Mechanism Of Abnormal Differentiation Of Bladder Urothelium

ObjectiveBladder cancer is a common malignant tumor of urogenital system.Firstly,we explored the method to differentiate human pluripotent stem cells(hPSC)to specific bladder urothelial cells(BUC)using a chemically defined system.The mechanism of bladder tissue regeneration and bladder cancer in vitro was studied by this differentiation scheme.Secondly,based on the unclear regulatory genes and mechanisms of tumor epithelial-mesenchymal transformation(EMT)and lacking of drugs and therapies targeting tumor EMT,we intend to sequence the clinical samples of bladder cancer by single cell sequencing to find out the highly EMT subpopulations and identify the characteristic genes and biological processes related to EMT in these cells.Meanwhile,we elucidated the molecular mechanism of the corresponding genes promoting the progression and metastasis of bladder cancer at the level of cell subsets.By further revealing the upstream related pathways and downstream targets and mechanisms of action,we can offer the supplement the biological mechanism of cancer EMT.MethodshESC were differentiated into endodermal cells(DE)and further differentiated into BUC cultured in the retinoic acid-treated keratinocyte-specific serum-free medium(KSFM).In order to optimize the differentiation stage of DE,two cell culture supplements,B27 and ITS,were added into serum-free medium to detect the effect of differentiation.In order to prove that DE cells can successfully differentiate into BUC by a chemically defined system,we used DMEM/F12 mixed medium to compare with KSFM.Total RNA content of specific biomarkers gene was determined by real time RT-PCR.Fluorescence immunoassay was used to determine the expression of specific genes in the final differentiated BUC.ResultsWe successfully step-by-step differentiated hESC into DE and BUC in a retinoic acid treated serum-free medium.The results showed that the cell death rate was slightly higher when B27 was used in the stage of DE differentiation,but both CXCR4 and SOX17 were higher than that when ITS was used.We also found that the expression of urothelial specific genes such as CK20,UPII and UPIb was significantly increased in KSFM group.Immunocytochemistry results showed that UPIIIA and CK8/18/20 were significantly expressed in differentiated BUC.In addition,the basic characteristics of 4 patients with high-grade invasive bladder cancer were identified by single-cell sequencing and thedata was analyzed.It was confirmed that alpha-glucosidase 2(GANAB)is a regulatory gene highly associated with EMT,which significantly promotes the migration,invasion and characteristics of cancer stem cells.In addition,we will further reveal its upstream pathways,downstream targets,and the biological mechanism of EMT,which will provide theoretical supporting for the clinical prevention and control of bladder cancer.ConclusionshESC were successfully differentiated into DE and BUC in a retinoic acid treated serum-free medium.We identified that alpha-glucosidase 2(GANAB)is a regulatory gene highly associated with EMT,which significantly promotes the migration,invasion and stem cell characteristics of bladder cancer.Figure 9 table 1 reference 66...