Infection with a Hypervirulent Strain of Helicobacter Pylori Primes Gastric Cells Toward Intestinal Transdifferentiation

doi:10.21203/rs.3.rs-866653/v1

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Infection with a Hypervirulent Strain of Helicobacter Pylori Primes Gastric Cells Toward Intestinal Transdifferentiation

https://doi.org/10.21203/rs.3.rs-866653/v1

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published 01 Dec, 2021

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Background: Intestinal metaplasia, gastric-to-intestinal transdifferentiation, occurs as a result of the misexpression of certain regulatory factors, leading to genetic reprogramming. Here, we have evaluated the H. pylori-induced expression patterns of these candidate genes.

Methods: The expression levels of 1)tissue-specific transcription factors (RUNX3, KLF5, SOX2, SALL4, CDX1 and CDX2), 2)stemness factors (TNFRSF19, LGR5, VIL1) and 3)tissue-specific mucins (MUC5AC, MUC2) were evaluated by quantitative real-time PCR in gastric primary cells (GPCs), in parallel with two gastric cancer (MKN45 and AGS) cell lines, up to 96h following H. pylori infection.

Results: Following H. pylori infection of GPCs, RUNX3 declined at 24h post infection (PI) (-6.2±0.3) and remained downregulated for up to 96h. Subsequently, overexpression of self-renewal and pluripotency transcription factors, KLF5 (3.6±0.2), SOX2 (7.6±0.5) and SALL4 (4.3±0.2) occurred. The expression of TNFRSF19 and LGR5, demonstrated opposing trends, with an early rise of the former (4.5±0.3) at 8h, and a simultaneous fall of the latter (-1.8±0.5). This trend was reversed at 96h, with the decline in TNFRSF19 (-5.5±0.2), and escalation of LGR5 (2.6±0.2) and VIL1 (1.8±0.3). Ultimately, CDX1 and CDX2 were upregulated by 1.9 and 4.7-fold, respectively. The above scenario was, variably observed in MKN45 and AGS cells.

Conclusion: Our data suggests an interdependent gene regulatory network, induced by H. pylori infection. This interaction begins with the downregulation of RUNX3, upregulation of self-renewal and pluripotency transcription factors, KLF5, SOX2 and SALL4, leading to the downregulation of TNFRSF19, upregulation of LGR5 and aberrant expression of intestine-specific transcription factors, potentially facilitating the process of gastric-to-intestinal transdifferentiation.

Cancer Biology

Cell Communication and Signaling

Applied & Industrial Microbiology

Helicobacter pylori

Wnt signalling

intestinal metaplasia

gastric cancer

transdifferentiation.

Helicobacter pylori infects the majority of the population in the developing world ¹. This infection induces a number of complications, ranging from chronic active gastritis to peptic ulcers and gastric cancer (GC) ^2,3. Gastric cancer is the fourth most common cancer worldwide and ranks as the second leading cause of cancer-related deaths ⁴. Histologically, according to Lauren’s classification, intestinal type gastric cancer is one of the major subtypes of gastric adenocarcinoma with precancerous processes, clinical features and epidemiology, different from the diffuse subtype ^5,6. Intestinal type GC develops from a multi-step alteration process, which begins with superficial gastritis and can progress to chronic and then atrophic gastritis, leading to intestinal metaplasia (IM) and ultimately intestinal type GC ⁷. Accordingly, the annual incidence of GC, within the five years post diagnosis, is 2.5-fold higher in those with IM ⁸.

The gastric-to-intestine metaplasia is a postnatal ‘transdifferentiation” process, in which terminally differentiated gastric cells, are irreversibly replaced by adult intestinal cells ⁹. Both the stomach and the intestine originate from the same endodermal lineage in the early embryo ¹⁰, and their fate is decided by a gradient of induction factors and specific gene signatures for each tissue ¹¹. Normally, “master control genes” encoding transcription factors, control this development rather than a multitude of genes ¹². During gastric-to-intestinal transdifferentiation, a major alteration in gene expression, especially of tissue-specific transcription factors, result in switching off one set of downstream genes and switching on another ¹³.

On the other hands, there is a worldwide correlation between H. pylori infection and the incidence of GC, mostly of the intestinal subtype ¹⁴. Several pathways are involved in gastric carcinogenesis, amongst which the Wnt signalling pathway plays a fundamental role ¹⁵. This pathway is a critical regulator of the gastrointestinal development and maintenance, employing multiple arrays of molecules, which interact to adjust, activate or suppress different subsets of transcription factors, leading to stem cell identities and various differentiation fates ¹⁵. In other words, the Wnt signalling pathway maintains the homeostasis of the normal gastric epithelium by balancing stem cell maintenance and proliferation, the dysregulation of which plays a critical role in the development of more than 30% of gastric tumors and also observed in gastric cancer cell lines ^16–18. Numerous genes such as KLF5, TNFRSF19, LGR5, CDX1 and CDX2 are affected by the canonical Wnt signalling pathway ¹⁹. H. pylori is, indeed, a major disruptive factor in this pathway ²⁰. Notably, H. pylori, via the Wnt/β-catenin pathway, induces gastric stem cell generation and expansion, promoting gastric cancer initiation and progression. Through its oncogenic protein, CagA, H. pylori can induce aberrations in stemness gene expression, particularly leucine-rich repeat-containing G-protein coupled receptor (LGR)5-positive stem cell proliferation ²¹. Conversely, the expression of a member of the tumor necrosis factor receptor superfamily called TNFRSF19 ¹⁹, is believed to function as a negative modulator of the Wnt pathway, in particular on LGR5-positive stem cells ¹⁹. Another inhibitory molecule on this pathway is RUNX3, which is also modulated by H. pylori infection ²². Moreover, SOX2 is one of the key transcription factors in the foregut region known to promote stemness by upregulating genes involved in self-renewal and pluripotency, and suppressing those involved in the development of mature differentiated cells ²³. It has been reported that SOX2 is also overexpressed in cancer stem cells (CSCs) ²³. Finally, once the process of gastric IM occurs, the type of cell surface mucins will be altered to match the substituted (intestinal) tissue. Mucins are heavily glycosylated glycoproteins which constitute the major components of the protective mucous gel covering the surface epithelial tissues ²⁴. Immunohistology staining of the normal gastric mucosa shows cell type-specific expression of MUC1, MUC5AC, and MUC6, with first two mucins found in the superficial epithelium and MUC6 in the deep glands ²⁵. MUC2 is the main mucin of the intestinal mucosa ²⁶, and fully absent in the normal gastric epithelium ²⁷.

