City, CA, United States) with specific probes for each gene and miRNA for relative quantification. The reactions were analyzed using the StepOnePlus real-time PCR System (Applied Biosystems, Foster City, CA, United States). Molecular diagnosis of H. pylori was performed by nested PCR for gene HSP60. In addition, we also used a bioinformatic tool ‘miRNA Data Integration Portal’ (http://ophid. utoronto.ca/mirDIP/) to build an miRNA:mRNA interaction network by using Cytoscape software (version 3.1.1).
Ours results showed up-regulation of TNFR2 receptor and its ligand TNF, as well as downstream genes of cellular survival as TRADD, TRAF2, CFLIP, and NFKB2, besides CASP8 and miR-34a in GC tissues, whereas mediators of apoptosis such as TNFR1 and CASP3 were down-regulated. Although we did not observe changes in the expression of most miRNAs, the miRNA:mRNA interactions network suggests a mechanism of regulation mainly by miR-19a and miR-103a, which targets a greater number of genes of the TNF-α pathway.
Our findings highlight the TNFR2 receptor as up-regulated in GC tissue, as well as its TNFA ligand, favoring the pro-tumoral effect of this cytokine and transcription of cell survival genes via TNFA/TNFR2 /NF-kB, and down-regulation of TNFR1 and CASP3 related to apoptosis evasion. Moreover, this pathway can be modulated by an intricate regulatory network of miRNA:mRNA.
The antitumor effect of TNF-α has been investigated as a form of cancer therapy associated or not with chemotherapeutic agents; however, its toxicity and adverse effects have limited its application. Another strategy for cancer therapy is to block the TNF receptors, so may increase the effectiveness of the TNF-α treatment and decrease its systemic toxicity. Therefore, in vitro functional studies may better elucidate the role of these receptors in gastric carcinogenesis.