Negative feedback loop in the activation of non-homologous end joining DNA repair pathway in Helicobacter pylori infected patients with gastritis

doi:10.21203/rs.3.rs-4718686/v1

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Negative feedback loop in the activation of non-homologous end joining DNA repair pathway in Helicobacter pylori infected patients with gastritis

https://doi.org/10.21203/rs.3.rs-4718686/v1

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Chronic gastritis if left untreated can develop into cancer. This study was done to investigate the activation of error-prone DNA repair pathway in response to Helicobacter pylori infection. Relative changes in the expression levels of genes involved in the non-homologous end-joining pathway (NHEJ) in H. pylori-infected (Cases) and non-infected patients (Controls) with chronic gastritis were measured. A significant increase in the relative expression level of TP53, and significant decrease in the relative transcription of lncRNA LINP1 and XRCC5 were detected in the case group. The transcription of Lig4 and XRCC6 was increased in the case group, which was not statistically significant. The Spearman’s Correlation Coefficient analysis showed a significant positive-correlation between the transcriptional levels of LINP1 and XRCC4/XRCC5/Lig4, and between XRCC5 and TP53/Lig4 both in the case and control groups. Moreover, a significant positive correlation between LinP1 and XRCC6 in the case, and a significant positive correlation between XRCC4 and Lig4, and a negative correlation between TP53 and LinP1/XRCC4/XRCC5 in the control group was detected. Collectively, our results suggest a protective role for TP53 against the assembly and stabilization of NHEJ complex, mainly through downregulation of LinP1 and XRCC5, in the H. pylori-infected patients with chronic gastritis.

Helicobacter pylori

Chronic Gastritis

Non-homologous end-joining

DNA repair system

DNA double-strand breaks

LncRNA in Non-Homologous End Joining Pathway 1

Chronic gastritis, as a primary inflammatory disease affecting the gastric mucosa, without treatment could progress and manifest as severe gastritis, metaplasia, dysplasia, and eventually cancer [1, 2]. Cancer, a primary cause of worldwide human mortality, has historically been linked predominantly to aberrations in the genetic composition of the host cells. Diverse forms of cancer, including gastric cancer, are recognized as conditions correlated with inflammation, frequently originating from enduring viral, bacterial, or parasitic infections [3]. Helicobacter pylori, a microaerophilic Gram-negative bacterium that inhabits the stomach of over half of the global population, could cause chronic gastritis [4, 5]. Though not fully elucidated, the pathways by which this bacterium induces the transformation of host cells into neoplastic entities share a common feature. Accordingly, the infection promotes the formation of DNA double-strand breaks (DSBs) within the host's genetic material. Unrepaired DSBs can prompt apoptosis or cellular senescence; however, mishandling of these breaks results in various mutations, thereby inducing genomic instability and fostering the initiation of cancer [6].

Regardless of H. pylori virulence genotypes and severity of the induced histopathological damages, chronic infection with this bacterium could increase the risk of non-cardia gastric cancer about six-fold in the infected patients 10 years after primary diagnosis [7]. The bacterium induces epigenetic and genetic alterations in the gastric mucosa, probably through interaction with host DNA-damage response system, which are associated with GC [8]. Human cells have developed the DNA-damage response (DDR) mechanism as an evolutionary adaptation to overcome DNA damage [9]. Mainly, two functionally distinct sets of pathways have developed for the repair of DNA double-strand breaks (DSBs), namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). HR relies on the identification and alignment of disrupted DNA ends with undamaged homologous sequences found either on a sister chromatid, an allelic site, or potentially at an ectopic position within the genome. NHEJ entails the modification and fusion of the fractured DNA ends with minimal or absent sequence similarity, frequently resulting in minor deletions or insertions. While NHEJ remains active across the cell cycle, its significance is particularly pronounced during the G1 phase [10]. Within the process of NHEJ, the initial recognition of the DSB occurs through the Ku70–Ku80 heterodimer (Ku), functioning as a foundational platform or loading protein. Additional NHEJ proteins are subsequently recruited as required to facilitate the alignment and joining of DNA ends. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exhibits a strong binding affinity for Ku-associated DNA ends and collaborates with Ku to establish the DNA-PK complex [11]. The kinase function of DNA-PK stands as a pivotal element in the NHEJ process. DNA-PK engages in phosphorylation events involving various proteins, such as Artemis, which interacts with DNA PKcs, thereby activating its endonuclease activity toward both 3’ and 5’ overhangs. Direct ligation of DNA ends is facilitated by XLF/DNA ligase IV/XRCC4, post-interaction with DNA PK. Multiple proteins, including Artemis and members of the Polymerase X family, such as Pol µ and λ, are implicated in the processing of these DNA ends. Consequently, the joining of DSBs through NHEJ often leads to the deletion or addition of a few nucleotides, creating short complementary sequences at the break site. These sequences presumably aid in aligning the DNA ends and contribute to rendering NHEJ an error-prone repair pathway [12].