The goal of this study was to assess the regulation of the above-mentioned cascade of gene signatures during the course of time, following H. pylori infection of gastric primary cells (GPCs), as compared to two gastric cancer (MKN45 and AGS) cell lines. For this purpose, the three categories of tissue-specific transcription factors (RUNX3, KLF5, SOX2, SALL4, CDX1 and CDX2), the stemness markers (TNFRSF19, LGR5 and VIL1) and tissue-specific mucins (MUC5AC and MUC2) were traced over time post infection.

2.1. Baseline gene expression patterns over time

We, primarily, compared baseline expression of our target genes in two gastric primary cells from two different donors (GPCs1 and GPCs2), at two different time points (1st and 3rd months). The gene signatures of both cells remained consistent over time and were located in the same cluster as the normal gastric tissue. [Euclidean distances, (GPCs1:1st month: 15.4 and 3rd month: 15.1) and (GPCs2: 1st month: 17.3 and 3rd month: 17.2)] and apart from the duodenum or Tumor [Euclidean distances, (GPCs1:1st month: 20.5 and 3rd month: 20.8) and (GPCs2: 1st month: 23.7 and 3rd month: 23.1)]. Therefore, GPCs1 was selected for further studies and was referred to as GPCs, throughout the study (Supplementary Fig. 1). Next, we assessed baseline expression of the target genes in GPCs, MKN45, and AGS cells up to 96 h of culture. Accordingly, the baseline mRNA levels and relative gene expression for the three groups of 1) tissue-specific transcription factors (RUNX3, KLF5, SOX2, SALL4, CDX1 and CDX2), 2) stemness factors (TNFRSF19, LGR5, VIL1) and 3) tissue-specific mucins (MUC5AC, MUC2) were found to be consistent over time (Fig. 1A). According to their comparative gene expression profiles, the three cell lines behaved as follows: Of the first category of genes, SOX2, SALL4, and CDX1 were similarly expressed amongst the three cell lines. Whereas, RUNX3 and KLF5 were produced at lower and higher levels in AGS and GPCs, respectively (Fig. 1A). The levels of CDX2 expression were observed in the following sequence: AGS > MKN45 > GPCs. Of the second category of genes, GPCs and AGS cells were TNFRSF19 and VIL1 -high and -low producers, respectively and LGR5 was equally expressed amongst the three cell lines (Fig. 1A). In the category of mucins, GPCs were high producers, particularly of MUC5AC, followed by MKN45 and AGS cells, respectively (Fig. 1A).

A heatmap was depicted in order to classify the cells according to the proximity of their baseline gene expression profiles to the gastric versus intestinal origins (Fig. 1B). As a result, GPCs appeared closer to the normal gastric tissue (Euclidean distance of 3.82) and apart from the other two (AGS and MKN45) cells, as well as the tumor tissue (Euclidean distance of 8.22). Whereas, AGS and MKN45 cells appeared closer together (Euclidean distance of 1.09) and in the vicinity of the intestinal type tumor (Euclidean distance of 1.76), followed by the duodenal tissue. Therefore, due to their gastric nature, the GPCs were selected as the primary model system for assessment of time-dependent gene expression, following H. pylori infection, against which the corresponding profiles of the MKN45 and AGS cancer cell lines were compared.

2.2. Characterization of the hypervirulent H. pylori strain

The selected hypervirulent strain of H. pylori (C142LD) strain for cell culture infections, was identified as a gram-negative, helically-shaped, urease, catalase, and oxidase-positive bacterium, which was positive for H. pylori ureC (glmM) gene (Fig. 2A). Its cagA, vacA and babA genotypes were determined as cagA-positive (ABCCC-type), vacA s1m1i1 and babA AA type, respectively (Fig. 2A). This strain expressed the CagA/VacA/BabA gene products (Fig. 2B) and was able to induce cellular vacuolation and the hummingbird phenotype as early as 24h PI, which intensified at 48h PI (Fig. 2C). Therefore, the C142LD strain was considered as hypervirulent ²⁸.

2.3. Characterization of H. pylori coculture

During the co-culture assay, the interactions between the hypervirulent H. pylori strain and gastric cells, were investigated by the following assays: 1) induction of morphologic changes: vacuolation and hummingbird phenotypes were detected in GPCs, as well as AGS, and MKN45 cells (Supplementary Fig. 2A), at 24h PI, 2) the effect of H. pylori infection on cell viability was examined during the entire assay (0-96h). Accordingly, in all three cells, the OD₄₅₀ increased over time, which was a sign of cells growing during the course of the experiment (Supplementary Fig. 2B), 3). At 24h PI, IL-8 production was detected in GPCs (529.8 ± 5.4 pg/10⁶ cells) ²⁹, MKN45 (883.5 ± 12.7 pg/10⁶ cells) and AGS (302.2 ± 9.7 pg/10⁶ cells), as compared to uninfected controls (P < 0.0001, Supplementary Fig. 2C).

2.4. Gene signatures following H. pylori infection

The expression patterns of our three categories of target genes (tissue-specific transcription factors, stemness markers and tissue-specific mucins), suspected to be involved in the process of gastric-to-intestinal transdifferentiation were then assessed, in GPCs, as the primary model, against which the corresponding profiles of two gastric cancer (MKN45 and AGS) cell lines were compared, up to 96h following H. pylori infection.

2.4.1. Tissue-specific transcription factors

H. pylori infection of GPCs, resulted in an early reduction in RUNX3 and a significant escalation of KLF5, SOX2 and SALL4 genes expression, in reference to untreated cells (Fig. 3). In other words, following a sudden peak at 8h PI (2.9 ± 0.3, P < 0.0001), the expression level of RUNX3 declined by more than 7 times (-6.2 ± 0.3, P < 0.0001), at 24h PI and despite an increasing trend in 48h (-1.9 ± 0.3, P < 0.0001) to 96h (-0.8 ± 0.5, P < 0.0001), as compared to 24h PI, it remained significantly downregulated, throughout the experiment as compared to uninfected cells (Fig. 3A). In contrast, the expression levels of KLF5 increased and peaked at 8h PI hours (3.6 ± 0.2, P < 0.0001), followed by SOX2 (7.6 ± 0.5, P < 0.0001) and SALL4 (4.3 ± 0.2, P < 0.0001) at 24h PI, all of which remained elevated for up to 96h, in reference to untreated cells (Fig. 3B-D). In parallel, a small peak of CDX1 expression was detected at 8h PI (2.0 ± 0.4, P < 0.0001), which dropped to baseline, at 24h and remained there until another slight rise at 96h PI (1.9 ± 0.1, P = 0.004, Fig. 3E). Alongside CDX1, CDX2 gene expression developed a rising trend, at 48h PI (2.5 ± 0.3, P < 0.0001) and reached nearly 5 fold upregulation, at 96h (4.7 ± 0.1, P < 0.0001), as compared to the uninfected cells (Fig. 3F).

