The Expression And Function Of Programmed Death Reception Ligand-1 In CD133~+ Human Liver Cancer Stem-like Cells

Objective:To investigate the expression and function of of PD-L1 in CD133~+liver cancer stem-like cells(LCSLC).Methods:LCSLC was obtained by sphere-formating culture;Expression of stemness markers was detected by flow cytometry,Expression of stem cell specific molecules was detected by Western blot and RT-q PCR respectively,The biological function of LCSLC were detected by functional assay in vitro and in vivo;Western blot and RT-q PCR were used to detect the expression of PD-L1 in LCSLC,The signaling pathways which regulate expression of of PD-L1 were investigated by specific cell signaling pathway inhibitors;siRNA was used to knocked-down the expression of PD-L1 in LCSLC,Expression of stem cell marker and tumor biological functions of LCSLC were investigated after PD-L1knockdown respectively.Results:1.Expression of CD133 was significantly increased in LCSLC than that in Hep G2(p<0.01),Expressions of Nanog,Oct4A and Snai1 in LCSLC were also higher than those in Hep G2 cells(p<0.05),The spheres formation,migration,invasion,clonal colony formation,proliferation and tumorigenicity were significantly enhanced in LCSLC cells than those in Hep G2(p<0.05 or p<0.01),Cell cycle assay showed that cell cycle of LCSLC was likely to be blocked in the G0/G1 phase;The expression of PD-L1,p-STAT3 and p-Akt were significantly up-regulated in LCSLC,Expression of PD-L1 was significantly decreased(p<0.05)by inhibition of the phosphorylation of STAT3 and Akt,The expression of p-ERK1/2 on LCSLC was down-regulated in LCSLC and the expression level of PD-L1 was not affect by inhibiting phosphorylation of ERK1/2(p<0.05);Expression of CD133,Nanog,Oct4A and Snai1 were significantly decreased by PD-L1 knockdown(p<0.05),Functional assays showed that sphere formation,migration,invasion,colony formation,proliferation and tumorigenicity of LCSLC were inhibited by PD-L1 knockdown,oppositely,transformation of LCSLC from G0/G1 phase to S phase and apoptosis of LCSLC were increased as a result of PD-L1 inhibition(p<0.05 or p<0.01),Moreover,the phosphorylation of STAT3 and Akt also were inhibited by PD-L1 knockdown.Conclusions:We enriched CD133~+LCSLC by serum-free ball culture method,CD133~+LCSLC has higher stem molecule expression and stronger tumor biological functions in vivo and in vitro,comparing normal hepatocellular carcinoma cell line Hep G2,It showed that the CD133~+LCSLC we obtained has the characteristics of cancer stem cells;The expression of PD-L1 is significantly increased in LCSLC,and this effect should be mediated by JAK/STAT3 and AKT signaling pathways;PD-L1 in LCSLC can promote the expression of CD133 and other stem molecules,maintain the tumor stem cell characteristics of LCSLC,also promote its tumor biological functions in vitro and in vivo;PD-L1 can further activate the JAK/STAT3 and AKT signaling pathways in LCSLC,which may be the potential molecular mechanism to maintain the tumor stem cell characteristics of LCSLC and promote its tumor biological functions in vitro and in vivo.