The Role Of UHRF1 And TRIB2 In Esophageal Squamous Cell Carcinoma And Its Underlying Mechanism

Part1 The role of UHRF1 in esophageal squamous cell carcinoma and its underlying mechanismPurpose: Esophageal squamous cell carcinoma(ESCC)accounts for approximately 90% of esophageal cancer cases in China,but the five-year survival rate for patients is less than 10% due to limited clinical approaches for early diagnosis and treatment.Ubiquitin-like with plant homeodomain and ring-finger domains 1(UHRF1)plays an essential role in DNA methylation,and the overexpression of UHRF1 is associated with poor prognosis in various cancers.However,the role of UHRF1 in ESCC remains unclear,our study aimed to investigate the expression of UHRF1 in ESCC,and elucidate its biological role in ESCC development.Methods: UHRF1 expression in ESCC and normal esophageal tissues was examined using immunohistochemical staining,followed by analysis of the correlation between UHRF1 expression and clinical features.In addition,the effects of lentivirus-mediated RNA interference of UHRF1 on global DNA methylation,cell proliferation,cell cycle progression and apoptosis and were investigated in ESCC cells.Results: UHRF1 was overexpressed in ESCC tissues and was an independent prognostic factor for ESCC patients.In ESCC cells,knockdown of UHRF1 caused global DNA hypomethylation,inhibited cell proliferation and induced apoptosis.Furthermore,UHRF1 depletion induced cell cycle arrest at the G2/M phase,accompanied by activation of Wee1 and DNA damage response pathway.We further observed that knockdown of UHRF1 inhibited in vivo xenograft growth.Conclusions: UHRF1 is a promising prognostic marker for ESCC and may be a potential therapy target for ESCC patients with elevated UHRF1 expression.Part2 The role of TRIB2 in esophageal squamous cell carcinoma and its underlying mechanismPurpose: Cancer stem cells(CSCs)are identified as a sub-population of cancer cells with self-renewal and differentiation potential,which can generate heterogeneous cancer cells.CSCs are responsible for tumorigenesis,metastasis,drug resistance and recurrence.However,the characteristics,molecular mechanism and the role in carcinogenesis of esophageal cancer stem cells are still unclear.TRIB2 is an important serine/threonine pseudokinase of eukaryotic cells,which plays an important role in immune function,lipoprotein metabolism,cell differentiation and proliferation.This study aimed to study the isolation,gene expression of esophageal cancer stem cells and the role of TRIB2 in ESCC.Methods: Esophageal cancer stem cells were enriched by sphere formation assay.The stemness was verified by measuring the expression of stem cell markers through q RTPCR and CD133 staining.Then,we analyzed the differentially expressed genes of esophageal cancer stem cells and esophageal cancer cells by RNA-seq,and found that TRIB2,with significantly differential expression,may be a potential regulator of the characteristics of esophageal cancer stem cells.The differential expression of TRIB2 in esophageal cancer stem cells and ESCC tissues was confirmed using q RT-PCR and bioanalysis.We constructed TRIB2 stable knockdown ESCC cells using lentiviral transfection.Then,the important role of TRIB2 in self-renewal,proliferation,cell cycle and migration were respectively examined by sphere formation assay,CCK8 and colony formation assay,PI staining and Transwell assay.Furthermore,the molecular mechanism of TRIB2 in ESCC cells was examined by q RT-PCR and Western blot.Results: Stem cell markers were high expressed in cells enriched from sphere formation assay,indicating that these cells possessed stemness.TRIB2 was high expressed in ESCC cells and ESCC tissues,while the expression of TRIB2 in esophageal cancer stem cells was higher than in ESCC cells.TRIB2 knockdown significantly inhibited the self-renewal ability,induced cell cycle arrested in G0/G1 phase,inhibited cell proliferation and migration in ESCC cells.Moreover,TRIB2 knockdown inactivated p-Smad2 and inhibited the expression of SOX2.Conclusion: TRIB2 is an important gene regulating the characteristics of esophageal cancer stem cells,and its effect may be achieved by regulating the expression of SOX2.