The expression pattern of RUNX3 in H. pylori-infected MKN45 cells similar to GPCs, demonstrated a decrease, but at a much smaller scale (Fig. 3A). It, then, returned to near baseline expression at 48-96h PI (Fig. 3A). Very similar to the pattern of KLF5 expression in GPCs, that of MKN45 also had a rising trend, with an initial peak at 8h, followed by a steady state of expression at 24h to 96h PI (Fig. 3B). The same occurred for SOX2 expression, with an early peak at 8h, which was nearly double that of GPCs, at that time point. But then, declined at 24h. Nevertheless, it remained upregulated up to 96h PI (Fig. 3C). This increasing pattern was also observed for SALL4 expression in H. pylori-infected MKN45 cells, as compared to uninfected cells, throughout the experiment. However, initially there was an upward trend similar to GPCs, but then unlike GPCs, it took a downward trend (Fig. 3D). In terms of the intestinal transcription factors, the expression of CDX1 in MKN45 was highly upregulated, compared to uninfected cells, throughout the experiment and maintained a rising trend (Fig. 3E). The expression of CDX2 in these cells was also upregulated (but to a lesser degree than CDX1), as compared to uninfected cells throughout the experiment (Fig. 3F). Except for the initial peaks, the observed rising trend was quite similar to that of GPCs.

The expression of RUNX3 in H. pylori-infected AGS cells, unlike GPCs, declined early post infection, and returned to near baseline expression at 48 -96h (Fig. 3A). Very similar to the pattern of KLF5 expression in GPCs and MKN45 cells, that of AGS cells also had a rising trend, with an initial peak at 8h, followed by a steady state overexpression at 24h to 96h PI (Fig. 3B). The expression of SOX2 in AGS cells, was upregulated, compared to uninfected cells, throughout the experiment but, unlike GPCs, had an overall reducing trend. A 1.5 times initial overexpression of SOX2, compared to that of GPCs, at 8h PI, was observed, followed by a decreasing trend at 24-96h PI (Fig. 3C). The same occurred for SALL4 expression, with an early peak at 8-24h PI, which was 1–4 times that of GPCs. But then, declined at 48-96h PI (Fig. 3D). The expression of the intestinal transcription factors, CDX1, in AGS had an early fall and a late peak, which was almost double that of GPCs, at 96h PI (Fig. 3E). The expression of CDX2, however, in AGS cells had a rising trend, throughout the experiment, which nearly coincided with that of GPCs at 48-96h PI (Fig. 3F).

2.4.2. Stemness markers

In GPCs, the expression of the stemness markers, in particular TNFRSF19 and LGR5, demonstrated an opposing trend, during the time course of H. pylori infection (Fig. 3G-H). In other words, the former depicted an early rise (4.5 ± 0.3, P < 0.0001, Fig. 3G) at 8h PI, coinciding with a simultaneous fall of the latter (-1.8 ± 0.5 at 8h, P < 0.0001, down to -11.1 ± 0.7 at 24h, P < 0.0001, Fig. 3H). Whereas, this trend was reversed at the latest time point (96h PI), such that the decline in TNFRSF19 (-5.5 ± 0.2, P < 0.0001, Fig. 3G) coincided with the escalation of LGR5 (2.6 ± 0.2, P < 0.0001, Fig. 3H). Throughout this time, expression of VIL1 experienced two separate peaks at 24h (2.7 ± 0.2, P < 0.0001) and 72h (2.4 ± 0.4, P < 0.0001), as compared to the untreated cells (Fig. 3I).

The expression of TNFRSF19 in H. pylori-infected MKN45 cells, similar to that of GPCs, was biphasic, with an early peak and a late drop, as compared to uninfected cells (Fig. 3G-H). LGR5 expression, however, was mostly up-regulated, with two peaks at 8h and 72-96h PI (Fig. 3H). Similar to GPCs, however, the final peak in LGR5 in MKN45 cells at 96h PI, coincided with the down-regulation of TNFRSF19 (Fig. 3G-H). The expression of VIL1 in MKN45 cells, on the other hand, contrasted with its early peak in GPCs, though coinciding with the late peak at 96h PI (Fig. 3I).

TNFRSF19 expression in H. pylori-infected AGS cells was upregulated, as compared to uninfected cells throughout the experiment, which was similar to that of GPCs, but with lower extent, up to 72h PI, after which unlike that of GPCs, it remained upregulated up to 96h (Fig. 3G). LGR5 expression, in AGS cells, in contrast to that of GPCs, was upregulated at the beginning, but returned to baseline by the 96h (Fig. 3H). The expression of VIL1 in AGS cells, also remained upregulated throughout the experiment, which was similar to that of GPCs, at 24 and 72h PI (Fig. 3I).

2.4.3. Tissue-specific mucins

The expression patterns of gastric-specific (MUC5AC) and intestine-specific (MUC2) mucins of H. pylori-infected GPCs are illustrated in Fig. 3J-K, which indicate a significant peak for MUC5AC expression, following 24h PI (2.9 ± 0.2, P < 0.0001), which declined by the end (at 96h, 1.4 ± 0.2, P = 0.5, Fig. 3J). This coincided with a sharp decline in MUC2 (-5.2 ± 0.2 at 8h PI), which picked up at 96h (-0.3 ± 0.5, P < 0.0001, Fig. 3K) post infection, though remained below baseline.

The expression of MUC5AC, in H. pylori-infected MKN45 cells, was very similar to that of GPCs. Such that it was up-regulated throughout the experiment, although with a much higher intensity than in GPCs (Fig. 3J). MUC2 expression in MKN45 cells, however behaved the exact opposite of GPCs, with the upregulation in the former versus down-regulation in the latter cell line (Fig. 3K).

