| Esophageal cancer is one of the malignant tumors of the digestive system.It ranks 7th in the incidence and 6th in mortality of malignant tumors in the world.Pathological types of esophageal cancer are divided into esophageal adenocarcinoma and esophageal squamous cell carcinoma,of which 90%are squamous cell carcinoma(ESCC).Several sequencing data have revealed genomic changes of ESCC,which provides a solid theoretical basis for the development of esophageal cancer.However,because most patients with esophageal cancer are diagnosed at an advanced stage,the driver genes of ESCC and their timing from early to late stage is hard to find.Available animal models of rat esophageal carcinoma have access to pathological tissue and sequencing data on the dynamics of ESCC from hyperplasia to papilloma to esophageal carcinoma,but their long cycles(>35 weeks)and high tumor mutation burden make it difficult to distinguish true driving events.Therefore,we urgently need an in vitro model with clear background,stable genome,easy to operate,and can reflect the real situation in vivo to carry out relevant research on esophageal cancer.Three-dimensional organoids refer to the proliferation and differentiation of stem cells or organ-specific progenitor cells into 3D structures that are functionally similar to in vivo organs under specific culture conditions,and are a good alternative model for organ-level correlation studies in vivo.In this study,we will establish immortalized normal esophageal epithelium cell lines and its 3D organoids culture system based on rats,and investigate the regulatory mechanism of normal esophageal epithelium proliferation and differentiation homeostasis in vitro to provide a basis for the etiology of esophageal cancer.In our study,we successfully established immortalized normal rat esophageal epithelial cell lines D3,F3 and A1,and the epithelial properties of D3 cell line were more similar to the primary cells.The ROCK inhibitor Y27632,which is required for 2D and 3D organoids culture,mainly affects the biological processes such as cell proliferation,cell differentiation,TGF-? signaling pathway,WNT pathway and focal adhesion,and has no significant effect on BMP signaling pathway.Removal of Y27632 resulted in decreased cell proliferation capacity,changes in cell morphology,decreased 3D organoids formation rate,upregulation of differentiation-related gene expression?In the absence of Y27632,senescence-related gene p16 protein expression was upregulated,differentiation-related gene p63 expression was downregulated,and ELF3 expression was upregulated.Knockdown of TP63 showed decreased 2D proliferative capacity of D3,changes in cell morphology,decreased the 3D organoids formation rate,and undifferentiated or poorly differentiated organoid.Marker expression was disrupted in the basal and upper basal layers of organoids lacking P63 expression,SOX2 expression was decreased,E-cad expression was lost in the outermost cell membrane in contact with the matrix gel.Correspondingly,differentiation-related genes NOTCH 1 and NOTCH3 were down-regulated and ELF3 expression was up-regulated.Knockdown of ELF3 had no significant effect on the expression and nuclear localization of P63 and no significant effect on proliferation.But knockdown of ELF3 resulted in upregulation of the expression of genes related to the differentiation pathway,such as NOTCH 1,NOTCH3 and RBPJ,chip-seq data showed that they were the target genes directly bound by ELF3.In conclusion,this study has successfully established rat normal esophageal squamous epithelial cell lines and three-dimensional organoids culture system,which can be used as an in vitro substitute for esophageal tissues and provide a basis for esophageal-related research on differentiation regulation of esophageal,markers of esophageal stem cells and etiology of esophageal cancer.Our study revealed that TP63 and ELF3 co-regulate the proliferation and differentiation homeostasis of esophageal squamous epithelium for the first time. |
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