Although the accumulation of mutations in different GC-related genes due to H. pylori infection has been described before, no clear evidence is available to support the involvement of H. pylori infection in the promotion of these mutations by the activation of error-prone DNA repair pathway. Within non-coding regions of the genome, lie a diverse array of regulatory and functional components, among which the most abundant are the loci responsible for encoding long non-coding RNAs (lncRNAs), which are longer than 200 nucleotides and are involved in the regulation of many aspects of cells, including cell differentiation and development, physiological processes, control of chromatin architecture, gene enhancing and silencing, tumorigenesis, and metastasis [13, 14]. Although mechanisms of lncRNAs-H. pylori interactions are not well understood, the role of a lncRNA, known as lncRNA in non-homologous end joining (NHEJ) pathway 1 (LINP1), in the p53-mediated response to DNA damage was described. LINP1, which functions as an RNA scaffold, interacts with Ku70–Ku80 and DNA-PKcs, thereby facilitating and promoting NHEJ repair processes [15]. To understand the role of H. pylori infection in the activation of error-prone DNA repair system in the gastric tissue of patients with chronic gastric before the formation of preneoplastic lesions, we investigated the expression profiles of genes associated with the non-homologous end joining (NHEJ) pathway, alongside the expression patterns of the lncRNA LINP1 and its correlation with the regulatory transcription of TP53. This assessment was conducted among patients afflicted with mild or moderate gastritis due to H. pylori infection, in comparison to the non-infected patients who present similar histopathological characteristics.

1.1. Patients and samples

This case and control study was approved by the ethical committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.RICH.REC.1402.028) and Research Ethics Committee of Islamic Azad Tehran Medical Sciences University- Pharmacy and Pharmaceutical Branches Faculty (IR.IAU.PS.REC.1401.354). This study was conducted in compliance with the Declaration of Helsinki. No personal or clinical details of the patients are presented in our study that compromise anonymity and no medical intervention was done for the patients in addition to the usual medical diagnosis.

The biopsy samples were obtained from adults with digestive problems referred to the endoscopy department of Laleh Hospital in Tehran, Iran. All the patients filled out an informed consent form and a questionnaire related to demographic and medical data. The biopsy samples were collected from the antrum and used for the microbiological, histopathological, and molecular tests. According to the test results, a total of 50 samples of H. pylori-infected patients with chronic gastritis (Case group) and 50 samples of H. pylori-non-infected patients with chronic gastritis (Control group) were selected out of 195 patients by simple random sampling. Sample size calculation was done according to binary outcome formulas, considering the frequency of 20% for H. pylori infection and 58% for chronic gastritis. The inclusion criteria were as follows: Adult patients with mild or moderate chronic gastritis subjected to endoscopic and pathological analysis of the gastric mucosa, and no usage of antibiotics in the preceding 4 weeks. The exclusion criteria included: patients with acute gastritis, those with gastric cancer (GC) at different stages or GC history in first-degree relatives, those with stomach surgeries, patients who were treated with effective antibiotics against H. pylori, and those being treated with chemotherapy drugs.