The expression of MUC5AC in H. pylori-infected AGS cells, similar to that of GPCs, had an early rise at 8h PI, but underwent a drastic fall at 48h, which picked up again at 72-96h PI, but remained below baseline (Fig. 3J). As for MUC2 expression in AGS cells, it followed the same direction as that of GPCs, except that it crossed the baseline at 48h PI, and remained overexpressed up to 96h PI (Fig. 3K).

2.5. Time-course of gene expression

In order to better understand the patterns and trends of gene expression following H. pylori infection in each cell line, we defined gene expression patterns, as “altered” if variations occurred at > 100% higher or lower, than that of the uninfected cells, as presented in the collective gene signatures of each cell line, during early (0–24 h) and late (24-96h) stages post infection (Fig. 4). Amongst, tissue-specific transcription factors, the expression of RUNX3, was consistently and strongly downregulated in GPCs (Fig. 4A). But in MKN45 and AGS cells, its underexpression occurred, only early after infection, with lesser intensity in both cancer cell lines (Fig. 4B-C). In contrast, KLF5, SOX2 and SALL4 were consistently upregulated in all three cell lines, throughout the experiment (Fig. 4A-C). As for intestinal type transcription factors, CDX1 was strongly upregulated in MKN45 (early and late, Fig. 4B) and AGS (late, Fig. 4C) cell lines and to a much lesser degree in GPCs (early, Fig. 4A) post infection. CDX2 however, was strongly over-expressed, in all 3 cell lines, late post infection (Fig. 4A-C). In the stemness marker group, TNFRSF19 was first upregulated in GPCs and MKN45, then down regulated (Fig. 4A-B). This coincided with an opposite trend for LGR5 in GPCs (throughout, Fig. 4A) and late after infection for MKN45 cells (Fig. 4B). However, these two stemness factors (TNFRSF19 and LGR5) were both upregulated in AGS cells, albeit still an alternating sequence (Fig. 4C). As for VIL1, it was mostly upregulated in GPCs and AGS cells (Fig. 4A & C). But in MKN45 cells, it was initially downregulated, then upregulated (Fig. 4B). Regarding tissue-specific mucins, the expression of MUC5AC and MUC2 had opposing trends in GPCs and AGS cells, although in reverse order. Whereas, they were both extensively upregulated in MKN45 cells (Fig. 4A-C).

2.6. Comparative gene expression signatures in gastric primary cells (GPCs) vs. cancerous cell lines (MKN45 and AGS)

The above-stated results indicate similarities and differences, in their pattern of gene expression, following H. pylori infection, amongst gastric primary cells, as our model system, in reference to gastric cancer cell lines. The results of the heatmap and correlation clustering showed that GPCs’ gene expression signature starts near the profile of normal gastric tissue early (8-24h) after infection, and moves toward that of the intestinal type tumor tissue, late after H. pylori infection (Fig. 5A). As a result, GPCs at 48-96h PI appeared closer to the intestinal type tumor (Euclidean distances: 11.7, 14.2 and 12.1) and apart from the normal gastric tissue (Euclidean distances: 19.5, 21.5 and 22.8). On the other hand, MKN45 (Euclidean distance: 14.1, 12.1, 11.3, 11.5 and 12.3) and AGS (Euclidean distances: 9.9, 9.9, 9.7, 10.4 and 12.7) cells, at both early (8-24h PI) and late (48-96h PI) stages post infection, remained as united clusters and in the vicinity of intestinal type gastric tumor (Fig. 5B-C).

The hypothesis of this study was that in the process of gastric-to-intestinal transdifferentiation, due to H. pylori infection, a terminally differentiated gastric cell undergoes a reduction in gastric-specific and an escalation in progenitor/intestine-specific factors, respectively.

H. pylori infection, in the harsh acidic milieu of the stomach, induces a cascade of pathologic changes, to which a series of virulence factors contribute ^28,30. These predominantly include CagA ^31–33, VacA ³⁴ and BabA adhesin ^35,36. Such that patients infected with cagA+, s1m1, babA2 + H. pylori, are at more than 23 fold increased risk of severe gastrointestinal complication, as compared to cagA-, s2m2, babA2- strains ³⁰, and the risk of gastric cancer in the former group is 6.4 times higher than that in the latter ²⁸. In our study, we have used a cagA+ (ABCCC-type), vacA s1m1i1 and babA AA, hypervirulent H. pylori strain, which was also capable of inducing cellular elongation and vacuolation, as well as IL-8 production. This hypervirulent strain was co-cultured with gastric primary cells (GPCs) and two commonly used (MKN45 and AGS) cancer cell lines, for 8, 24, 48, 72, and 96 hours and the expression profiles of the following 3 categories of genes were assessed: 1) tissue-specific transcription factors (RUNX3, KLF5, SOX2, SALL4, CDX1 and CDX2), 2) stemness factors (TNFRSF19, LGR5, VIL1) and 3) tissue-specific mucins (MUC5AC, MUC2).

First and foremost, to understand the essence of the studied cells, we examined their gene expression and clustered them by drawing gene expression charts and heat maps. Of these cells, the non-transformed (non-cancerous) primary cells (GPCs ²⁹) clustered with the normal gastric tissue. Whereas, the essence of MKN45 and AGS cells was closer together and clustered with the intestinal type gastric tumor and duodenal tissue. In recent years, several attempts have been made to elegantly build a suitable model for in vitro infection of H. pylori, ranging from one-dimensional gastric cancer cell lines ³⁷ to three-dimensional, patient-derived organoids ³⁸. The former group include MKN45 ³⁹ and AGS ⁴⁰ cancer cell lines, which are already transformed, as manifested by their prominent expression of intestine-specific genes, i.e. CDX2, MUC2, TFF3 ⁴¹, VIl1, LGALS4 and LI-cadherin ⁴². By definition, these cancer cell lines have already undergone gastric-to-intestinal transdifferentiation (transformation), evidenced by the down regulation of p53 gene, CDK2 and G1 cyclins expression, homozygous deletion of the p16 and p15 genes or p27 gene rearrangement, promoter mutation of E-cadherin, etc. ^42,43. According to these reports and in line with our results, gene expression patterns of these cancerous cell lines (MKN45 and AGS) are closer to the intestinal type tumor and the duodenum, rather than the normal stomach ⁴². Therefore, assessing alteration in gene expression patterns, subsequent to H. pylori infection in gastric primary cells (GPCs), relative to these cell lines, may provide a more realistic insight.

