As a precursor event of gastric cancer, IM can dramatically increase the incidence of gastric cancer in patients [
36]. Generally, many etiological factors affect the progression of IM, including Hp infection, alcohol intake, cigarette smoking, radiation and bile acid reflux. However, the molecular mechanism of phenotypic transformation in stomach epithelium with IM remains largely elusive.
As a homeobox transcription factor, CDX2 is essential for intestinal differentiation and is mainly expressed in the intestinal epithelium. Previous studies in transgenic mice have demonstrated that ectopic CDX2 expression may be the early event that triggers the progression of IM in the gastric mucosa [
10,
37]. Our results confirm that bile acid DCA can activate CDX2 and its downstream intestinal markers, including KLF4, HNF4α and cadherin 17, in gastric cell lines. Similar to our results, several studies on Barrett’s esophagus also indicated that CDX2 expression induced by bile acid reflux plays an important role in phenotypic change in the esophageal mucosa [
27,
30]. In the present study, we found that CDX2 and several known CDX2 target genes could be induced by bile acid in gastric cell lines in a dose-dependent manner. Additionally, CDX2 expression was increased in IM tissue.
The transcription factor SOX2 is a member of the SRY-related HMG box family. SOX2 is involved in the regulation of embryonic development and in the determination of cell fate. In addition to being required for stem cell maintenance in the central nervous system, SOX2 also regulates gene expression in the stomach [
38]. Aberrant SOX2 expression has been reported to be associated with various types of cancer, especially gastric cancer [
39‐
41]. However, due to conflicting results, it is still unclear whether SOX2 is an oncogene or functions as a protective gene in cancer development. For instance, one study found that SOX2 was increased in gastric cancer, and tumors expressing high levels of SOX2 showed more extensive invasion, higher TNM stages, and worse prognoses [
42]. However, another study found that compared with SOX2-positive gastric cancer, decreased SOX2 expression in gastric cancer was associated with an increased extent of tumor invasion, higher rates of lymph node metastasis and shorter survival [
38]. In IM, a precancerous lesion of gastric cancer, most studies showed that SOX2 expression was absent [
3,
16,
40]. Another study showed that SOX2 expression was maintained in CDX2-induced intestinal metaplastic mucosa even though the gastric phenotype was completely lost, which indicated that CDX2 expression is sufficient and the loss of SOX2 expression is not necessary for the progression of IM [
34]. Interestingly, ectopic expression of SOX2 in the intestinal epithelium could convert the cell fate of already committed intestinal cells into stomach-like cells. CDX2 expression was maintained in SOX2-induced animals, while several known CDX2 target genes were clearly downregulated [
33]. In the present study, we first demonstrated that the expression of SOX2 was decreased in bile acid-treated gastric cells, which might reveal a potential cause of SOX2 loss in IM tissue. To investigate the functions of SOX2 and CDX2 in IM, we coinfected SOX2- and CDX2-overexpressing lentiviruses into GES-1 cells. Our results showed that SOX2 inhibited CDX2-induced intestine-specific markers (KLF4, HNF4α and cadherin 17) in CDX2-overexpressing gastric cells, while there was no direct regulatory relationship between SOX2 and those markers in cells that lacked CDX2 expression. This result suggested that CDX2 is the molecular trigger in the progression of IM; in addition, although SOX2 was suppressed during this process, it might function as a protective factor by indirectly inhibiting intestine-specific factors. Our result was consistent with other researchers’ opinion that SOX2 might interfere with the function of CDX2 by suppressing its downstream DNA-binding capacity [
33]. However, the molecular mechanism by which SOX2 interferes with the function of CDX2 is unclear. In this study, we demonstrated that SOX2 could strongly suppress the transcriptional effect of CDX2 on its genomic target sites using a dual-luciferase reporter assay. In addition, Co-IP was performed to investigate the relationship of these two factors at the protein level. Our results suggested that SOX2 and CDX2 could form protein complexes in the nucleus. Thus, the formation of a SOX2-CDX2 complex might be one of the most important mechanisms contributing to the subsequent suppression of intestine-specific markers in IM. Moreover, we found that bile acid-induced KLE4 was inhibited by overexpression of SOX2. Considering that bile acid is an exogenous stimulator of CDX2, this result verified our view that SOX2 inhibits the progression of intestinal phenotype conversion by interfering with the function of CDX2.
miRNAs are endogenously expressed small noncoding RNAs that play a vital role in the development of cancer and premalignant lesions [
21‐
23]. Among those miRNAs, we focused on miR-21 because a recent study showed that miR-21 suppresses the expression of SOX2 in human MSCs [
35]. Additionally, a previous study reported that bile acid appeared to accelerate the expression of the “oncomir” miR-21 in the laryngeal mucosa [
43]. In the present study, to investigate the regulatory effect of miR-21 on SOX2 in gastric cells, miR-21 loss- and gain-of-function experiments and dual-luciferase reporter assays were performed to demonstrate that miR-21 regulates SOX2 by directly binding its 3′-UTR. Furthermore, qRT-PCR results suggested that miR-21 expression could be induced by bile acid in a dose-dependent manner. In addition, the ISH results showed that miR-21 expression was significantly increased in the IM sample compared with that in normal gastric tissue. Finally, we found that knockdown of miR-21 partially rescued the inhibition of SOX2 in bile acid-treated gastric cell lines. Taken together, these results suggest that miR-21 plays an important role in the progression of bile acid-induced IM by regulating the expression of SOX2.