1.2. Microbiological and histopathological analysis

Endoscopic findings, screening of H. pylori infection by rapid urease test, microscopic examination, and histological information were used to select cases and controls. To prevent RNA degradation, one of the biopsies were transferred in a liquid nitrogen container and kept in a -70 ^oC freezer for molecular investigation in the microbiology laboratory of the Pediatric Infections Research Center. RNAase-free and DNase-free containers and microtubes were used in all steps during the sampling and transfer to the laboratory. To screen urease activity in the gastric biopsies, one of the biopsies was placed in the rapid urease test medium and change in the reaction was followed up in an incubator for 24 hours due to the over-the-counter administration of proton pump inhibitors (PPIs) that causes delayed urease activity. The histological analysis was done in the Histology Department of Laleh Hospital and Arvand Pathobiology laboratory. The microscopic analysis of the urease-positive and urease-negative biopsies was done on a high-power field after Gram-staining using Carbol Fuchsin as a counter stain. RNA extraction from the urease-positive and microscopically or histopathologically confirmed samples was done according to the standard protocols as described below.

1.3. Molecular investigations

The RNSolH protocol was followed for RNA extraction. Accordingly, 800 µl of RNSolH solution, a mono-phasic solution of phenol and guanidine isothiocyanate (Roje Technologies, Iran), was added to each biopsy in a microtube. The biopsy was completely lysed and pipetted in the resulting solution. The homogenized solution was kept at room temperature for 5 minutes. Chloroform was added to remove cell lysates, DNA, and proteins. The isopropanol and alcohol precipitation (Ethanol, 75%) was used for the RNA pellet formation. In the final step, a 50 µl volume of RNase-free deionized water was slowly added to each tube and pipetting was done repeatedly until the sediment was completely dissolved. The tube was placed in a Bain-Marie at 60°C for 10 minutes to dissolve the sediment. The extracted RNA was kept at − 70°C until cDNA synthesis. NanoDrop spectrophotometer (Thermo Scientific, USA) was used to control the quality and determine the concentration of extracted RNA for each sample. cDNA synthesis was done using Easy™ cDNA Synthesis Kit (Parstous Biotechnology, Iran). Total cDNA for human genes was synthesized using random hexamer and oligo dT primers. cDNA synthesis for the analysis of H. pylori transcription was done using glmM specific primers. Adjustment of RNA concentration was done to minimize diversity among the samples. cDNA was synthesized using a thermocycler according to the manufacturers’ protocol. Relative quantitative Real Time PCR method was used to investigate the change in gene expression using ready to use SYBER Green master mix reagents (Ampliqon, Denmark). Amplification of the cDNA products was done by Real time PCR using exon-exon primers (Synthesized by SinaClon Company, Tehran, Iran). All the primers were checked in the Ensembl Genome database to prevent unappropriated results (Supplementary Table 1). B2M gene was selected as the endogenous control gene. To measure primer efficiency, 5 different dilutions of samples with suitable Ct value were prepared (1, 1⁄10, 1⁄100 and 1⁄1000 and 1⁄10000) and Real-Time PCR reaction was done for each pair of the primers at the same conditions in ABI StepOnePlus machine (Applied Biosystem, Foster, USA). By drawing a standard curve, the amplification efficiency of each primer pair was calculated. In all the assays, a negative control sample was used to ensure the absence of contamination. All the experiments were done in duplicate. The reaction mixture contains 12.5 µL Real time PCR SYBR Green Master Mix (Ampliqon, Denmark), 2 µL cDNA, 0.5 µL of forward and Reverse primer (5 pmol/µL), and 9.5 µL ddH2O. Real Time PCR reaction program for all genes, except LINP1, was as follows: 1 cycle of 95 °C for 15 minutes, 40 cycles of 95 ^oC for 15 seconds, 60 ^oC for 5 seconds, and 72 ^oC for 30 seconds. The melting curve analysis was done after 1 cycle of 95 °C for 15 seconds, and melting at a range of 60°C to 95°C by 0.3 ^oC gradual increase, every 15 seconds. Due to the difference in melting temperature of the primers of the LINP1 gene, the following program was used: 1 cycle of 95 °C for 15 minutes, 40 cycles of 95 ^oC for 15 seconds, 58 ^oC for 30 seconds, 72 ^oC for 30 seconds, and melting curve analysis (1 cycle of 95 °C for 15 seconds, and melting at a range of 58°C to 95°C by 0.5 ^oC gradual increase, every 15 seconds).

