Transcriptional Profile of Helicobacter pylori Virulence Genes in Patients with Gastritis and Gastric Cancer

Introduction Numerous molecular epidemiology studies have been performed about the frequency of Helicobacter pylori virulence genes in patients with H. pylori infection so far. This study was conducted to detect transcriptional profile by cDNA of H. pylori virulence genes in gastric biopsy samples of gastritis and gastric carcinoma patients. Materials and Methods In a case-control study, based on the prevalence of gastritis and gastric cancer in Sanandaj city during 2018 and 2019, 23 and 11 gastric antral biopsy samples with H. pylori infection were collected from gastritis and gastric carcinoma patients by the consecutive and available sampling method. Pathological characters, including tumor grades and tumor areas for gastric carcinoma biopsy samples prepared from gastric cancer areas, were determined by the pathologist. Total RNA of gastric antral biopsy samples was extracted, and their cDNA was synthesized by TaKaRa kit. H. pylori virulence genes' cDNA using specific primers and PCR was detected. This study's results were analyzed by SPSS version 25 and statics chi-square tests for determination of relationship and correlation between cDNAs of H. pylori transcriptional profile and clinical outcomes of H. pylori infection, including gastritis, gastric carcinoma, tumor grades, and tumor area. Results The positive statistical correlations were observed between transcripts of cagA, cagA-EPIYAC, cagE, and cagY genes and H. pylori infection clinical outcomes (P < 0.05). Conclusion Detection of the H. pylori virulence genes' cDNA in gastric biopsy samples can help provide the prognosis of clinical outcomes.


Introduction
Microbe factors, including toxins, enzymes, and structural antigens, induce the host's immune system [1]. e most common cause of human chronic infection is Helicobacter pylori, which is considered host-microbe interactions [2][3][4]. H. pylori chronic infection is followed by chronic mild gastritis with or without clinical manifestations, which progresses to gastric cancer in 1-2% of patients with H. pylori cagApositive genotypes infection [5]. e molecular epidemiology studies show a positive correlation between H. pylori virulence genes' frequency and the clinical outcomes [6,7]. e results of previous studies show that the clinical outcomes are related to H. pylori virulence genes' expression. In parallel with host immunologic effects on gastritis progression, there are the effects of different ambient conditions on H. pylori virulence genes' expression in host cells [8][9][10][11][12][13][14]. As the relevant studies, salt and acidic pH change the transcription of the H. pylori virulence genes through the bacterial ARS two-components system [9,13]. Some studies have concluded that the H. pylori persistence infection is due to the antigenic phase variations in H. pylori outer membrane proteins, including Hop, Bab, Sab, Oip, Dup, and Alp proteins [15][16][17][18][19][20].
In the early stages of H. pylori infection, Bab adhesin attaches to Lewis B antigens located on gastric epithelial cells' surface by a tight attachment between the bacterial type 4 secretory system and gastric epithelial cells [20]. e babA2 gene, with 60% frequency, encodes bab adhesin in H. pylori strains related to gastric cancer [21].
Besides, the persistence infection is stabilized by attaching Sab antigens to sialyl-Lewis x receptors on gastric epithelial cells [17,18]. e previous studies show a high frequency of sab genes among H. pylori strains related to severe clinical manifestations [22].
Alp adhesin of H. pylori involves proinflammatory signals in gastric mucosal epithelial cells by attaching to type IV collagen and laminin on gastric epithelial cells (GECs) and activation of MAPK and NFKB cell signaling pathways [23]. e results of relevant studies show that all clinical H. pylori strains express the alpA/B gene [24].
Like Alp adhesin, Oip adhesin in the company with the type 4 secretory system contributes to bacterial colonization and inflammation by increasing IL-8 production [25,26]. e upregulation of the oip gene induces apoptosis and changes in the GECs cytoskeleton, causing severe clinical manifestations, including gastric cancer [22]. Molecular epidemiology studies show a frequency of oip gene expression up to 70% among H. pylori clinical strains [25].
H. pylori vacuolating cytotoxin A pierces the mitochondrial cytoplasmic membrane and activates the apoptosis cell signaling in GECs [13][14][15]. H. pylori vacA gene sequence is involved in the s, i, and m variable regions that show statistical correlations with clinical outcomes of H. pylori infection and geographical distribution [27]. e vacA gene regularly expresses because it is located adjacent to the 16s rRNA conserved gene area [13]. Some studies show that vacA gene expression is induced after H. pylori colonization on GECs, and fluctuations in vacA gene expression rate among H. pylori clinical strains can be expectable [28,29]. e cag pathogenicity island consists of 31 genes that express type 4 secretory system (T4SS) proteins [30,31]. e T4SS conducts the injection of CagA (cytotoxin-associated gene A) protein into GECs [30,31]. e CagA protein induces GECs proliferation through JAK/STAT3 and MAPK kinases pathways [30]. e surface connecting complex of T4SS has been comprised of CagY, CagL, CagI, CagC, and CagH proteins [30]. e ɑ5β1 integrin receptor on the gastric epithelial cells is the ligand of CagY protein [32]. e results of relevant studies show a few variable regions with high recombination tendency in the cagY gene [33]. e recombination between forward and middle repeated sequences of the cagY gene changes its expression and, consequently, the cagY protein's affinity to integrin receptors [32]. CagY protein contacts GECs receptors and changes the host mucosal immune response's balance toward reducing mucosal immune and progression of the H. pylori persistent infection [32].
e previous studies show that cagY gene expression is regulated separately from other cag pathogenic island genes [33,34]. e results of relevant studies state the modulation role of CagY protein in the GECs immune system in the progression of an H. pylori active chronic infection [32][33][34][35]. e previous studies show the positive correlations between CagT and CagE proteins and the H. pylori pathogenicity [36,37]. CagE, as an NTPase enzyme, involves the secretion of cagA [30]. CagT, as a lipoprotein, connects the T4SS core proteins complex to the bacterial outer membrane [30]. Both CagT and cagE are essential for the injection of CagA into GECs [30].
Various molecular mechanisms of gene expression control have been shown in H. pylori. e detection of bacterial virulence genes alone does not indicate genes' permanent expression [24][25][26][27][28]. In this regard, sab gene expression changes by turning on and off an operator called the switching mechanism [24]. e slipped-strand repair mechanism controls oipA gene expression through repetitive CT sequences at the promotor's 5′ end [22,23]. hopZ gene has a repeat sequence in the signaling region that shows high recombination frequency and antigenic phase changes [38]. e changes in the stomach acidity flow and the constant contact of H. pylori to the gastric epithelial cells increase the expression of H. pylori adhesin's genes such as hopZ [38]. ese statements mean that microbe and host interaction depends on ambient variations on microbes' virulence genes expression.
In the current study, we surveyed H. pylori virulence genes' transcript profile and their relation with clinical outcomes by detecting cDNAs in gastric antral biopsy samples collected from gastritis and gastric cancer patients H. pylori infection referred to hospitals of Sanandaj city.

