Deoxycholic Acid(DCA) Confers An Intestinal Phenoype Onesophageal Squamous Epithelium Via Induction Of The Stemness-associated Reprogramming Factors OCT4 And SOX2

Barrett's esophagus(BE)is essentially a metaplasia in which the normal stratified squamous epithelium is replaced by columnar epithelium.BE is known as a precancerous lesion of esophageal adenocarcinoma(EAC).Molecular mechanisms underlying the development of BE was widely studied in recent years because the incidence of EAC and BE has dramatically increased in the whole world,especially in the western countries in the past decade.The cellular origin of BE remain unclear.There are two types of hypothesesfor the source of the Barrett's esophagus cell,which aretransdifferentiation andtranscommitment,but the both hypotheses are not entirely convincing due to lack of direct evidence.BE has been considered to be a complication of gastroesophageal reflux disease(GERD),and the detection rate of BE in patients with gastroesophageal reflux is as high as 10%-15%.Combined with previous studies which showed that deoxycholic acid(DCA)plays an important role in the occurrence and carcinogenesis of BE,we propose a hypothesis that DCA stimulation might regulate the expression of the stemness-associated reprogramming factors OCT4 and SOX2 and lead to retrodifferentiation of mature esophageal squamous epithelial cells into cells with partial differentiation potential,which form columnar epithelium through irreversible direct phenotypic conversionunder the stimulation of environmental factors.Aims: The aim of this study was to investigate the role of OCT4 and SOX2 in the process of DCA induced formation of BE.This work may provide new theoretical basis fornovel molecular biomarker and therapeutic target of BE.Methods:1.Evaluation of OCT4 and SOX2 expression and expression of intestinal cell markers Cdx2,MUC2 and squamous differentiation marker p63 in BE versus normal esophageal squamous epithelium and esophagitis tissues with IHCin human and surgical bile acid reflux rat model.2.Het-1A cells were exposed to 200 ?M DCA for 2,4,8 and 12 hours,then OCT4 and SOX2 expression and expression of intestinal cell markers Cdx2,MUC2 and squamous differentiation marker p63 were examined by real-time PCR and Western Blot.3.Over-expression and knock-down the expression levels of OCT4 and SOX2 in esophageal epithelial cells via lentiviral construct over-expressing and RNAi approach to investigate the effects of modulations in the expression levels of OCT4 and SOX2 on DCA induced intestinal metaplasia.4.Evaluation of the interaction between OCT4 and SOX2 in Het-1A cellsthrough over-expression and knock-down the expression levels of OCT4 and SOX2.Results:1.OCT4 expression is negtive in normal esophageal squamous epithelium and weak in the esophagitis.Increased expression of OCT4 and decreased expression of SOX2 was seen in BE compared with normal esophageal tissues and the esophagitis.2.Cdx2,MUC2 expression was increased in BE compared with normal esophageal tissues and the esophagitis,whilep63 showed a decreased nuclear expression in BE.3.We successfully established a surgical rat model of BE through esophagoduoden-ostomy plus gastrectomy.Expression of OCT4 and SOX2 and intestinal cell markers Cdx2,MUC2 and squamous differentiation marker p63 in rat model are consistent with human body specimen.4.In vitro experiments confirmed that DCA treatment can up-regulate the expression of OCT4 in squamous epithelial cells and down-regulate the expression of SOX2 in time-dependent mannersandinduce the expression of intestinal type marker molecules Cdx2 and MUC2.5.Down-regulation of OCT4 expression by si RNA abrogated DCA-induced expression of Cdx2 and MUC2,but over-expression of OCT4 alone was insufficient to up-regulate the intestinal hallmark Cdx2 and MUC2.6.Knockdown of SOX2 significantly up-regulated the expression of both Cdx2 and MUC2 and significantlyenhanced DCA-induced Cdx2 protein expression,and SOX2 up-regulation could conversely decrease Cdx2 and MUC2 expression.7.Neither knockdown nor over-expression of OCT4 affected expression of SOX2.Similarly,neither knockdown nor over-expression of SOX2 affected the expression of OCT4 in Het-1A cells.Conclusions:1.OCT4 and SOX2 may play an important role in the transition from squamous epithelium to the intestinal phenotype triggered by gastro-esophageal reflux.2.In rat model,single bile acid reflux can induce Barrett's esophagus.3.DCA induces conversion of esophageal squamous epithelium into intestinalized columnar epithelium through up-regulation of stemness-associated reprogramming factor OCT4 and down-regulated SOX2.4.OCT4 may have a new partner in esophageal epithelial cells other than SOX2.