B2M was used as house-keeping gene (HKG) in all the calculations. The expression ratio of each gene of interest (GOI) in each sample was calculated according to the efficiency corrected calculation (Pfaffl model) based on the following formula: (E_GOI) ^{ΔCt GOI}/(E_HKG) ^{ΔCt HKG}. The fold change (Rq) was measured using 2^^−ΔΔCt formula as described before: 2^^{− (ΔCt of HP infected group – average Δct of HP non−infected group)} [16]. Median of the Rq values, standard deviation, and 95% of confidence intervals were calculated for each gene.

1.4. Statistical analysis

Statistical analysis was done in GraphPad Prism version 10.2.3 (GraphPad Software, La Jolla, CA, USA). The expression levels of genes were measured using the comparative ΔCt method as described above. Unpaired t-test or non-parametric test (Mann–Whitney U test) was used to determine differentially expressed genes between the patients and control groups. Correlations between the expression levels of genes were measured using the Spearman’s rank correlation coefficient. The receiver operating characteristic (ROC) curves were depicted to evaluate the diagnostic power of the expression levels of differentially expressed genes. The statistical significance limit was considered less than 0.05.

2.1. Patients and samples

A total of 195 biopsy samples were collected from symptomatic patients in the age ranges of 21 to 86 years old during the endoscopy procedure in Gastroenterology Department of Laleh Hospital in Tehran, Iran. Based on the pathological (Report of chronic gastritis) and microbiological findings (Positive rapid urease test, the microscopic and RT-real time PCR confirmation) and according to the inclusion and exclusion criteria, 50 samples from the cases (Women, 29/50; men: 21/50) and 50 samples from the controls (Women: 29/50, men: 21/50) were selected for further investigations. Details of the patients’ demographics are shown in Table 1.

Table 1

Demographic and clinical findings of adult patients in this study.
		Control	Case
Age groups
	15–24	1	0
	25–34	3	3
	35–44	5	6
	45–54	11	9
	55–64	14	15
	65–74	13	14
	> 75	3	3
Gender
	Women	29	29
	Men	21	21
PPI usage
	Yes	26	24
	No	37	13
Bismuth usage
	Yes	2	9
	No	41	48
Endoscopic findings^a
	Chronic Gastritis (CG) 50		50
	CG + Peptic ulcer 2		2
	CG + Duodenal ulcer 3		3
	CG + Erosion	5	4
^a. The endoscopic findings are related to 50 cases and 50 controls with chronic gastritis.

2.2. Transcriptional analysis

The melting curve (Unique specific product) and primer efficiency analyses (0.95-1 for all the primers) confirmed the specificity of the products and the condition used for the transcriptional analysis. The results of gene expression ratios and measured fold changes are shown in Fig. 1, Table 2 and supplementary Table 2. A significant difference in the transcription of XRCC5 (p-value = 0.0001), TP53 (p-value = 0.002), and LINP1 (p-value = 0.03) was detected between the case and control groups. Analysis of the measured relative fold change in the transcription of GOI showed an increase in XRCC6, TP53, and LIG4, no change in XRCC4, and decrease in LinP1 and XRCC5 in the gastric biopsy of H. pylori-infected patients compared with the non-infected group (Table 2). Analyzing the correlation between expression levels of the studied genes showed the most robust correlations between LinP1 and XRCC4/XRCC5/LIG4, XRCC4 and XRCC5, XRCC6 and TP53/LIG4, and TP53 and Lig4 in both the case and control groups. The correlation between LinP1 and XRCC6 was detected only in the case group, while transcription of XRCC4 and Lig4 was correlated only in the control group. Negative correlation was found between TP53 and LinP1/XRCC4/XRCC5; however, it was not statistically significant (Fig. 2, Table 3). AUC values for the GOI ranged from 0.72 to 0.50 (XRCC5 to XRCC6, respectively) (Fig. 1). Remarkably, XRCC5 was found to have the highest sensitivity value for differentiation of the cases from the controls. Moreover, TP53 and LinP1 showed a relatively similar sensitivity for the differentiation of the H. pylori infected in comparison to non-infected patients (Sensitivity values of 0.67 and 0.62, respectively).

