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Helicobacter pylori induces caudal‐type homeobox protein 2 and cyclooxygenase 2 expression by modulating microRNAs in esophageal epithelial cells

Dysregulation of microRNAs (miRNAs) has been linked to virulence factors of Helicobacter pylori. The role of H. pylori in esophageal disease has not been clearly defined. We previously reported that H. pylori esophageal colonization promotes the incidence of Barrett's esophagus and esophageal adenocarcinoma in vivo. Here, we studied the direct effects of H. pylori on the transformation of esophageal epithelial cells, with particular focus on whether H. pylori exerts its effects by modulating miRNAs and their downstream target genes. The normal human esophageal cell line HET‐1A was chronically exposed to H. pylori extract and/or acidified deoxycholic acid for up to 36 weeks. The miRNA profiles of the esophageal epithelial cells associated with H. pylori infection were determined by microarray analysis. We found that chronic H. pylori exposure promoted acidified deoxycholic acid‐induced morphological changes in HET‐1A cells, along with aberrant overexpression of intestinal metaplasia markers and tumorigenic factors, including caudal‐type homeobox protein 2 (CDX2), mucin 2, and cyclooxygenase 2 (COX2). Helicobacter pylori modified the miRNA profiles of esophageal epithelial cells, particularly aberrant silencing of miR‐212‐3p and miR‐361‐3p. Moreover, in biopsies from Barrett's esophagus patients, esophageal H. pylori colonization was associated with a significant decrease in miR‐212‐3p and miR‐361‐3p expression. Furthermore, we identified COX2 as a target of miR‐212‐3p, and CDX2 as a target of miR‐361‐3p. Helicobacter pylori infection of esophageal epithelial cells was associated with miRNA‐mediated upregulation of oncoprotein CDX2 and COX2. Our observations provide new evidence about the molecular mechanisms underlying the association between H. pylori infection and esophageal carcinogenesis.


| INTRODUCTION
Chronic inflammation can promote the initiation and progression of many cancers, 1 as exemplified by the gastroesophageal reflux disease (GERD) to Barrett's esophagus to esophageal adenocarcinoma sequence. Chronic gastroesophageal reflux plays a crucial role in the development of Barrett's esophagus, a condition of columnar (intestinal) metaplasia of the esophageal squamous epithelium. 2 It has been widely accepted that this esophageal metaplasia is a precursor of esophageal adenocarcinoma. 3 The risk of developing esophageal adenocarcinoma in patients with Barrett's esophagus is 30-125 times higher than in the normal population. 4 Helicobacter pylori is a risk factor for the development of gastritis, peptic ulcer disease, and gastric cancer. 5 The association between H. pylori infection and Barrett's esophagus or esophageal adenocarcinoma is controversial. A number of studies have concluded that H. pylori infection tends to protect against Barrett's esophagus or esophageal adenocarcinoma. [6][7][8] In contrast, a meta-analysis showed no convincing association between H. pylori eradication and the development of GERD, 9 and eradication of H. pylori might halt the progress to esophageal adenocarcinoma in patients with GERD and Barrett's esophagus. 10 However, these studies did not evaluate the impact of H. pylori colonization sites on the development of esophageal disease. Our previous studies have indicated that, in a rat model of chronic gastroesophageal reflux, H. pylori colonization in the esophagus increased the severity of esophageal inflammation and the incidence of Barrett's esophagus and esophageal adenocarcinoma, whereas H. pylori colonization in the stomach had no influence on the esophageal mucosa. 11,12 Thus, the effect of H. pylori on the esophagus varies with the colonization site. Both chronic gastroesophageal reflux and esophageal H. pylori infection could play important roles in the development of inflammation and Barrett's esophagus-associated carcinogenesis. Reflux of gastric contents, including acid and bile, induces metaplasia of esophageal mucosa and facilitates colonization of H. pylori in the distal esophagus, and therefore aggravates esophageal injury. Clinical data have shown the high prevalence of H. pylori in the esophagus, and its presence is correlated with signs of inflammation. 13 In Barrett's esophagus patients, the presence of metaplasia within the esophagus is a prerequisite for H. pylori colonization, and H. pylori may exacerbate inflammation in Barrett's epithelium. 14 However, the molecular mechanisms involved in the effects of H. pylori in the GERD-Barrett's esophagus-esophageal adenocarcinoma sequence remain largely unknown. We have found that H. pylori colonization was associated with overexpression of cyclooxygenase 2 (COX2) in esophageal mucosa. Celecoxib, a selective COX2 inhibitor, significantly inhibited Barrett's esophagus-associated carcinogenesis. 11 The mechanisms for the regulation of H. pylori on COX2 expression in esophageal mucosa may still need to be further investigated.
MicroRNAs (miRNAs) regulate various cellular functions, including proliferation, differentiation, and apoptosis. 15 Aberrant miRNA expression has been associated with human diseases such as inflammation and cancer. 16 Several miRNAs have been identified as being involved in the development and progression of Barrett's esophagus and esophageal adenocarcinoma. 17 Helicobacter pylori infection can modify the expression of more than 50 miRNAs in the gastric mucosa, and these miRNA levels can be restored to normal after H. pylori eradication. 18 Recent studies also show that the association of miRNA with esophageal adenocarcinoma prognosis may be influenced by H. pylori infection status, suggesting that miRNA-H. pylori interactions play an important role in the prognosis of esophageal adenocarcinoma. 19 However, none of the earlier studies examined the expression signature and the role of miRNA in H. pylori-infected esophageal epithelial cells.
In this study, we determined the long-term and direct effects of H. pylori on the phenotype of esophageal epithelia cells by using a well-established in vitro model. 20 We particularly focused on whether H. pylori exerts its effects through modulating miRNAs and their downstream target genes.

