| Background: Bladder cancer is a common malignant tumor among the urinary system.It can be divided into muscle invasive tumor and non-muscle invasive tumor.The main treatment method to bladder cancer is surgical resection,supplemented by radiotherapy and chemotherapy.However,due to postoperative recurrence and chemotherapy resistance,it often leads to poor prognosis.Genetic and environmental factors are recognized as carcinogenic factors,such as age,smoking,and industrial compound products.Smoking is considered to be the most important independent risk factor for bladder cancer.Epithelial-mesenchymal transition(EMT)is a process in which epithelial cells acquire interstitial properties and plays an important role in tumorigenesis and development.Cancer stem cells(CSCs)are multi-directional differentiation potentials,mostly in quiescent tumor-initiating cells.The main features including: drug resistance,self-renewal,cloning into spheres,multi-directional differentiation and high-efficiency tumorigenicity.Recently,studies have shown that the acquisition of cancer resistance is closely related to cancer stem cells.There is also a great connection between CSCs and EMT,which can promote the expression of CSCs during EMT.A large number of studies have shown that smoking promotes bladder cancer proliferation and EMT,and the promotion of smoking on cancer stem cells has also been confirmed in pancreatic cancer and oral cancer.Objective: The effect of smoking on bladder CSCs has not been reported.The main purpose of this study was to investigate the effects of smoking on the stemness and EMT of bladder CSCs.Methods: We used serum-free medium(SFM)culture system to enrich the bladder cancer stem cells from bladder adherent cells EJ and UM-UC-3.The obtained spheroid cells have stem cell characteristics and high expression of bladder CSCs markers(CD44,CD133,Oct4,ALDH1A1,Nanog).Flow cytometry was used to detect the content of CD44 positive cells and the cell cycle in spheroid cells and adherent cells.Based on previous literature methods,we established an in vitro smoking model to obtain a cigarette smoke extract(CSE).Then we used different concentrations of CSE to treat bladder cancer stem cells,and detected the expression of stem cell markers,CD44 positive cells,EMT marker expression and cell proliferation related proteins.At the same time,we examined the expression of the Sonic Hedgehog signal pathway related proteins in this process and further validated the role of the Sonic Hedgehog signal pathway using the specific inhibitors of the pathway,Vismodegib and Gli1-si RNA.Finally,we examined the expression of bladder CSCs and Sonic Hedgehog signal pathway related proteins in smoker and non-smoker bladder cancer specimens.Results: In the present study,bladder cancer UM-UC-3 and EJ cells were maintained in serum-free medium(SFM)for acquiring tumorspheres characteristic of bladder CSCs.Investigation about the effects of cigarette smoke(CS)on bladder CSCs stemness and EMT demonstrated that CS enhanced tumorsphere formation capacity,increased the expression of CSCs markers(CD44,CD133,ALDH1A1,Oct4,Nanog),elevated CD44+ cell population and promoted EMT process.It was further illustrated that Sonic Hedgehog(Shh)pathway regulated CS-triggered EMT and stemness.Moreover,we also revealed that higher expression of bladder CSCs markers and Shh pathway related-proteins were detected in smoker bladder cancer specimens than in non-smoker cancer specimens.Conclusion: Findings from this study could provide new insights into the critical role of Shh pathway in CS-promoted stemness and EMT of bladder CSCs as well as the potential target for bladder cancer intervention. |
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