Table 2

The Relative fold change of genes linked to the nonhomologous end joining (*NHEJ*) DNA repair pathway and related long non-coding RNA, LINP1, in the *Helicobacter pylori*-infected in comparison to non-infected patients.
	LinP1	XRCC4	XRCC5	XRCC6	TP53	Lig4
Fold change^a.
Median	0.382724	0.824696	0.453511	2.085849	2.693027	5.280001
STD	190.2216	2.41E + 09	401847.7	6.959462	7.191116	1691296
p-value	0.03	0.108	0.0001	0.0582	0.0028	0.196
95% CI of median	0.234–1.465	0.1819–3.784	0.2537–1.113	1.297–3.35	1.526–5.728	3.528–8.907
95% CI of geometric mean	0.3682–1.358	1.101–97.55	0.3059–2.574	1.404–2.809	1.247–3.765	2.431–20.02
a. The fold change was measured using 2^-ΔΔCt formula. Gene expression ratios in the case and control groups were used for the calculation of p-values and 95% confidence interval. Significant p-values are shown in bold.

Table 3

Correlation in RNA expression levels among genes linked to non-homologous end joining DNA repair pathway in the *Helicobacter pylori*-infected and non-infected patients with gastritis.^a
	LinP1		XRCC4		XRCC5		XRCC6		TP53		Lig4
	HP-non-Infected	HP- Infected	HP-non-Infected	HP- Infected	HP-non-Infected	HP- Infected	HP-non-Infected	HP- Infected	HP-non-Infected	HP- Infected	HP-non-Infected	HP- Infected
LinP1	1	1	0.832***	0.861***	0.445**	0.598***	0.237	0.294^*	-0.031	0.263	0.443**	0.332^*
XRCC4			1	1	0.519***	0.663***	0.181	0.174	-0.069	0.158	0.298*	0.277
XRCC5					1	1	0.0878	0.123	-0.128	0.0329	0.0938	0.070
XRCC6							1	1	0.310*	0.329^*	0.375**	0.4205**
TP53									1	1	0.356*	0.496***
Lig4											1	1
^a. The correlation between transcriptional levels of genes were measured using the Spearman’s correlation coefficient. p-value < 0.05, * p-value < 0.01, *** p-value < 0.001.

Previous studies showed that H. pylori infection introduces DSB in the gastric tissue [17]. In a study by Hartung et al., activation of the NHEJ DNA repair pathway as a consequence of H. pylori induced DSB was elucidated. They showed that the siRNA-mediated knockdown of XRCC4 and P53 binding protein 1 (53BP1), but not the homologous recombination repair factor BRCA1, can reduce the cells’ repair capability, which supported that H. pylori-induced DSBs are preferentially addressed by the NHEJ pathway [18]. Baseline expression of TP53 is low in normal histological status but increases greatly upon cellular stresses, like DNA damage. Although the role of TP53 mutations in chromosomal instability, amplification of oncogenes, and deletion of tumor suppressor genes is well described in different types of cancers, its interplay with chronic gastritis and H. pylori infection, and involvement in the progression of pre-neoplastic lesions is not well known. As was shown in our study, overexpression of wild type TP53 in the gastric tissue of H. pylori infected patients with chronic gastritis seems to be linked to the induction of the inflammatory pathway and DNA damage [19, 20]. DNA damage can cause the stabilization and accumulation of TP53 in the nucleus leading to cell cycle arrest, which supports the activation of DNA repair systems [20]. miRNAs–MDM2–P53 feedback loop is probably the main mechanism for the overexpression and activation of TP53 in response to a stress signal, like DNA damage [21]. Overexpression of TP53, itself could play an inhibitory effect on the error-prone NHEJ pathway [22]. It seems that by counteracting with the error-prone repair system, TP53 plays a stabilizing role against genomic instability. This protective role will be lost due to a decrease in the TP53 levels, as observed in the late stages of the disease. This reduction is associated with increased mutation rates, proteasome degradation, and post-transcriptional modification, all linked to the cell proliferation and progression toward gastric cancer [20].

