The Effect Of BMP4 Signaling Pathway And KLF4 On The Forming Of Barrett's Esophagus

Background and ObjectiveBarrett's esophagus is a metaplastic lesion,in which the distal normal esophageal squamous epithelium was replaced by the columnar epithelium.Based on a few experiments reported in the literature,gastricesophagitis reflux diseas characterized by bile acid is the main risk factors for BE(Barrett's esophagus).Refluxed bile acid can lead to chronic inflammation and injury on the mucosal epithelium.Through a series of molecular biology events,normal esophageal squamous epithelium was transformed into BE which may lead to the occurrence of esophageal adenocarcinoma eventually.However,the cellular and molecular mechanisms involved are poorly understood.A few studies used serial analysis of gene expression,a technique to compare the expression profile of BE with a normal squamous esophagus,to identify genes specifically involved in the switching process of normal squamous esophageal mucosa to metaplastic BE.Bone morphogenetic protein 4(BMP4)was detected to be highly and uniquely expressed in the SAGE library of BE but not in a normal squamous epithelium.BMP4,a member of the transforming growth factor(TGF)-? family,and its downstream targets,including the BMP4 receptor,p-Smad 1/5/8(phosphorylated Smad 1/5/8),and Smad 4,comprise the BMP4 signalling pathway.The p-Smad 1/5/8 protein,in combination with Smad 4,forms a complex that translocates into the nucleus,where certain target genes can be transcribed.The BMP4 signalling pathway plays an important role in cell proliferation,tissue differentiation,and embryonic development.Previous study have shown that the BMP pathway could play a role in the transformation of normal oesophageal squamous cells into columnar cells.Krüppel-like factor 4(KLF4)belongs to the Krüppel-like factors(KLFs)family of conserved zinc finger-containing transcription factors,which are widely expressed in different organs and regulate numerous biological processes,including proliferation,terminal differentiation,and apoptosis.The function of KLF4 has been investigated in detail in cancer formation and the induction of pluripotent stem cells.However,the role of KLF4 in BE had been neglected.Based on the above studies,we hypothesized that KLF4 and BMP4 cooperatively promote a Deoxycholic acid(DCA)-induced intestinal phenotype of an oesophageal squamous epithelium.The aim of this study was to investigate the roles of KLF4 and BMP4 in the pathogenesis of Barrett's epithelium.Methods1.Esophageal biopsies were obtained from healthy volunteers and from individuals with BE.Fresh endoscopic biopsy specimens were fixed in 10% formalin.Paraffin sections(4 ?m thick)were routinely stained with haematoxylin.2.BE model is established by SD rats.Fresh endoscopic biopsy specimens were fixed in 10% formalin.Paraffin sections(4 ?m thick)were routinely stained with haematoxylin.3.Normal squamous epithelium tissue and BE tissue was performed in paraffin embedded sections.target gene expression in paraffin-embedded tissues is feasible by SP immunohistochemistry.Paraffin specimen histological slides were estimated blindly and independently by two experienced gastrointestinal pathologists.SPSS18.0 software was performed on the data.4.HET-1A,TE-1,OE33 cells were cultured in a BEBM and RPMI-1640 medium supplemented with matched cytokines.Cells were incubated with 200?M DCA for 4?8?12 hours,respectively.5.Design interference sequence according to Gene Bank.We screened 3 effective siRNAs against KLF4 to obtain the KLF4-si RNAs mixture.stand at room temperature for 20 minutes.The optimal concentration of siRNA added to transfection reagents for transfection and time for KLF4 inactivation(48 hours)were assessed.HET-1A cells were equally plated in six-well tissue culture plates.6.KLF4 bacteria was cultured in solid medium added whith kanamycin.Put culture plate upside down in half an hour.7.Follow the Sigma instructions,washing,centrifugal,separation,enrichment,cracking,extraction of plasmid.8.The cDNA coding for KLF4 was cloned into the lentiviral vector(LV-KLF4).Lentiviral vectors were packaged by transfecting into 293 T cells using Enhanced Infection Solution according to the manufacturer's instructions.HET-1A cells were transduced with the lentivirus containing KLF4 by polybrene.Forty-eight hours after infection,2 ?g/ml of puromycin was added to the media for 2 weeks to select the lentivirus infected cells.9.Incubation of HET-1A cells with either recombinant human BMP4 or Noggin was performed.10.Monoclonal antibodie was added into cultured cells.We detected target gene expression using immunofluorescence cytochemistry.11.q RT-PCR and Western blot results were used to detected target gene expression.Results1.Immunohistochemistry of biopsy specimens demonstrated that BMP4 was only detected in human BE tissue(positive rate 94%),but in the normal squamous epithelium tissue(positive rate 9.1%)of patients.p-Smad1/5/8 protein expression increased in BE tissue(positive rate 96%)compared with the normal squamous epithelium tissue of patients.BMP4 expression in BE was typically localized in the cytoplasm of the cell,while p-Smad 1/5/8 and KLF4 were localized in the nuclei of the surface villi.There was statistical difference between these two groups(P<0.05).2.Immunohistochemistry of biopsy specimens demonstrated that BMP4 was only detected in rats BE tissue,but not in the normal squamous epithelium tissue of rats.p-Smad1/5/8 protein expression increased in BE tissue compared with the normal squamous epithelium tissue of patients.BMP4 expression in BE was typically localized in the cytoplasm of the cell,while p-Smad 1/5/8 and KLF4 were localized in the nuclei of the surface villi.3.Our results showed that DCA can enhance the expression of BMP4,p-Smad 1/5/8 and KLF4.The BMP4 can up-regulate the expression of KLF4,CDX2,MUC2 and MUC5 ac and that after KLF4-siRNA,BMP4 cannot up-regulate the expression of CDX2,MUC2 and MUC5 ac.Furthermore,the BMP4 antagonist Noggin inhibits the expression of p-Smad 1/5/8,KLF4,CDX2,MUC2 and MUC5 ac,but it cannot inhibit expression of CDX2,MUC2 and MUC5 ac proteins in cells transfected with the KLF4 expression vector.Conclusion1.DCA can enhance the expression of BMP4,p-Smad 1/5/8 and KLF4.2.BMP4 can effect KLF4,CDX2,MUC2,and MUC5 ac expression in HET-1A cell3.Up-or down-regulation of KLF4 can effect CDX2,MUC2,and MUC5 ac expression in HET-1A cell3.BMP4 promotes a Deoxycholic acid(DCA)-induced intestinal phenotype of an oesophageal squamous epithelium via up-regulation of KLF4...