The Methodology Of Culturing Huh7 Liver Cancer Stem Cells And The Molecular Mechanism Of Celastrol Inhibits Huh7 Liver Cancer Stem Cells

Objective Exploring the methods on culturing Huh7 liver cancer stem-like cells and searching the molocular mechanism of celastrol inhibits stem-like cells Huh7.Methods1.Inducing the Huh7 cells by cultured it in serum-free conditions for 16 days,then reveal its stemness.(1)Primary hepatoma cells Huh7 and induced pluripotent stem cells Huh7 were dyed by Hoechst33342 and Rhodamine123,then analysised by flow cytometry.(2)The cell cycle of primary hepatoma cells Huh7 and induced pluripotent stem cells Huh7 were detected by flow cytometry.(3)The invasion ability of primary hepatoma cells Huh7 and induced pluripotent stem cells Huh7 were detected.(4)The relative expression levels of stemness protein on primary hepatoma cells Huh7 and induced pluripotent stem cells Huh7 were detected by western blot.(5)The xenograft ability of primary hepatoma cells Huh7 and induced pluripotent stem cells Huh7 were detected.2.The molecular mechanism of celastrol inhibits stem-like cells Huh7.(1)Huh7 and stem-like cells Huh7 were treated with different concentrations of celastrol(16,8,4,2,1,0.5μM and 32,16,8,4,2,1μM)for 24 h,48h and 72 h,then the cell viability was measured by CCK-8.(2)Huh7 were treated with celastrol(0,1.5μM),then cell cycle were detected by flow cytometry.(3)Huh7 were treated with different concentrations of celastrol(0,1,2,4μM),then cell apoptosis were detected by flow cytometry.(4)Huh7 were treated with different concentrations of celastrol(0,1,2,4μM),then cell spheres were detected.(5)Stem-like cells Huh7 were treated with different concentrations of celastrol(0,3,6,12μM)for 48 h.Western blot were used to detect the expression levels of Sox2,Klf4,Oct4.(6)Stem-like cells Huh7 were treated with different concentrations of celastrol(0,3,6,12μM)for 48 h.Western blot were used to detect the expression levels of Wnt/β-Catenin relevant signaling molecules.Results1.Comparing with primary hepatoma cells Huh7(1)Induced pluripotent stem cells Huh7 show less sensitive to Hoechst33342 and Rhodamine123.(2)Induced pluripotent stem cells Huh7 have higher rate on G0 cell cycle.(3)Induced pluripotent stem cells Huh7 have a stronger ability on invasion.(4)Induced pluripotent stem cells Huh7 stemness protein relative expression levels are higher.(5)Induced pluripotent stem cells Huh7 have a stronger ability on xenograft.2.(1)Celastrol displayed concentration-and-time-dependent growth inhibitory effects on Huh7 and stem-like cells Huh7.(2)Celastrol treatment led to a notable accumulation of S cell cycle arrest.(3)Celastrol can induce Huh7 cells apoptosis.(4)Celastrol can reduce Huh7 cell spheres.(5)Celastrol can inhibits the expression of Sox2,Klf4,Oct4.(6)Celastrol inhibits Wnt/β-Catenin signaling pathway.Conclusions1.The Huh7 cells which were cultured in serum-free conditions have the properties of cancer stem cells.2.Celastrol can inhitits cancer stem-like cells Huh7 by Wnt/β-catenin.