SOX2 Interferes With The Function Of CDX2 In Bile Acid-induced Gastric Intestinal Metaplasia

?Background? Gastric cancer(GC)is the second most common malignancy and the second leading cause of cancer-related death in China.The development of gastric cancer,especially intestinal type,usually occurs through chronic gastric inflammation,atrophic gastritis and intestinal metaplasia(IM).Helicobacter pylori(Helicobacter pylori,Hp)is considered the most important etiological factor in both the precursor event and subsequent gastric cancer development.However,a number of studies have shown that prolonged bile reflux also promotes the development of gastric IM.As a homeobox transcription factor,CDX2 is essential for intestinal cell growth and differentiation and is mainly expressed in the colon and small intestine.Previous studies from other groups and our group also indicated that bile acid could induce CDX2-and IM-related gene expression in vitro.Nevertheless,the exact molecular network that promotes CDX2 upregulation in IM development is still not completely understood.In contrast to CDX2,SOX2 is a member of the SRY-related HMG Box(SOX)family and was identified as a critical transcription factor for esophageal and gastric differentiation.A number of studies have found a converse expression pattern between SOX2 and CDX2 in IM tissue.However,the relationship between SOX2 and CDX2 is still controversial.MicroRNAs(miRNAs)are endogenously expressed small noncoding RNAs that play important gene-regulatory roles through binding to the 3'-untranslated regions(3'UTRs)of target mRNAs.To date,a number of studies have indicated that miRNAs are involved in the pathogenesis of many types of cancer,including gastric cancer.Among these miRNAs,miR-21 is one of the most common and highly upregulated miRNAs in gastric cancer and preneoplasia lesions.The expression of miR-21 is significantly related to tumor size,metastasis and later-stage disease in patients with gastric cancer.Based on these findings,we hypothesized that miR-21 may also mediate the phenotypic changes of gastric cells in the progression of IM.In this study,we aimed to explore the relationship between SOX2 and CDX2 and the role of miR-21 in bile acidtreated gastric cell lines.?Objectives? 1.To detect the expression of SOX2 and CDX2 in gastric IM.2.To explore the regulatory effect of SOX2 on CDX2.3.To study the regulation of bile acid-induced miR-21 on SOX2.?Methods? 1.Gastric cell lines GES-1,AGS,AZ-521 and MKN45 were treated with deoxycholic acid(DCA)in a dose-dependent manner for 24 hr.Western blotting was employed to dected the expression of SOX2,CDX2 and several intestinal markers(KLF4,Cadherin 17 and HNF4?).2.Real-time PCR was used to detect the expression of CDX2 and SOX2 in paired IM specimens from 8 individuals in Xijing Hospital of Digestive Diseases.Immunohistochemistry(IHC)was used to detect the expression of CDX2 in gastric tissue microarray.3.Co-immunoprecipitation(Co-IP)and immunofluorescence(IF)were performed to ascertain the interaction of SOX2 and CDX2.Luciferase reporter assays were used to detect the transcriptional activity of CDX2.SOX2 and CDX2 gain-and loss-of-function models are established via stable overexpressing lentiviral vector infection and siRNA transfection.Real-time PCR and western blotting were performed to test the expression of SOX2,CDX2 and intestinal markers.4.Real-time PCR was used to detect the expression of miR-21 in gastric cell lines and paired IM specimens.In situ hybridization(ISH)was performed to detect the expression of miR-21 in gastric tissue microarray.miR-21 gain-and loss-of-function models were established via transient mimic and antagomir transfection.Real-time PCR and western blotting were used to identify the influence of miR-21 on SOX2.To further investigate the regulatory relationship between miR-21 and SOX2,a dual-luciferase reporter assay was performed in HEK293 T cells.?Results? 1.Western blotting results indicated that CDX2 and intestinal markers(KLF4,Cadherin 17 and HNF4?)were substantially upregulated after 24 hr of DCA treatment at 100-200 ?M in GES-1.DCA could strongly suppress the expression of SOX2 at a 200 ?M concentration in AGS,AZ-521 and MKN45 cells.2.Western blotting and Real-time PCR results indicated that CDX2 could significantly upregulate KLF4,Cadherin 17 and HNF4?,while there was no direct regulatory relationship between SOX2 and those three intestine-specific factors.IHC result showed that the level of CDX2 was increased in IM tissues compared with that in normal tissues.And there was a strong correlation between the upregulation of CDX2 and IM status(P < 0.01).Real-time PCR results indicated CDX2 was overexpressed while SOX2 was downregulated in paired IM specimens from 8 individuals(P < 0.01).3.Western blotting results indicated that compared with that in the control group,the expression of KLF4,HNF4? and Cadherin 17 was induced by CDX2 and reduced by SOX2 in GES-1 cells coinfected with SOX2-and CDX2-overexpressing lentiviruses.Furthermore,Real-time PCR also confirmed that SOX2 suppressed KLF4 expression in CDX2 lentivirusinfected GES-1 cells.Knockdown of SOX2 in AGS cells expressing endogenous SOX2 and CDX2 had the opposite effect.Western blotting and Real-time PCR showed that DCAinduced KLF4 expression was decreased in SOX2-overexpressing GES-1 cells compared with that in the control group.As expected,the expression of KLF4 was further increased in SOX2 knockdown AGS cells at the protein level.Dual-luciferase reporter assay indicated that SOX2 could strongly suppress the transcriptional activity of CDX2.4.Co-IP analysis revealed that endogenous SOX2 and CDX2 could form protein complexes in AGS cells.Furthermore,IF staining for SOX2 and CDX2 was performed in AGS and AZ-521 cells.The IF results proved that SOX2 and CDX2 colocalized in the nucleus in AGS and AZ-521 cells.5.Western blotting results revealed that ectopic miR-21 expression reduced the protein levels of SOX2 in AGS and AZ-521 cells,while miR-21 knockdown increases SOX2 expression in AGS and MKN45 cells.Real-time PCR analysis showed that SOX2 mRNA levels were maintained in most groups transfected with miR-21 mimics or antagomirs.Dual-luciferase reporter assay confirmed that miR-21 could suppress the expression of SOX2 by directly binding to the SOX2 3'UTR.As Real-time PCR analysis indicated,after treatment with DCA for 24 hours,the expression of miR-21 increased significantly in a dose-dependent manner in both GES-1 and AZ-521 cells.Real-time PCR of miR-21 in 8 pairs of matched human IM specimens indicated the expression of miR-21 was higher in IM samples(P < 0.01).The expression of miR-21 was measured in three tissue microarray chips containing 141 cases of IM and 62 cases of normal gastric tissue by ISH.The results indicated that miR-21 expression was significantly upregulated in IM tissue compared with that in normal gastric tissue.Western blotting results indicated that the inhibition of SOX2 by bile acids in AGS cells was partially rescued by miR-21 knockdown.?Conclusions? SOX2 inhibits the transcriptional activity of CDX2 by forming protein complexes in gastric cells.miR-21 downregulates the expression of SOX2 by binding to its 3'-UTR directly.After treating with DCA,the expression of CDX2 is upregulated in gastric cells while SOX2 is inhibited by DCA-induced miR-21 which further promotes the development of IM.