Methods
Cochran's statistical formula was used in a case-control study to determine the number of required samples based on gastritis and gastric cancer patients' prevalence in Sanandaj city [39]. Accordingly, 50 gastritis (control group) and 30 gastric carcinoma biopsy samples (case group) were collected by the consecutive and available sampling method from patients referred to Tohid and Shaheed Ghazi hospitals in Sanandaj city for 18 months from September 2018 to March 2019. 23 (46%) and 11 (66.36%) patients had been infected with H. pylori in gastritis and gastric carcinoma patients, respectively. After obtaining ethical consent for publishing research results without patient details, data of geography for each patient, including age, sex, and results of a urea breath test for rapid diagnosis of H. pylori infection, were recorded in questionnaire forms. e exclusion criteria included the patients with chemotherapy against H. pylori infection, smoking, and alcohol consumption to eliminate study interventions. Endoscopic observations of gastroenterologists diagnosed gastritis and gastric carcinoma. e histological and pathological characters' data for gastric carcinoma samples were collected from the Pathology Laboratory of Tohid Hospital in Sanandaj city. According to the manufacturer's RNALater solution protocol, a gastric antral biopsy sample was obtained from each gastritis and gastric cancer patient with H. pylori infection to detect cDNAs of H. pylori virulence genes by using the PCR method. Gastric antral biopsy samples were dropped into RNALater solution (Roche Co., Germany) immediately. e total RNA of biopsy samples was extracted according to the manufacturer's protocol (all in one mini preps kit, Bio Basic Canada Inc.). e 28S and 18S rRNA bands had evaluated the RNA integrity on an agarose gel at a concentration of 1.2%. e absorbance ratio at 260 nm and 280 nm was assessed for the purity of RNA by the NanoDrop ® 2000 spectrophotometer machine ( ermo Fisher Company, Germany). Total RNA was stored at −70 degree centigrade. At the next step, according to the manufacturer's protocol of PrimeScriptTM RT reagent Kit (TaKaRa Co.), total RNA was converted to cDNA. To assess cDNAs' purity, PCR was performed using H. pylori 16s rRNA-specific primers; then, PCR products were run on agarose gel 1.5%. Forward and reverse specific primers for detecting H. pylori virulence genes' cDNA were designed using Primer3 online software (version 0.4.0). Table 1 shows the characteristics of all used specific primers in this study that include the sequence of primers, annealing temperature, and product size. e cDNAs of H. pylori virulence genes were detected using the gradient thermocycler PCR machine (BioRad Company, Germany). PCR master mix included buffer 10x (2.5 microlitres), DNA Taq polymerase 5 U/microliter (0.25 microlitres), dNTPs 10 mM (0.5 microlitres), MgCl 2 50 mM (1 microlitre), cDNA (2 microlitres), forward and reverse specific primers 10 picoliters (each one 0.5 microlitres), and RNase-free water (17.75 microlitres) in final volume 25 microlitres. e thermal cycling PCR steps were involved an initial denaturation at 94°C for 5 minutes, a denaturation at 94°C for 30 seconds, a primer annealing for 45 seconds (primers temperatures have been shown in Table 1), an extension at 72°C for 45 seconds, and a final extension at 72°C for 5 minutes [27]. e denaturation through the extension step was repeated for 30-35 cycles. e PCR products of H. pylori virulence genes' cDNA were run on 1.5% agarose gel. e results were analyzed using SPSS software version 25 and statics chi-square tests. We used the exact Fisher static test and confidence interval 95% to analyze subgroups with a few numbers. Table 2 shows the demographic data of patients. e static results did not show any significant difference between disease (gastric and gastric cancer) and H. pylori infection (P � 0.487). As the same result, there was no significant static correlation between H. pylori infection and sexuality of patients (P � 0.518). However, there was a robust static correlation in the prevalence of gastric carcinoma in men (P � 0.005).