Based on the behaviour of GPCs, the obtained data suggest an interdependent gene regulatory network, induced by H. pylori infection. This interaction begins with the downregulation of RUNX3, upregulation of self-renewal and pluripotency transcription factors, KLF5, SOX2 and SALL4, leading to the downregulation of TNFRSF19 and upregulation of LGR5 and aberrant expression of intestine-specific transcription factors, particularly CDX2, thereby facilitating the process of gastric-to-intestinal transdifferentiation.

This network of interactions is activated by H. pylori infection, which affects the Wnt pathway in three aspects. It primarily lifts the inhibitory role of RUNX3, by causing its gene hypermethylation (inactivation) and mislocation ⁴⁴. Secondly, it disrupts the E-cadhein/β-catenin interaction between epithelial cells, by injecting CagA oncoprotein into the cells and causing excessive accumulation of β-catenin in the cytoplasm, part of which enters the nucleus ⁴⁵ and transcribes the Wnt responsive genes, such as cell cycle control, pluripotent ⁴⁶, and self-renewal genes of cancer and stem cells ⁴⁷ and ultimately, aberrant expression of CDX1 and CDX2 and occurrence of intestinal metaplasia ⁴⁸. Since, we have, herein, used a hypervirulent strain of H. pylori, the reduction in RUNX3 gene expression in GPCs was clearly observed. This phenomenon was also observed in MKN45 and AGS cancer cell lines, but with less intensity coinciding with or earlier than GPCs, respectively, which returned to baseline expression at late time points. This observation may be due to the earlier onset of RUNX3 down regulation in these two cells, as they have already experienced this process in their previous malignant transformation, as evidenced by the cytoplasmic localization of RUNX3 in MKN45 ⁴⁹ and its inactivation in AGS ⁵⁰.

A recent study on 192 GC patients, whose tumor tissues were analysed by methylation-specific PCR and IHC, demonstrated significant RUNX3 promoter hypermethylation (40.6%, 78/192) and subsequent protein underexpression (51.65%, 99/192) ⁵¹. This pattern was found closely associated with H. pylori infection ⁵¹. In addition, a study of 154 healthy volunteers revealed that RUNX3 CpG island methylation was significantly higher (5.4 to 303-fold) in H. pylori-positive versus negative subjects ⁵². On the other hand, several meta-analyses have confirmed the close association between RUNX3 gene downregulation and gastric cancer development ^53–55 and disease progression ⁵⁶. How H. pylori manages to downregulate RUNX3 gene expression may be due to its indirect activation of DNA methyltransferases via inflammatory mediators ⁵⁷, and/or the direct effect of CagA ⁵⁷. Nevertheless, when RUNX3 is downregulated, its inhibitory role is lifted and aberrant activation of the Wnt signalling pathway, leading to spontaneous epithelial-mesenchymal transition (EMT) and production of tumorigenic stem cell-like subpopulations, occurs ⁵⁸. In line with this scenario, we observed SOX2, SALL4 and KLF5, upregulation, at early time points, which mostly remained as such, in the other two cell lines, albeit to a lesser extent, throughout the experiment.

SOX2 expression plays an important role in regulating tissue development and cell differentiation, as this factor is highly expressed in the foregut region, during embryonic development, giving rise to the stomach and generating the boundary between the posterior stomach and the proximal intestine ⁵⁹. Generated Sox2-GFP indicator mice from embryonic stem cells (ESCs) reveal that, in adulthood, SOX2 + cells are located in the base of the pyloric and corpus glands, capable of generating surface mucous, chief, parietal, and enteroendocrine cells of the gastric units ⁶⁰, ablation of which results in the disruption of the physiological renewal of the gastric epithelium ⁶⁰. The role of SOX2, in gastric cancer development, however, remains obscure. Studies can be categorized in three groups, as those having provided evidence in support of ^61–65 or against ^65,66 its role in gastric carcinogenesis, as well as those which have found no significant association ⁶⁷. In particular, analysing normal gastric mucosae, intestinal metaplasia and tumor tissues of 68 gastric carcinoma patients by IHC, showed SOX2 as moderately expressed in sites with intestinal metaplasia ⁶². In the gastric tumors, however, SOX2 is mainly expressed at sites with high proliferation rates ⁶⁸. Therefore, SOX2 is considered functionally active in cancer stem cells, maintaining their self-renewal capacity ⁶⁸. Considering these results, the elevated expression of SOX2 in GPCs, may primarily be due to the formation of a subpopulation of progenitor cells, further supported by the simultaneous/subsequent upregulation of SALL4 and KLF5 expression. SALL4 ⁶⁹ and KLF5 ⁷⁰ are considered as oncogenes and distinguished as stemness-related reprogramming factors. SALL4 is aberrantly expressed through the Wnt/β-catenin pathway in several human malignancies, such as oesophageal squamous cell carcinoma ⁷¹, osteosarcoma⁷², leukaemia ⁷³, and gastric cancer ⁷⁴. Its expression is closely correlated with a poor outcome and resistance to therapy ⁷¹. SALL4 expression in the normal stomach is limited to the proliferating and stem cells areas, though its expression in gastric tumor tissues is also detected ⁷⁵. Likewise, the expression of KLF5 increases in the process of gastric carcinogenesis, and is particularly detected in tissues with intestinal metaplasia and dysplasia ⁷⁶. This transcription factor is critical in maintaining the integrity of intestinal stem-cells ⁷⁷. Expression of these two stemness-related reprogramming factors indicates the emergence of a subpopulation of progenitor cells, which may play an intermediary role in the gastric-to-intestinal transdifferentiation process. H. pylori infected MKN45 and AGS cancer cell lines also showed initial KLF5, SOX2 and SALL4 overexpression, which remained elevated throughout the experiment, respectively. Studies show that MKN45 and AGS cells contain large populations of cancer stem cells capable of spheroid and tumor formation in vitro and in vivo ^78,79. Thus, their initial higher expression of KLF5, SOX2 and SALL4 may be due to the presence of larger starting population of cancer stem cells in MKN45 and AGS cells, prior to infection.

