Effect On Expressions Of KLF4and NOTCH1in Barrett Esophagus Induced By Deoxycholic Acid

Backgroud:Barrett esophagus (BE) is a pathological phenomenon about metaplasia of columnarepithelium in esophagus, it is the most important precancerous lesion which could induceesophageal adenocarcinoma(EAC). The malignant adenocarcinoma,EAC,has fast growthrate companied with high lethality in recent years, that makes BE become the first riskfactor attracting wide attentions. The molecular mechanism is unclear in BE induced byreflux esophagitis and its origin is always a hotspot issue. There are many hypotheses, butnone of them carry total conviction.The influence of cellular reprogramming on the development of the disease graduallyhas aroused people’s concerns. In brief, it is the reversed differentiation of mature cells. IPS(Induced pluripotent stem cells) cells are reprogrammed directly into pluripotent cellswhich resembles embryonic stem cells. Barrett’s esophagus is one kind of this phenomenoncalled as columnar metaplasia, the past researches have confirmed that the normalesophageal squamous epithelia treated by bile acid can express classical intestinal stem cellmarker Cdx2, and other cytokines such as Sox2, Bmp4associated with cell pluripotency.As a result, we assume that Barrett’s esophagus comes from the normal esophagealsquamous epithelial cells, the normal cells are reprogrammed directly into pluripotent cellsunder the stimulation of the external environment like gastroesophageal reflux, then thepluripotent cells differentiate into columnar epithelium.Klf4, known as intestinal Krüppel zinc finger transcription factor, it is expressed inendothelial cells in vivo and in vitro, also play an role in maintaining plastic state of stemcells. Notch1is one member of the Notch signal pathway, This family can regulate celldifferentiation、proliferation and apoptosis, which affects the normal tissues and cells.In addition, it is also involved in a wide variety of tumor such as breast cancer, liver cancer,etc. Recent studies show that the Notch pathway activation also plays an important role inthe regulation about stem cell self-renewal and differentiation. It is one of the symbols of cell pluripotency.In this study, we chose deoxycholic acid to stimulate immortalized normal esophagealsquamous epithelial cells in order that we can create an environment similar to humane BE.Then we observed the expression change of Klf4and Notch1in this process. At the sametime, we still trace the expression of two molecules in three distinct humane esophagealtissues including normal esophageal tissue, esophagitis tissue and Barrett tissue. This workmay provide a new theoretical basis to clarify the human BE source.Methods:1. Human simple collection and immunohistochemical SP staining.We collect97subjects from digestive department in The Third Military MedicalUniversity Affiliated Southwest Hospital from March2012to Decembe2012.They include26cases of esophagitis,22cases of BE and49cases of healthy people.Make tissuespecimen as experimental group and control group, respectively. The expression of Klf4and Notch1were determined in all specimens by immunehistochemical staining. Theresults were analysized by SPSS18.0data analysis system for statistics.2. Cultivate the immortalized normal esophageal squamous epithelial cells Het-1Aand treatment.The Het-1cells coming from the ATCC (American Type Culture Collection) growwith special epithelial cultured medium, in this process,these cells were treated withdeoxycholic acid of different concentrations (0μmol//l,100μmol//l,200μmol//l) in4,8,12hours, respectively. Finally we observe cells growth.3. RT-PCR and Western blot detection。After the process of Het-1A cells in different concentration of deoxycholic acid ofthree time points,we detected the expression of Klf4and Notch1by RT-PCR and Westernblot,respectively. The results were analyzed by SPSS18.0data system for statistics.Results：1. The expressions of Klf4was stronger in Barrett esophagus compared withesophagitis tissues, the level of Klf4is the lowest in normal esophagus tissues, there wassignificant difference among those groups(P <0.05);The NOTCH1level was showed nodifference among three group tissues.2. The mRNA level of Klf4in HET-1A cells has increased with the concentrations of DCA and the extension of treatment time, The mRNA level of Notch1in HET-1A cells hasalso presented the same trend; there was significant difference among three groups(P <0.05). Meanwhile, The protein expressions of Klf4and Notch1has increased with theconcentrations of DCA and the extension of treatment time, the difference is significant(P<0.05).Conclusions：1. The expressions of Klf4was stronger in Barrett esophagus and esophagitis tissuescompared with normal esophagus tissues; the mRNA and protein level of Klf4in HET-1Acells has increased with the concentrations of DCA and the extension of treatment time.Allof this suggested that Klf4could play an important role in the development of Barrettesophagus.2. The expressions of Notch1was showed no difference among three group tissues; themRNA and protein level of Notch1in HET-1A cells has increased with the concentrationsof DCA and the time of treatment extending. All of this suggested that Notch1could playan important role in the development of Barrett esophagus. In combination with the changeof Klf4and the role of both two in pluripotent cells, it provides the theoretical basis thatBarrett’s esophagus comes from the normal esophageal squamous epithelial cells which arereprogrammed directly into pluripotent cells under the stimulation of the externalenvironment like gastroesophageal reflux, then the pluripotent cells differentiate intocolumnar epithelium.