The expression and function of PD-L1 in CD133(+) human liver cancer stem-like cells / 中华肿瘤杂志

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.

Expression and role of programmed cell death ligand-1 in sorafenib-resistant hepatocellular carcinoma cells / 第二军医大学学报

Objective To explore the expression and role of programmed cell death ligand-1 (PD-L1) in sorafenib-resistant human hepatocellular carcinoma cells. Methods Sorafenib-resistant human hepatocellular carcinoma cell lines (Hep3B-SR, HepG2-SR) were established by the procedure of stepwise increase in sorafenib concentrations. CCK-8 assay was used to detect half inhibition concentration (IC50). The expressions of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and PD-L1 were examined by Western blotting and qPCR. The expression of PD-L1 was silenced by transfecting siRNA into the drug-resistant cells, and the silencing efficiency was tested. After silencing the expression of PD-L1 in the drug-resistant cells, CCK-8 assay was used to detect IC50, the drug-resistance index was calculated, and the expressions of P-gp and MRP1 were examined. Then cell proliferation assay, wound healing assay, colony formation assay and flow cytometry were applied to examine cell proliferation, migration, clone formation and apoptosis, respectively. Results The drug-resistance indexes of Hep3B-SR and HepG2-SR were 5.4 and 5.2, respectively. The expressions of P-gp, MRP1 and PD-L1 in the drug-resistant cells were significantly up-regulated in comparison with the parental cells (all P0.01). After inhibiting the expression of PD-L1, the drug-resistance indexes of Hep3B-SR and HepG2-SR decreased to 1.8 and 1.5, respectively, and the expressions of P-gp and MRP1 were down-regulated (all P0.01). Silencing the PD-L1 expression could significantly inhibit the proliferation, migration and colony formation of drug-resistant cells, and could significantly promote the apoptosis (all P0.01); and these effects were strengthened by combining sorafenib. Conclusion PD-L1 is highly expressed in sorafenib-resistant hepatocellular carcinoma cells. Inhibition of PD-L1 expression can partially reverse the drug-resistance of the cells and significantly enhance the anticancer effect of sorafenib. Inhibiting the expression of PD-L1 can effectively repress the growth of sorafenib-resistant hepatocellular carcinoma cells, which is more if combining with sorafenib.