On a different note, using immunohistochemistry, we have previously reported an inverse trend between TNFRS19 downregulation and LGR5 upregulation in gastric cancer tumours in humans, as well as in response to H. pylori infection in mice ⁸⁰. Other studies also support a potential negative modulatory role for TNFRSF19 on LGR5 expression ¹⁹ and the Wnt signalling pathway ⁸¹. For instance, TNFRSF19 overexpression in MKN45 cells, leads to their decreased clonal expansion ⁸¹. LGR5 is a stem cell surface receptor and gastric LGR5 + cells have a structure similar to the undifferentiated stem cell population, with large nuclei, limited rough endoplasmic reticula and absence of secretory granules ⁴⁶. H. pylori is known to affect gastric precursor cells, leading to increased proliferation and expression of LGR5 + cells ²¹, which is higher prevalent in the gastric tumor than the non-cancerous surrounding tissues, as a marker of dedifferentiation ⁸². Furthermore, it has been revealed that LGR5 is considered as a marker of intestinal stem cells ⁸³, and its overexpression at the end of our experiment may due to the appearance of an intestinal progenitor subpopulation. We have, herein, observed that a reduction in TNFRSF19 coincided with the escalation of LGR5, at the latest time point in GPCs. A similar pattern was also observed for H. pylori-infected MKN45 cells. This inverse trend between TNFRSF19 and LGR5, was also slightly observed in the consistently upregulated AGS cells. In regards to the development of intestinal progenitor subpopulations, Villin, which is the building block of the intestinal microvilli ⁸⁴ and has also been designated as an indicator of dormant gastric stem cells ⁸⁵, is also under focus. Our result showed that VIL1 gene expression experienced late peaks, in all three cell lines, which further conforms with this concept.

Finally, in the gastric to intestinal transdifferentiation process, it is ultimately expected that the intestine-specific transcription factors undergo upregulation. Accordingly, we observed that both CDX1 and CDX2, had an increasing trend in GPCs, as well as MKN45 and AGS, but with varying patterns. The expression of CDX1 peaked but declined in GPCs, early and late after infection, respectively. Whereas, in MKN45 cells this rise began early and persisted up to late stages, when, AGS cells joined in. The Caudal homeobox gene family is responsible for differentiating the endoderm into the posterior endoderm and are distinguished as intestine-specific transcription factors ⁸⁶. The activities of the two CDX1 and CDX2 transcription factors are limited to the middle and posterior intestinal region ⁸⁷ and both are essential in the regulation of the intestinal cell proliferation and differentiation ⁸⁸. It has also been shown that the undifferentiated cells of the intestinal crypts express CDX1 predominantly, whereas the differentiated cell of intestinal villi, mostly express CDX2 ⁸⁸. On the other hand, the aberrant expression of CDX1 is detected in areas of the stomach and oesophagus affected by intestinal metaplasia ⁸⁹. Studies have shown that the Wnt signalling pathway regulates both CDX1 ⁹⁰ and CDX2 ⁹¹ and that H. pylori increases their expression ^92,93. Given these findings and the results obtained herein, it seems that following infection with a hypervirulent strain of H. pylori and activation of the Wnt pathway, the progenitor subpopulations initiate the gastric-to-intestinal transdifferentiation process, by aberrant expression of CDX1 and CDX2. In GPCs, with normal gastric nature, undifferentiated intestinal subpopulations expressing CDX1 and differentiated intestinal subpopulation expressing CDX2, appeared at early and then late time points, respectively. Whereas in MKN and AGS ells, with cancerous and mostly intestinal nature, a mixture of CDX1 expressing undifferentiated and CDX2 expressing differentiated cells are formed earlier or at the final stages, respectively.

According to our hypothesis, the above interdependent network of gene expression would supposedly translate into downregulation of gastric-specific (MUC5AC) and upregulation of intestine-specific (MUC2) mucins. However, in GPCs, we observed a consistent upregulation of the former and downregulation (yet with a rising trend) of the latter. In MKN45 cells, both MUC5AC and MUC2 were upregulated throughout the experiment. In AGS cells, an opposing trend was observed in the expression of these two tissue-specific mucins. As the colonization of Le^b-binding H. pylori strains, including our herein used hypervirulent strain, is dependent on MUC5AC ⁹⁴, the upregulation of this receptor upon infection may be induced for better colonization. It is also hypothesized that, injection of CagA into gastric epithelial cells triggers an array of molecular cascades ⁹⁵ leading to transient escalation in MUC5AC expression ⁹⁶. Accordingly, we observed an initial peak in MUC5AC expression, in all three cell lines. AGS cells, however, was the only cell line in which the previously expected pattern occurred, namely MUC5AC downregulation coincided with MUC2 upregulation. In support of the observed pattern in AGS cells, a meta-analysis of 7 case-control studies (1997–2012) concluded that the presence of H. pylori decreases gastric epithelial expression of MUC5AC, by more than half ⁹⁷ and its eradication restores its levels to some degree ⁹⁸. MUC5AC downregulation was also evident in gastric preneoplastic lesions, including areas with atrophic gastritis, intestinal metaplasia ^27,99 and dysplasia ⁹⁹. As for MUC2, immunohistochemical and RNA northern and slot-blot analysis of normal and neoplastic human tissues confirmed its lack of expression in normal gastric epithelium, thereby restricting it to the intestines ¹⁰⁰. Consequently, the level of MUC2 is substantially increased, in intestinal metaplasia and intestinal type gastric cancer ²⁷, particularly following H. pylori infection ¹⁰¹. Histopathological and histochemical studies have divided intestinal metaplasia into two types: 1) complete or type I, which is characterized by the presence of absorptive, Paneth, and goblet cells and corresponds to the small intestine phenotype and 2) incomplete or types II and III, which are characterized by the presence of columnar and goblet cells ¹⁰². It has been shown that in type I intestinal metaplasia “gastric” mucins (MUC1, MUC5AC, and MUC6) are decreased, while MUC2 is aberrantly expressed. In contrast in types II and III intestinal metaplasia, “gastric mucins” (MUC1, MUC5AC, and MUC6) are co-expressed with “intestinal” mucin (MUC2) ¹⁰³. Hence, our results may suggest that following H. pylori infection of MKN45 cells, the upregulation of both MUC5AC and MUC2 may represent incomplete or types II/ III of intestinal metaplasia cells, in which copresence of intestinal differentiated cells, columnar and goblet cells, are expected. In contract in AGS cells, the inverse trend of MUC5AC/MUC2 expression, may be due to the formation of complete or type I intestinal metaplasia.