In this study, a comparison in the transcription of XRCC4, XRCC5, XRCC6, Lig4, TP53 and LINP1 genes was done between the H. pylori infected and non-infected patients with chronic gastritis. The results showed an increase in the transcription of TP53, Lig4 and XRCC4 and a decrease in the transcription of LinP1 and XRCC5 in the case group. Consistent with these results, in our previous study 5.0-fold increase in transcription of TP53 was reported in gastric biopsies of H. pylori infected compared with non-infected patients with chronic gastritis [23], which was accompanied by overexpression of other mediators of NHEJ and HR pathways. The overexpression of TP53 was also confirmed by Mahmoudzadeh Sagheb et al. in the gastric tissue of H. pylori patients with pre-neoplastic histological changes [24].

DNA Ligase 4(IV) protein plays an essential role in V(D)J recombination and DNA double strand break repair through non-homologous end joining. This protein forms a complex with XRCC4 and interacts with DNA-dependent protein kinase. In the present study, the expression of Lig4 showed an increase of 5.28 times in the case group compared with the median transcription value of the control group. According to our knowledge, the relationship between the expression level of Lig4 and an inflammatory state of the gastric tissue in the H. pylori infection is presented for the first time in this study; however, a significant increase in the expression level of Lig4 was shown in some non-gastric cancers, like prostate and breast cancer [25]. Lig4 plays its unique function in the NHEJ DNA repair system, which supports the involvement of H. pylori infection in the induction of DSB and the promotion of error-prone DNA repair system. The LIG4 complex (Lig4, XRCC4, and XLF) mediates ligation as the last step of the NHEJ repair. XRCC4 can stimulate Lig4, which supports the observed correlation between these two mediators among controls in our study [26]. In the current study, the relative increase in the transcription of this gene in H. pylori infected patients was correlated with increased transcription of LinP1, XRCC6 and TP53 genes. The observed correlation between TP53, as a tumor suppressor gene, and Lig4 in both the case and control groups proposed the existence of a feedback loop against the activation of NHEJ DNA repair system in the patients with chronic gastritis.

Although transcriptional alteration of LinP1 in patients with chronic gastritis was investigated for the first time in our study, its role as long non-coding RNA and scaffold, linking XRCC6 and DNA-PKcs in NHEJ pathway, was already described in breast cancers [27]. LINP1 is negatively regulated by TP53 through mir-29 and LINP1 could partially reverse the inhibitory effects of TP53 on proliferation and migration [28]. The positive correlation of Lig4 transcription with LinP1 and XRCC4/5, as was shown in both the case and control groups, proposed the activation of a regulatory pathway associated with DNA repair at the early stages of chronic gastritis regardless of the infection status. A positive correlation between the transcription of this lncRNA and XRCC6 was shown only in the case group, which highlights the role of H. pylori infection in the induction and the formation of a primary complex of NHEJ DNA repair system following the formation of DNA DSB. The higher amount of TP53 transcription in the H. pylori infected patients at this stage of the disease proposed a protective role against the progress of NHEJ pathway. Knockout of TP53, micro RNAs and LncRNAs which play regulatory roles in the activation of NHEJ pathway, and study of their effects on the activation of this pathway in response to H. pylori infection could better reveal this interplay at different stages of gastritis.

In the current study, while a correlation was detected among LinP1, XRCC4, XRCC5, and Lig4 in the case and control groups, we found a significant difference in the transcriptional levels of LinP1, TP53, and XRCC5 in the case in compare to the control group. Accordingly, infection with H. pylori caused an increase in the transcription of TP53 and a decrease in the transcription of XRCC5 and LinP1. The overexpression of LinP1 in esophageal squamous cell carcinoma and the impact of its Knockdown in the suppression of esophageal cancer tumorigenesis through an increase in the apoptotic rate was established in a study by Lu et al [29]. Low expression of LinP1, which plays a role as a scaffold for the localization of XRCC5 and DNA-PKcs at the site of DSB, in correlation to a relatively lower level of XRCC5 expression in the H. pylori infected cases, proposed the existence of a negative feedback loop that may be linked to the increased levels of TP53, Lig4, and XRCC6 in the infected tissue. This negative feedback loop emphasized the role of H. pylori in this interplay. Overexpression of TP53 in the pre-neoplastic tissue highlights the protective role of this protein as the “guardian of the genome” against the introduction of new mutations and genomic instability in response to damaged DNA that is linked to gastric cancer. The impact of miR-29-TP53 on transcriptional regulation of LinP1 as negative feedback was described by Sakthianandeswaren A. et al [30]. TP53 positively regulates the expression of miR-29, and TP53–miR-29 through degradation of LinP1 may play their negative regulatory mechanism to restrict NHEJ activity in gastric tissue of patients with chronic gastritis [31, 32].

