H. pylori infects the stomach of about half of the world's population, while gastritis and its progresses to gastric cancer is occurs in low percentages of the infected patients (11). Although this infection is considered as a risk factor for gastric cancer, mechanisms mediating the disease in the gastric tissue are not well known. Chronic gastritis, deregulation of cell signaling pathways, mucosal damage and genomic instabilities seems critical in this interplay. Chronic inflammation is estimated to be the cause of approximately 25% of human cancers (12). In the case of gastric tissue, it is assumed that H. pylori infection could induce gastritis by two different mechanisms, either through toxins that are secreted by different bacterial secretory systems (e.g., CagA, VacA) or by invasion and induction of the epithelial cells to release pro-inflammatory mediators (13, 14). The induced chemokines can promote neutrophil infiltration and also T lymphocytes to reinforce reactive oxygen and nitrogen species (RONS), which play key role in DNA damage and mutagenesis (15, 16). Although there is a growing body of evidence supporting direct or indirect involvement of H. pylori in genomic instability and mutagenesis, molecular mechanisms that link DNA damage to erroneous DNA repair system, which will affect oncogenes and tumor suppressor genes, is not well known. To show this relationship, we addressed activation of NHEJ in the gastric tissue of H. pylori infected compared with non-infected patients. Our findings provide initial evidences of the activation of the error-prone repair pathway (NHEJ) in patients infected with H. pylori.
Activation of the NF-κB transcription factor, overexpression of IL8, and release of free oxygen radicals that are related to oxidative stress (ROS) in the gastric epithelial cells are associated with mutagenesis through DSBs and activation of DDR systems (17). DSBs are detected by the DNA damage sensors, namely the MRN complex (Mre11-Rad50-Nbs1), which successfully manipulates other components of the DDR pathway, including activation of ATM and ATR leading to H2AX histone phosphorylation that is followed by activation of the transducers Chk1 and Chk2, resulting in p53 activation. Cell cycle arrest through activation of TP53 is a trigger for the repair of DNA breaks (18). So far, not many studies have been done on the role of this bacterium in causing DSBs in human gastric cells. Findings from in vitro examinations suggest a possible role for VacA and CagA in DSBs development (6). Despite these shreds of evidence, it remains unclear how this pathogen may play a role in gastric carcinogenesis. Investigating a possible link between the activation of repairing mechanisms and the occurrence of errors that cause genetic mutations in the gastric tissue can clarify the relevance. In our study, among the studied genes, POLM and XRCC4 that are associated with the NHEJ repair pathway, showed the highest expression in the H. pylori infected patients, which indirectly suggests the formation of chromosomal mutations. Interestingly, in a study of prostatic adenocarcinoma cells, increased expression of the POLM gene was reported, which is in line with the results of the current study. It has also been suggested increased expression is because of the inefficiency of the HR repair pathway (19). Also, in studies on gastric and breast cancerous tissues, an increase in the XRCC4 gene expression was reported (20, 21). Kitagawa et al. introduced the expression of the XRCC4 gene as a biomarker to detect the recurrence of breast cancer (21). Overexpression of the DCLRE1C gene, which rises through recruitment of NHEJ repair pathway, was previously reported in lung cancer (22). On similar study, Farkas et al. reported a rise in the expression of the DCLRE1C gene in colorectal cancer (23). In current study, overexpression of DCLRE1C can be a result of activation in NHEJ pathway in terms of compensating the induced damage by H. Pylori.
In the current study, the TP53 gene from the DDR pathway showed the highest increase in expression levels in H. pylori infected patients, supporting a link between the cell cycle arrest and activation of the repair system in response to the induced damage on DNA. In a study on gastrointestinal cancer by Sun et al., an increase in the expression of this gene was reported in comparison to normal and benign tissues, which is to somehow in line with the current study (24). Paradoxically, in a study on H. pylori mediated gastric cancer by Calcagno et al., a decrease in expression of the TP53 gene at mRNA levels was reported. In this study, cancerous tissue was used instead of inflammatory tissue (25). It is plausible that decreased expression of the TP53 gene could be resulted from mutations that occur during the carcinogenesis process, which occur over time and can reduce the function of this gene. Another study by Nianshuang et al. examined the role of the TP53 protein in H. pylori infection and its association with gastric cancer. The study results suggested that H. pylori might use its pathogenic agents, such as CagA that can enter into the host cells, to promote degradation and reduction of TP53 protein. This could accelerate the occurrence of DSBs in host DNA (26). According to these findings, the increase in TP53 transcription that observed in our study could be described through degradation of the protein in the infected cells and its compensation by the cell after transcription.
Decreased CHEK2 gene expression was generally reported in studies on gastric, lung, colon, and breast cancers (27–30), which are in contrary to our findings. In a study performed by Bae et al. on animal models of normal gastric tissue to investigate the effect of H. pylori on CHEK2 gene expression, it was demonstrated that the expression of this gene was increased, which is in line with the results of our study. Their findings showed increased expression of the TP53 and CHEK2 genes simultaneously, indicating that the cell is headed for cell death after the cell cycle arrest due to DSB (31). Moreover, ATM gene expression has decreased in most studies on cancerous tissues (32, 33), which contrasts with the present study's findings. In general, the decreased expression of DDR-related genes in cancerous tissues may be due to multiple mutations occurs overtime which could give rise to the loss of function of these genes. The overexpression of ATM gene in current study could be explained due to the different essence of tissues and the mediation of H. pylori in this interplay.
We were the first to study the transcription of genes linked to NHEJ and DDR pathways in association to H. pylori infection in the precancerous gastric tissue, however, the mechanism of chromosomal mutations and genomic instabilities caused by this bacterium need further examinations. Absence of healthy individuals to measure the basic expression levels of target genes and comparing them with case group and impossibility to study all NHEJ genes and regulators, failure to determining DNA breaks at the chromosomal stages and possible shuffling and translocations, and finally, the impossibility of investigating the genome of bacterial strains in the studied specimen samples, in order to understand their relationship with the NHEJ pathway activation are among the limitations of the present study. Future studies can identify subtypes of pathogenic factors of H. pylori involved in DSBs of host cells in gastric tissue. It can also be studied to identify activators of other DSB-related signaling pathways by H. pylori and their interaction with DDR. Additional tests to understand the type of mutations associated with the NHEJ pathway and the identification of NHEJ pathway mediators that play a vital role in the repairment of DSB in gastric tissue, and the design of appropriate drugs for therapeutic purposes, might be topics for future studies.