In summary, GPCs as a non-cancerous primary cell culture model for H. pylori infection ²⁹ seems to undergo transdifferentiation by downregulating RUNX3 and TNFRSF19, up-regulating self-renewal and pluripotency transcription factors (SOX2, KLF5 and SALL4), which then lead to the up-regulation of LGR5 and aberrant expression of intestine-specific transcription factor, particularly CDX2. Aberrant expression of MUC2 did not occur in these cells, during the time course of our experiment and might appear in prolonged cocultures. In contract, MKN45 and AGS cancerous cells, infected with H. pylori may manifest the process of transdifferentiation by upregulation of self-renewal and pluripotency transcription factors, due to their prior downregulation of RUNX3. In both cancer cell lines, the upregulation of LGR5 and aberrant expression of CDX2 is observed. However, in MKN45 cells, the simultaneous upregulation of MUC5AC and MUC2 may represent incomplete types (II and III) intestinal metaplasia, whereas in AGS cells, their inverse trend may manifest the appearance of the complete type (I). In other words, upon H. pylori infection, the differentiated gastric cells in GPCs, seem to develop into naïve progenitor cells, which later take on the nature of intestinal progenitor cells. But as expected and in contrast to GPCs, in the two H. pylori- infected gastric cancer (MKN45 and AGS) cell lines, less naïve and intestinal progenitor cells and more intestinally differentiated cells become expanded. Thus, depending on the target gene of interest, and its potential therapeutic manipulations, the herein illustrated time course of gene expression, in three different cell lines, following H. pylori infection, will allow for an educated choice of cell line and time point, for future study designs.

4.1. Cell culture and tissue specimens

For this study, two gastric primary cells from two different donors²⁹ and two gastric cancer cell lines (MKN45 and AGS) were used. GPCs were developed as previously described ²⁹. Briefly, gastric tissues were obtained from H. pylori-negative consenting donors, with normal gastric mucosa, under endoscopy at Amiralam Hospital (Tehran, Iran). The tissue specimens were minced, enzymatically digested and used for gastric primary culture on mouse embryonic fibroblast (MEF). GPCs were expanded in advanced DMEM/F12 (Gibco, USA), supplemented with growth factors and small molecules (2-dimensional medium) ^29,104. AGS and MKN45 gastric cancer cell lines were obtained from the Iranian Biological Resource Center (IBRC) cell line collection (Tehran, Iran). Both cell lines were seeded and expanded in DMEM/F12 medium (Gibco, USA), supplemented with 10 mM L-glutamine, 10% (v/v) fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin and incubated at 37°C in a humidified incubator with 5% CO₂, for the duration of each experiment. All tissue specimens, including those of the gastric tumor, its normal counterpart and duodenum of a GPCs donor, were collected via gastroscopy. All experiments and methods were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by a named institutional/licencing committee. Specifically, human gastric tissue specimens were approved by the Committee on Ethical Issues in Medical Research, Pasteur Institute of Iran (IPI); Ref No. IR.PII.REC.1394.57. (Tehran, Iran). Informed consent was obtained from all subjects, and all methods were carried out in accordance with the relevant guidelines and regulations of IPI Ethics Committee.

4.2. Bacterial strain culture and characterization

4.2.1. H. pylori culture and isolation

An H. pylori (C142LD) strain, originally isolated from a gastric cancer patient, was selected for this study. As previously described ¹⁰⁵, the C142LD strain was cultured on H. pylori Special Peptone Agar (HPSPA) plates, and incubated at 37°C under microaerophilic conditions (10% CO₂, 5% O₂ and 85% N₂), for 7–10 days. The grown strain was confirmed as H. pylori by routine microbiological assays, including gram staining, urease, catalase and oxidase tests. Sweeps of grown bacteria were serially subcultured, in order to obtain single colonies.

4.2.2. Genomic DNA extraction and PCR

Whole genomic DNA extraction from C142LD H. pylori strain was carried out, using DNA Mini Kit (Qiagen DNeasy Blood & Tissue Kit, Germany), following the manufacturer’s recommendations. The extracted DNA was, then, checked for the presence of glmM, cagA, vacA, and babA/B genes by gene-specific PCRs (Supplementary Table-1).

4.2.3. Protein expression assays

The C142LD strain was evaluated for the expression of CagA, VacA, and BabA proteins, according to the previously published methods ¹⁰⁶. In brief, plates of full-grown bacteria were washed with PBS and centrifuged at 6000 rcf for 5 min, followed by resuspension in 500 µl reduced Laemmli sample buffer (Biorad, USA) and incubated at 100°C for 10 minutes. The samples were then centrifuged at 3000 rcf for 5 minutes. Bacterial lysates (70 µg/strain) were applied on 10% SDS-PAGE and analyzed by silver nitrate staining. Western blotting was performed using 1:1000 dilution of first antibodies (CagA- and VacA-specific polyclonal antibodies, Austral Biologicals, USA) and gift BabA antibody (generously provided by Prof. Thomas Boren, University of Umea, Sweden), all of which were detected by horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:5000, Dako, Denmark). Blots were developed by DAB (3,3-diaminobenzidine) solution (Sigma, USA).

4.4. H. pylori liquid culture

The C142LD strain was expanded in liquid culture, according to a previously optimized protocol ¹⁰⁷. In brief, a single colony of C142LD strain was grown in Brucella broth (Difco, USA), supplemented with 0.2% β-cyclodextrin (Fluka, USA), under microaerophilic conditions and continuous shaking at 120 rpm, for 24 hours at 37°C. When the turbidity of the bacterial suspension reached an optical density of ~ 1 unit at 550 nm, bacteria were collected by centrifugation (3000 g, for 15 minutes). The viability and possible contamination of the collected bacteria were checked, by wet mount, under light microscopy and cultured onto blood agar plates for 3 days, respectively.

