Expression And Functional Regulation Of Stemness Gene Lgr5 In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal carcinoma(EC)is the eighth most common cancer and the sixth leading cause of cancer death around the world.Esophageal squamous cell carcinoma(ESCC)is the main histopathological subtype of EC in East Asians,with a 5-year survival rate <30%.Although there are advanced therapeutic treatments for ESCC such as chemotherapy,radiotherapy,surgery and target therapy,many ESCC cases progress or recur,and prognoses is poor.Emerging evidence indicates that cancer stem cells(CSCs)contribute to tumor maintenance,tumor progression,therapy resistance,and distant metastasis.CSCs are defined as a rare subpopulation of undifferentiated cells with biological characteristics that include the capacity for self-renewal,differentiation into various lineages,and tumor initiation.Mounting evidence demonstrates that CSCs can be enriched and maintained using a spheroid body formation assay in serum-free medium at low adherence,and isolated using fluorescence-activated cell sorting(FACS)according to CSCs-specific cell surface markers.Lgr5 is a member of the G-protein-coupled receptor(GPCR)family of proteins,and is a regulated target of Wnt signaling.Lgr5 contains a large extracellular domain with 17 leucine-rich repeats and a seven-transmembrane domain typical of the rhodopsin family of GPCRs.Barker et al.discovered that Lgr5 expression is restricted to the crypt of the small intestine and colon and that single sorted Lgr5+ crypt base columnar cells can also initiate crypt-villus organoids.Therefore,Lgr5 is a marker of stem cells in the small intestine and colon.In recent years,studies have revealed that Lgr5 is overexpressed in various types of tumors,including colorectal cancer,hepatocellular carcinoma,ovarian cancer,glioma,basal cell carcinoma and gastric carcinoma.The Wnt/β-cateninsignaling pathway plays a critical role in the regulation of stem and cancer stem cells,and abnormally activated Wnt/β-catenin pathway is usually associated with tumorigenesis,including ESCC.Objective1.To explore the expression and clinical significance of Lgr5 in Esophageal squamous cell carcinoma(ESCC)tissues,ESCC cells and spheroid body-forming cells which were identified have high stemness in ESCC;2.Through the high expression of CSCs-related genes of ESCC cells and high tumorigenicity in vivo to identify high stemness in ESCC spheroid body-forming cells;3.To analyse the ability of proliferation,migration and invasion,the expression of CSCs-related genes and Wnt/β-catenin signaling,while silencing of Lgr5 expression in the ESCC cells by siRNA.MethodsThe expression level of Lgr5 was assessed in 280 cases of ESCC and 150 cases of normal Esophageal squamous epithelia tissues by immunohistochemistry;Kaplan-Meier plots were used for survival analysis of ESCC patients;We used the method of ultra low attachment culture to develop spheroid body-forming cells in ESCC cell line KYSE450;The expression levels of CSCs-related genes and Lgr5 in spheroid body-forming cells and parental cells were detected via Quantitative real-time PCR(qRT-PCR)and Western Blot;Parental cells and spheroid body cells were injected into BALB/c nude mices to investigate the stemness of spheroid body cells;The application of siRNA was used to transient transfect KYSE450 cell line,CCK8 assay and Transwell assay were used to measure proliferation,migration and invasion ability,qRT-PCR and western blot assay were used to explore the expression of CSCs-related genes and Wnt/β-catenin relative members.Results1.The expression level of Lgr5 in ESCC was significantly higher,compared to normal Esophageal squamous epithelia.The high expression of Lgr5 was correlated with lymph node metastasis,depth of invasion,tumor size,advanced differentiation,tumor stage and poorer survival;2.The expression of Lgr5 and CSCs-related genes were all high in ESCC spheroid body cells;3.Spheroid body cells display high tumorigenic potential in vivo;4.Silencing of Lgr5 inhibits the proliferation,migration and invasion of ESCC cells;5.Silencing of Lgr5 reduces the expression of CSCs-related genes,SOX2,LDH1A1,NANOG;6.Silencing of Lgr5 reduces activity ofβ-catenin and cyclinD1、c-Myc,which are important target genes of the Wnt/β-catenin signaling pathway.ConclusionLgr5 may be a biomarkof recurrence,progression,invasion and migration of ESCC,the overexpression of Lgr5 in ESCC may identify the ESCC’s stem-like properties,and Lgr5 may play a key role in maintaining ESCC stem-like cells through Wnt/β-catenin signaling.Thus,Lgr5 has the potential to be an important cell surface marker of ESCC stem cells and it may be used as a molecular target for the development of treatments for ESCC.