| Clinical samples
A total of 16 Barrett's esophagus patients with (n = 10) or without (n = 6) esophageal H. pylori colonization were enrolled in this study.    As an intestine-specific transcription factor, caudal-type homeobox protein 2 (CDX2) regulates goblet-specific MUC2 gene expression, resulting in the differentiation of intestinal epithelium. 21 Normal esophageal keratinocytes do not express CDX2, which has been reported to be a highly sensitive and specific marker of intestinal metaplasia and adenocarcinoma. 22,23 In agreement with the effect on cell morphology, treatment of HET-1A cells with H. pylori extract or acidified DCA significantly increased the mRNA expression of CDX2 and mucin 2 (MUC2), and these effects were evident at 8-12 weeks and maintained for up to 36 weeks (Figure 1Da,b). Moreover, CDX2 and MUC2 protein expression was upregulated in cells treated with these compounds at 36 weeks (Figure 1Da,b). Cytokeratin 5 (CK5) is expressed by normal esophageal keratinocytes but not by intestinal epithelium with metaplasia. 24 Coincidentally, with upregulated CDX2 and MUC2, the expression of CK5 also decreased in HET-1A cells treated with H. pylori extract or acidified DCA (Figure 1Dc). Importantly, the alteration of CDX2, MUC2, and CK5 was enhanced when HET-1A cells were exposed to both two compounds. As shown in Figure 1D, after 36 weeks, the expression of  Figure 1F).
Thus, prolonged exposure of HET-1A cells to H. pylori seems to aggravate the tumorigenic phenotype conferred by acidified DCA.

Helicobacter pylori infection
To determine the miRNA profiles of esophageal epithelial cells associated with H. pylori infection, we carried out a microarray analysis.
Comparison of HET-1A as well as OE33 cells treated with or without H. pylori revealed that 15 miRNAs were differentially expressed with at least 1.5-fold changes. A heat map representation of differentially expressed miRNAs is shown in Figure 2.

| DISCUSSION
The role of H. pylori in esophageal disease has not been clearly defined. Here, we showed that exposure to H. pylori promoted acidified DCA-induced transformation of esophageal epithelial cells. showing that H. pylori colonization can aggravate esophageal injury and promote the incidence of Barrett's esophagus and esophageal adenocarcinoma in a model of chronic gastroesophageal reflux. 11,12 Caudal-type homeobox protein 2 is a key factor for intestinal development. Ectopic expression of CDX2 occurs during the development and maintenance of Barrett's esophagus. 30 Bile acids can directly induce CDX2 expression and activation in Barrett's esophagus. 31 Our previous studies showed that H. pylori infection induced CDX2 expression in esophageal epithelium, playing an important role in the inflammation associated with Barrett's esophagus and tumorigenesis. 11,12 In this in vitro model, we found CDX2 was increased in esophageal epithelial cells in response to H. pylori or acidified DCA treatment, implying that the cells underwent transformation. Chronic expression of COX2 plays a crucial role in H. pylori-associated gastric carcinogenesis. 32 Moreover, the expression of COX2 is gradually increased during the neoplastic progression of the esophageal mucosa from Barrett's esophagus to adenocarcinoma. 33 In the present study, COX2 expression in esophageal epithelial cells Thus, alteration of these molecules may contribute to the transformation of the phenotype of the esophageal epithelial cells.
Helicobacter pylori infection could alter the expression and function of miRNAs through epigenetic regulations such as DNA methylation and histone modification. 34 The role of miRNA in the progression from normal esophageal mucosa to Barrett's esophagus and finally to esophageal adenocarcinoma has been established. 17 However, the contribution of H. pylori-related miRNA dysregulation to the transformation of esophageal epithelial cells remains unclear.
Our microarray data showed that 15  Beijing Natural Science Foundation (BNSF#7123232) awarded to W.

CONF LICT OF I NTEREST
The authors have no conflict of interest.