4.5. Co-culture assays

Twenty-four hours prior to H. pylori infection, the culture media on GPCs (antibiotic-free 2-dimensional ^29,104), MKN45 and AGS cells (DMEM/F12) were refreshed. On the day of the experiment, the liquid culture-harvested C142LD H. pylori was washed with phosphate-buffered saline (PBS), resuspended in each cell-specific growth medium, and diluted to a final concentration of 1 × 10⁸ CFU/mL. The viability of H. pylori cells was monitored by Dil (ThermoFisher) live staining, according to manufacturer’s instructions. In parallel, the cells (GPCs, MKN45, and AGS) were rinsed with PBS and fresh media were added. Thereafter, the bacterial suspension was added to the cell cultures (in triplicates), at the multiplicity of infection (MOI) of 1:1, for up to 96h. The MOI was determined according to approximate cell counts, which were based on standard curves, using a nontoxic cell counting solution (Orangu™; Cell Guidance System, UK). The cultures of infected and uninfected cells were sampled at 6-time intervals (0, 8, 24, 48, 72, and 96h) post infection.

4.6. Vacuolation and hummingbird assay

The vacuolation and hummingbird assays were performed to determine: 1) the functional activity of the C142LD H. pylori strain on AGS cell line and 2) investigation of GPCs, MKN45 and AGS cellular changes up to 96h, following H. pylori infection. The morphology of the cells was examined, using an inverted light microscope. Observation of vacuole formation in more than 50% of the cells, in 10 different microscopic fields at ×40 magnification, was defined as a positive vacuolation phenotype ¹⁰⁸. An elongated hummingbird phenotype (the ratio of the most extended protrusion to the shortest protrusion of greater than 2), was also scored in 10 different microscopic fields at ×40 magnification and the average elongation length was measured and documented ¹⁰⁹. H. pylori-untreated cells were assessed as negative controls.

4.7. IL-8 secretion

IL-8 secretion by H. pylori-infected cells was assessed, at 24h PI, to ensure bacterial-cell interaction. Cell supernatants were centrifuged at 15,000 rcf, for 10 minutes and stored at -80°C, until further use. IL-8 levels were measured using a commercial human IL-8 kit (CytoSet; Invitrogen Corporation, USA and DuoSet ELISA; R&D Systems, Canada), according to manufacturer’s instructions. IL-8 concentrations were determined based on a standard curve and recorded as pg/10⁶ cells.

4.8. Cell viability

The viability of the co-cultures was ascertained throughout the experiment, using Orangu assay, according to manufacturer instructions. Briefly, an optimum amount (10% of the culture medium) of Orangu solution (OR01-500, Cell Guidance Systems, UK) was added to the co-cultures, at 8 to 96h post treatment. The plates were then incubated at 37°C for 1 hour. The optical density (OD₄₅₀) of the cell supernatants were quantitated using a microplate reader. The assay was based on the reduction of water-soluble tetrazolium salt by dehydrogenase, which forms formazan dye in direct proportion with the number of live cells ¹¹⁰.

4.9. RNA extraction and quantitative real-time PCR

Total RNA was extracted from GPCs, MKN45 and AGS cells, as well as tissue specimens of intestinal type gastric tumor, its normal gastric counterpart and the duodenum, using Trizol reagent (Invitrogen, USA). According to the manufacturer's instruction ¹¹¹, complementary DNA strands (cDNA) were generated using the high-capacity cDNA reverse transcription kit (ABI, USA). Quantitative real-time PCR (qRT-PCR) reactions were performed (in duplicates), using SYBR green (ABI, USA) and gene-specific primers to amplify the target and house-keeping genes (glyceraldehyde-3-phosphate dehydrogenase; GAPDH) (Supplementary Table-2). qRT-PCR reactions were carried out using ABI One-Step 7500 Real-Time system (ABI, USA), at 50°C for 2 min, 95°C for 10 min, followed by 50 cycles at 95°C for 15s, and at 60°C for 1 min. The expression of GAPDH was used to normalize that of the target genes alteration by Qrel (2^−ΔCt) ¹¹² and 2^−ΔΔCt methods. Log2 transformed values were used to create more symmetrical and normally distributed data, in order to reduce statistical errors¹¹³.

4.10. Statistical analysis

Gene expression data from quantitative RT-PCR were expressed as mean ± standard deviation (SD) of three independent experiments and analyzed using GraphPad Prism (version 8.0.0 for Mac GraphPad Software, San Diego, California USA, www.graphpad.com). The differences between groups were evaluated by one-way analysis of variance (ANOVA), followed by post hoc contrasts, using the Bonferroni limitation for the statistical analysis. P values less than 0.05 were considered as statistically significant. To visualize hierarchical clustering of gene expression, the “Heatmap” package in R was used. Hierarchical clustering was performed in two steps: 1) calculating the distance matrix and apply clustering according to the absolute fold-changes in gene expression and 2) the correlation distance was defined as 1 – cor (x, y, method). The euclidean distance was calculated by “dist()” function ¹¹⁴.

Acknowledgement

This study was funded by Pasteur Institute of Iran: Grant/Award Number: #722; and #TP-9121 and Royan Institute: Grant/Award Number: 94000090

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Author Contributions Statement

SS and MM conceived the original idea and co-designed the study. SS carried out the experiments, did the analysis and wrote the manuscript. MM helped interpret the results and revised the manuscript. ME carried out patient sampling and laboratory processing. MT collected the gastric specimens. MEH supervised and carried out the gastroscopy and medical diagnosis. HB supported in providing the laboratory facilities. MM supervised the entire project.

Conflict of interest

None declared

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Infection with a Hypervirulent Strain of Helicobacter Pylori Primes Gastric Cells Toward Intestinal Transdifferentiation

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Abstract

Figures

1. Introduction

2. Results

2.1. Baseline gene expression patterns over time

2.2. Characterization of the hypervirulent H. pylori strain

2.3. Characterization of H. pylori coculture

2.4. Gene signatures following H. pylori infection

2.4.1. Tissue-specific transcription factors

2.4.2. Stemness markers

2.4.3. Tissue-specific mucins

2.5. Time-course of gene expression

2.6. Comparative gene expression signatures in gastric primary cells (GPCs) vs. cancerous cell lines (MKN45 and AGS)

3. Discussion

4. Methods

4.1. Cell culture and tissue specimens

4.2. Bacterial strain culture and characterization

4.2.1. H. pylori culture and isolation

4.2.2. Genomic DNA extraction and PCR

4.2.3. Protein expression assays

4.4. H. pylori liquid culture

4.5. Co-culture assays

4.6. Vacuolation and hummingbird assay

4.7. IL-8 secretion

4.8. Cell viability

4.9. RNA extraction and quantitative real-time PCR

4.10. Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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