Demographic Characteristics of Patients.
e frequency of gastric carcinoma in men was four times rather than in women (Table 2). e highest and lowest H. pylori infection frequency was 61-73 and 18-30 years old, with 11 (32.4%) and 2 (5.9%) patients (Table 3). ese results show that there is no significant correlation statistically between H. pylori infection and patients' age (P � 0.314). On the other hand, there was a robust static correlation between gastric carcinoma and patients' age (P � 0.000) ( Table 2). Like the other similar study, increasing age in patients' populations increases gastric carcinoma frequency.

Transcription Profile of H. pylori Virulence Genes.
Molecular detection of H. pylori infection in gastric biopsy samples was detected using the PCR method and 16s rRNAspecific primers. e vacA s2 gene's partial cDNA was sequenced (Bioneer Company, South Korea) and registered in GenBank with accession number MK642592.1 to confirm the PCR result. Table 4 shows H. pylori virulence genes' cDNA's frequency in biopsy samples of gastritis and gastric carcinoma patients with H. pylori infection. e frequency of H. pylori virulence genes' cDNA in gastric biopsy samples was different. Except for the cagT gene's cDNA, the frequency of cag pathogenicity island gene's cDNA including cagA, cagA-EPIYAC, cagY, and cagE genes had a significant difference statistically with gastritis and gastric carcinoma and H. pylori infection (Table 4). e remarkable result was the low frequency of cagA gene's cDNA (4.35%) in biopsy samples of gastritis patients. In contrast, the cagA gene's cDNA frequency was 45.5% in patients with gastric carcinoma. 52.2% of gastritis biopsy samples with H. pylori infection had cagT gene's cDNA (cagT + cDNA) (P � 0.001) ( Table 4). In contrast, 36.4% of gastric carcinoma biopsy samples with H. pylori infection had simultaneous cagA, cagT, cagY, and cagE genes' cDNA (cagA + /cagT + /cagY + / cagE + cDNA) (P � 0.001). Transcript profile of H. pylori outer membrane adhesin's genes showed that the frequency of sab and hop genes' cDNA was different between gastric biopsy samples of gastritis and gastric carcinoma patients. However, this difference was not significant, with an error level of 0.05 (Table 3). Our results show there was not any significant difference statistically in the frequency of vacAs1m1/s1m2, oipA, alpA/B, and IceA1/2 genes' cDNA in two groups of patients with gastritis and gastric carcinoma that had H. pylori infection (Table 3).