Study limitation

In this study, the impact of H. pylori infection on transcription of NHEJ gene mediators was investigated only in patients with chronic gastritis, and a comparison of our results with samples of patients with other pre-neoplastic lesions, like intestinal metaplasia and dysplasia, was not done. Lack of funds and facilities for doing a transcriptomic analysis, and study of animal models and cell lines carrying knockout genes of the NHEJ mediators, inability to track differences in protein levels, and the low number of patients in the case and control groups were among the problems in our study.

Overall, our results confirmed the role of H. pylori infection in the induction of double-stranded DNA breaks in the gastric mucosa and the activation of the non-homologous end-joining repair pathway in patients with chronic gastritis. Activation of Lig4, as a key mediator involved in the non-homologous end-joining pathway, and the detected correlation of LinP1/XRCC6 in the H. pylori infected patients proposed a role for this infection for promotion of DSB and accumulation of somatic mutations in the gastric mucosa. The relative increase in transcription of TP53 in the H. pylori infected patients highlights its protective role against genomic instability in the early phase of the disease in the stomach. This negative effect could be attributed to a negative feedback loop mediated by the mir-29 and LinP1 interaction. Investigating other mediators affecting the non-homologous end-joining pathway, such as MALAT1 and ATM, as well as characterization of regulators of guardians of the genome, like TP53, to play their protective roles against the activation of the error-prone repair pathway, can help introducing new therapeutics in preventing the progression of pre-neoplastic lesion to cancer.

Data availability statement

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Authors’ contribution

M. A. performed the laboratory investigations, formal analysis, and data curation. A. K. cooperated in the methodology, formal analysis, investigations, and data curation. N. E. D. supported the endoscopy procedures, clinical diagnosis, pathological interpretation, and biopsy sample preparation. A. R. cooperated in writing the original draft of the manuscript, its review, and editing. M. H cooperated in the study design. M. A. performed conceptualization, visualization, formal analysis, laboratory investigations, data curation, writing the original draft of the manuscript, and the study supervision. All authors reviewed the manuscript.

Declaration of competing interest

The authors declare that they have no competing interests.

Additional information

Supplementary Information. The online version contains supplementary material available at https:// ……………

Acknowledgements

The authors of this study would like to thank Prof. Abdollah Karimi who provides facilities for doing this study at the Pediatric Infections Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. The authors also thank the cooperation of the endoscopy and pathology units of Laleh Hospital and the pathology unit of Arvand Pathobiology Laboratory, Tehran, Iran. This study was part of an MSc thesis supported by research project ID 32897 in the Pediatric Infections Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Ethics declarations

This study was approved by the ethical committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.RICH.REC.1402.028) and Research Ethics Committee of Islamic Azad Tehran Medical Sciences University- Pharmacy and Pharmaceutical Branches Faculty (IR.IAU.PS.REC.1401.354). The biopsy samples were obtained from adults with digestive problems referred to the endoscopy department of Laleh Hospital in Tehran, Iran. All the patients filled out an informed consent form and a questionnaire related to demographic and medical data at admission time. The biopsy samples were collected according to the medical orders from the antrum and used for the microbiological, histopathological, and molecular tests. No additional sampling and medical interventions were done for the patients during the study period.

Consent to publication

To analyze the laboratory and demographic data, the patients signed an informed consent form at admission time according to the hospital regulations. No personal or clinical details of participants are presented that compromise anonymity.

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Negative feedback loop in the activation of non-homologous end joining DNA repair pathway in Helicobacter pylori infected patients with gastritis

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Abstract

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Introduction

Materials and Methods

1.1. Patients and samples

1.2. Microbiological and histopathological analysis

1.3. Molecular investigations

1.4. Statistical analysis

Results

2.1. Patients and samples

2.2. Transcriptional analysis

Discussion

Study limitation

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Ethics declarations

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