SOX2 Promotes The Proliferation And Invasion Of Esophageal Squamous Cell Carcinoma Via Wnt/β-catenin Signaling Pathway

Objective: To explore the mechanism of SOX2 via Wnt/β-catenin signaling pathway and the key protein of this pathway,β-catenin,in regulating the proliferation,migration and invasion of esophageal squamous cell carcinoma(ESCC).Methods:(1)Single-cell data of ESCC(GSE160269)were analysed by bioinformatics methods to observe the m RNA expression of SOX2 in single-cell data of ESCC;the differential changes of SOX2 expression at high/low levels affecting the related signalling pathway were explored in the TCGA database of ESCC data.(2)At the tissue level,the expression of SOX2,β-catenin,E-cadherin and vimentin in ESCC tissues and adjacent normal tissues were detected by immunohistochemistry,and the correlation between positive expression of SOX2 and β-catenin and clinicopathological parameters and prognosis of patients with ESCC was analyzed;SOX2,β-catenin,epithelial mesenchymal transition markers and correlation between the three subtypes of epithelial mesenchymal transition,and analyze their correlation with survival prognosis.(3)At the cellular level:(1)The background expression of SOX2 and β-catenin in different ESCC lines and normal esophageal epithelial cell lines were detected by Western-blot and q PCR,and the final experimental cell lines were screened;using lentiviral transfection technique,SOX2 and β-catenin were interfered in TE-1 cell line,respectively,by Western-blot,q PCR,CCK-8,cell scratch assay,flow cytometry cell cycle assay,flow cytometry apoptosis assay,Transwell and other assays to observe the changes in proliferation,migration and invasion ability of the cells after SOX2 and β-catenin expression changes.(2)Western-blot,immunofluorescence,dual-luciferase reporter and immunoprecipitation assays were applied to observe the interaction between SOX2 and β-catenin,and the relationship between the two and star molecules such as Wnt2 and GSK3β in the Wnt/β-catenin signalling pathway.(3)The β-catenin overexpressing stable cell line was second transfected with sh SOX2,and the proliferation,migration and invasion of tumor cells were observed by Western-blot,CCK-8,flow cytometry cell cycle detection and Transwell assays to see whether SOX2 could regulate β-catenin via Wnt/β-catenin signaling pathway.(4)Animal level:(1)Using subcutaneous tumour formation assay in nude mice to detect the tumourigenic ability of β-catenin and its role in regulating tumour proliferation and invasion.(2)Using HE staining,immunohistochemical staining and Western-blot of transplanted tumours to investigate the interaction between SOX2 and Wnt/β-catenin signalling pathway and β-catenin.Results:(1)Part I:(1)Single cell transcriptome sequencing analysis revealed that SOX2 was only expressed in esophageal epithelial cells and was highly expressed in esophageal squamous carcinoma epithelial cells,1.26 times more than normal esophageal epithelial cells.(2)There was an interaction between SOX2 and β-catenin protein.(3)In esophageal squamous carcinoma,SOX2 was associated with epithelial mesenchymal transition and Wnt/β-catenin signaling pathway.(2)Part II:(1)In esophageal squamous carcinoma tissues,SOX2 was expressed in both cancerous tissues and normal mucosa adjacent to cancer,and the positive expression in ESCC tissues was significantly higher than that in normal tissues adjacent to cancer(P<0.001);The positive expression of β-catenin in ESCC tissues was significantly higher than that in normal tissues adjacent to cancer(P<0.001).(2)The positive expression of SOX2 in ESCC expression was positively correlated with tumor size(r=0.382,P<0.001)and depth of infiltration(r=0.281,P<0.001);β-catenin positive expression was positively correlated with distant metastasis(r=0.181,P<0.05);E-cadherin positive expression was correlated with the degree of tumor differentiation(P<0.05);Vimentin positive expression was positively correlated with the depth of tumor infiltration(P<0.05).(3)SOX2 positive expression was positively correlated with β-catenin and vimentin positive expression,with correlation coefficients of r=0.305 and r=0.316,respectively(both P<0.05);β-catenin positive expression was positively correlated with vimentin and E-cadherin positive expression,with the correlation coefficients were r=0.187 and r=0.151,respectively(all P<0.05).(4)The results of KaplanMeier survival analysis showed that positive expression of SOX2,β-catenin and vimentin was negatively correlated with the overall survival time of patients with esophageal squamous carcinoma,i.e.patients with positive expression had a short survival and poor prognosis(all P<0.05).(5)According to the results of E-cadherin and vimentin expression classified epithelial-mesenchymal transition(EMT)into three subtypes,and the expression of the three subtypes correlated with the depth of infiltration,which was higher in the mesenchymal than in the epithelial type(P=0.003).(6)SOX2,β-catenin positive expression was positively correlated with EMT staging,with correlation coefficients of r=0.149 and r=0.252,respectively(both P<0.05).(7)Kaplan-Meier survival analysis showed that the three subtypes of EMT correlated with the overall survival prognosis of patients with ESCC(both P<0.05);patients with interstitial and mixed types had a poor prognosis.(8)Multifactor COX regression analysis: The results showed that for patients with ESCC,the expression of β-catenin and the degree of differentiation may be independent risk factors affecting their prognosis.(3)Part Ⅲ:(1)Both SOX2 and β-catenin can promote the proliferation,invasion,migration and EMT of ESCC.(2)SOX2 and β-catenin interact positively,SOX2 can affect the expression of β-catenin and regulate the proliferation,EMT,migration and invasion of ESCC through the Wnt/β-catenin signaling pathway.And SOX2 is a key gene in the SOX2/Wnt/β-catenin positive feedback loop.(3)Although β-catenin and SOX2 have a mutual binding effect,β-catenin does not directly bind the SOX2 promoter to promote increased SOX2 expression.(4)Part IV:(1)β-catenin promoted the development and progression of subcutaneous tumorigenesis in nude mice.(2)sh SOX2 partially inhibited tumor growth,EMT by suppressing β-catenin expression through the Wnt/β-catenin signaling pathway.(3)SOX2 and β-catenin positively reciprocally regulated the onset and development of subcutaneous tumorigenesis in nude mice.Conclusions:(1)SOX2 is a key differentially expressed gene in esophageal squamous carcinoma epithelial cells.(2)SOX2 and β-catenin promote proliferation,EMT,migration and invasion of ESCC.(3)SOX2 interacts with β-catenin via the Wnt/β-catenin signaling pathway to form a positive feedback loop to promote proliferation,EMT,migration and invasion of ESCC.(4)SOX2 is an important gene in the SOX2/Wnt/β-catenin signaling axis,which influences the proliferation,migration and invasion of ESCC.