Correlation of H. pylori Virulence Genes' Expression.
e results of Spearman's statistic test showed the low positive correlations between the frequency of bab genes' cDNAs (babA2 and babB) and other genes including cagA, cagA-EPIYAC, sab, hopQ, and alp genes' cDNAs in gastric biopsy samples (correlations coefficients 0.428, 0.343, 0.435, 0.462, and 0.397 with P � 0.012, 0.047, 0.01, 0.02, and 0.006, respectively). ere were the low positive correlation coefficients 0.357 and 0.362 (Spearman's static test) between the frequency of the hopQ gene's cDNA (hopQI and hopQII) and the frequency of cagA and cagE genes' cDNA in gastric biopsy samples, respectively (P � 0.038 and 0.035, respectively). ere was a negative correlation coefficient −0.344 (Spearman's static test) between the frequency of sab gene's cDNA (sabA and sabB) and the frequency of vacAs1m2 gene's cDNA (P � 0.046). ere were positive correlation coefficients 0.432, 0.460, 0.460, 0.440, and 0.406 (Spearman's static test) between the frequency of sab gene's cDNA (sabA and sabB) and frequency of cagA, cagA-EPIYAC, cagE, alp, and oip genes' cDNA in gastric biopsy samples (P � 0.011, 0.006, 0.006, 0.009, and 0.017, respectively). Our study showed that the alpA/B gene's cDNA's frequency had only a positive correlation of 0.440 with the frequency of sab gene's cDNA in gastric biopsy samples (P � 0.009). In sum, these

Pathological Characters of Gastric Carcinoma Biopsy
Samples. Table 5 shows the frequency of tumor regions in gastric carcinoma patients. e cardia region's frequency was 46.66% and the highest frequency compared to other tumor regions in gastric carcinoma, including the body, lesser curvature, and antrum regions. Pathology findings showed that out of 30 biopsy samples with gastric carcinoma, 11 cases (37.5%) had H. pylori infection. ese findings were similar to the urea breath test results and H. pylori 16s rRNA molecular detection. e present study results showed no significant difference statistically between gastric carcinoma tumor regions and H. pylori infection (P � 0.276). e absence of the alpA/B gene's cDNA in the body tumor region significantly differed from other gastric carcinoma tumor regions (P � 0.024). e frequency of G1, G2, G3, and G4 tumor grades in gastric carcinoma samples were 7 (23.33%), 14 (46.66%), 8 (26.66%), and 1 (3.33%), respectively. All cases of H. pylori infection were detected in patients with the G1 and G2 tumor stages. 63.63% of gastric carcinoma tumors with H. pylori infection were in the G2 tumor stage (P � 0.047). e frequency of oipA, cagT, and iceA genes' cDNAs was similar in gastric carcinoma samples with G1 and G2 tumor grades (P � 0.047) ( Table 6). is study showed a significant correlation statistically between the frequency of the cagY gene's cDNA and G2 tumor grade in gastric carcinoma samples (P � 0.007).

Discussion
Our study results showed some positive correlations and relationships among transcripts of H. pylori virulence genes detected in gastric biopsy samples of gastritis and gastric carcinoma patients with H. pylori infection. We surveyed statistical aspects of H. pylori virulence genes' cDNA's frequency in gastric biopsy samples and their relationship with clinical outcomes, including gastritis, gastric carcinoma, gastric carcinoma tumor region, and tumor grade.
Numerous studies have shown the correlation of H. pylori vacA s1 and cagA genotypes with clinical outcomes [27,[40][41][42][43][44][45][46]. On the other hand, some studies have shown the opposite effects of cagA and vacA on gastric epithelial cells [46]. Vacuolating cytotoxin A inhibits the production of hummingbird phenotypes in gastric epithelial cells that are created by CagA toxin [46]. e EPEYC motif of CagA protein via stimulating SHP-2 (Src homolog2 domaincontaining tyrosine phosphatase) phosphatase creates needle-like protrusions on the surface of epithelial cells that are called hummingbird phenotypes with a high rate of growth and proliferation rather than normal cells [46]. However, the results of some molecular epidemiology studies show that cagA + strains likely are vacAs1 + genotype simultaneously [46]. Our study results show an inverse correlation coefficient −0.465 (Spearman's static test) between the frequency of vacAs1m2 and cagA genes' cDNA in gastritis gastric carcinoma samples with H. pylori infection (P � 0.01).
Some studies show that H. pylori cagA + /vacAs1 + /babA2 + genotypes significantly correlate with peptic ulcers and gastric cancer [47]. babA gene expression in European strains of H. pylori is 40-70%, but in East Asian and American strains, it is 70-100% [7,21,43,44]. ere is a high homology between the nucleic acid sequences of 5 and 3 ends of the babA, babB, and babC genes, which lead to recombination among these three genes and consequently turn on or off gene expression [43,48]. e result of our study showed that the frequency of the bab gene's cDNA is 63.6% and 24.8% in gastric cancer and gastritis biopsy samples with H. pylori infection, respectively (Table 4). ere were positive correlation coefficients between the frequency of the bab gene's cDNA and cagA, cagA-EPIYAC, sab, hopQ, and alp genes' cDNA (P < 0.05). hopQI + genotypes are predominant in East Asia and are associated with cagA + genotypes, whereas hopQII + genotypes are predominant in West and Europa and have no association with cagA + genotypes [27,42]. Some studies have demonstrated HopQ adhesin's effects on the injection and entry of cagA protein into gastric epithelial cells in the company with T4SS [42]. e progress of gastric mucosa Table 7: e combined cDNA detected in gastric antral biopsy samples. Some samples had single cDNA that we did not mention. ese findings were collected of 23 and 11 gastric antral biopsies in gastritis and gastric cancer samples with H. pylori infection, respectively. ere were no significant correlations between H. pylori virulence genes' cDNA combinations and clinical outcomes (P > 0.05).    [7,25,26]. e results of some studies show that oipA gene expression is directly related to the expression of cagA and vacA genes [25,26]. Our study results show that the oipA gene's cDNA's frequency had a positive correlation coefficient of 0.406 with the frequency of the sab gene's cDNA (P � 0.017). Studies show that AlpAB lipoprotein adhesin in H. pylori Western strains leads to different cell signaling in gastric epithelial cells than Eastern strains [23]. e study results showed a positive correlation of 0.349 between the frequency of alp + /oip + /hopQ + cDNA and gastritis (P � 0.049).

alp/oip
As a remarkable result, there was a low frequency of cagA gene's cDNA in gastric biopsy samples with H. pylori infection versus gastric carcinoma biopsy samples (P � 0.008). Four samples of the cagA gene's cDNA in gastric carcinoma biopsy samples had an EPIYAC sequence (P � 0.029). e cagY and cagE genes' cDNA frequency had a significant difference statistically between gastric biopsy samples with H. pylori infection and gastric carcinoma biopsy samples with H. pylori infection (P � 0.008 and 0.002, respectively). We compared all frequency of cag pathogenicity island genes' cDNA between two groups of patients with gastritis and gastric cancer in this study (Table 7). e results showed there are positive correlations between cag genotypes and clinical outcomes. e genotypes with transcripts of cagA-EPIYAC + /cagT + /cagY + /cagE + had a positive strong correlation of 0.692 with gastric carcinoma (P � 0.005). On the other hand, the strains of H. pylori without any transcript of cag pathogenicity island genes and cagT + had a low positive correlation of 0.320 with gastritis (P � 0.065). Although H. pylori did not correlate with clinical outcomes, transcripts of cagY, cagE, oipA, and IceA genes' cDNA had a significant correlation statistically with G2 tumor grade in gastric carcinoma biopsy samples (Table 6). e gastric biopsy samples without any alp gene's cDNA correlated with the body tumor area in gastric carcinoma (P � 0.024) ( Table 5).
e results showed a remarkable difference in H. pylori virulence genes' expression between body area tumor and other areas tumor of the stomach. It can be due to differences between the origin and pathophysiology of H. pylori infection. In this regard, based on previous studies, most of the gastric cancers in patients with H. pylori infection are in the antral and cardia areas [49][50][51].
To explain the low frequency of some H. pylori virulence genes' cDNA in this study, we would say that multiplying control mechanisms reduce gene expression [52][53][54]. e two-components ArsRS system, which is affected by acidic pH, regulates H. pylori genes' expression, including sabA and cagA [52]. Changing the ORFs direction of cag pathogenicity islands' promotors leads to an unequal gene expression [31,32]. On the other hand, the molecular mechanisms such as single strand mispairing and the activity of nine types of methyltransferases regulate and limit H. pylori genes' expression.

Conclusion
e results' statistics analysis shows that some separate and combinatorial transcripts of H. pylori virulence genes are related to clinical outcomes.

Data Availability
e data used to support the findings of this study are included within the article.

Additional Points
(i) Transcript's profile of H. pylori virulence genes, a bacterium with a high level of genetic diversity, is remarkable in clinical strains of gastritis and gastric cancer patients (ii) is study shows a high H. pylori strain frequency in gastric antral biopsy samples of gastritis patients without the cagA gene's cDNA to assess cagA gene expression (iii) Evaluation of pathological features of gastric biopsy specimens with Helicobacter pylori infection shows positive correlation coefficients between the frequency of H. pylori virulence genes' cDNA and clinical outcomes, including gastritis, gastric adenocarcinoma, tumor grades, and gastric tumor area Ethical Approval e institutional review board has approved the study as no published patients' names were involved in the research project.

Disclosure
is study results from Doctor Manouchehr Ahmadi Hedayati' (Ph.D. of Medical Bacteriology) thesis.

Conflicts of Interest
e authors declare that they have no conflicts of interest.