顺铂对肝细胞癌Hep3B细胞程序性死亡配体1表达及药物敏感性的研究

Study on programmed death ligand 1 expression and drug sensitivity of cisplatin in hepatocellular carcinoma Hep3B cells

目的探讨顺铂对肝细胞癌Hep3B细胞程序性死亡配体1(PD-L1)表达以及PD-L1对顺铂敏感性的影响。方法使用不同浓度的顺铂(0、5、10、20 mg/L)处理Hep3B细胞,利用q PCR、Western blotting法检测细胞中PD-L1的表达情况。设置对照组(加入PBS)、顺铂组(5 mg/L)、MK2206组(AKT抑制剂,5μmol/L)和顺铂+MK2206组(5 mg/L顺铂处理前1 h给予5μmol/L MK2206),分别干预Hep3B细胞,Western blotting检测AKT、p-AKT和PD-L1的表达情况。si RNA-PD-L1转染Hep3B细胞,Western blotting检测PD-L1和上皮-间质转化标志物(E-cadherin和N-cadherin)表达变化,CCK-8、细胞划痕实验和Transwell实验检测细胞功能变化。结果顺铂可上调Hep3B细胞中PD-L1的蛋白表达水平(P<0.05)。与对照组相比,5 mg/L的顺铂显著促进Hep3B细胞中PD-L1的m RNA水平(P<0.05)。MK2206可抑制顺铂对p-AKT和PD-L1的上调(P<0.05),而AKT的蛋白水平差异无统计学意义。si RNA-PD-L1可抑制顺铂对Hep3B细胞PD-L1表达的上调(P<0.05),增强顺铂对Hep3B细胞增殖、迁移和侵袭的抑制,以及对E-cadherin的表达上调和N-cadherin的表达下调(P<0.05)。结论顺铂可促进肝细胞癌Hep3B细胞PD-L1表达,其机制可能与上调了AKT通路蛋白的磷酸化水平相关;抑制PD-L1表达可增强肝细胞癌Hep3B细胞对顺铂的敏感性。

Objective To investigate the effect of cisplatin on the expression of programmed death ligand 1(PD-L1)in hepatocellular carcinoma Hep3B cells and the effect of PD-L1 on cisplatin sensitivity.Methods Hep3B cells were treated with 0,5,10,20 mg/L cisplatin,and the expression of PD-L1 was detected by qPCR and Western blotting.Hep3B cells were treated in control group(PBS only),cisplatin group(5 mg/L),MK2206 group(AKT inhibitor,5μmol/L)and cisplatin+MK2206 group(5μmol/L MK2206 was administered 1 hour before cisplatin treatment),and the expressions of AKT,p-AKT and PD-L1 were detected by Western blotting.siRNA-PD-L1 was transfected into Hep3B cells.Western blotting was used to detect the expression of PD-L1 and epithelial-mesenchymal transition markers(E-cadherin and N-cadherin).CCK-8,scratch assay and Transwell test were used to detect the changes of cell function.Results Cisplatin could up-regulate the protein level of PD-L1 in Hep3B cells(P<0.05).Compared with the control group,5 mg/L cisplatin significantly increased the mRNA level of PD-L1 in Hep3B cells(P<0.05).MK2206 could attenuate the promoting effect of cisplatin on p-AKT and PD-L1 expression in Hep3B cells(P<0.05).There was no significant difference in the protein level of AKT.siRNA-PD-L1 could attenuate the promoting effect of cisplatin on PD-L1 expression in Hep3B cells(P<0.05),enhance the inhibitory effect of cisplatin on proliferation,migration and invasion of Hep3B cells,enhance the promoting effect of cisplatin on E-cadherin expression and the inhibitory effect of cisplatin on N-cadherin expression in Hep3B cells(P<0.05).Conclusions Cisplatin can promote the expression of PD-L1 in hepatocellular carcinoma Hep3B cells,and its mechanism may be related to the up-regulation of phosphorylation level of AKT pathway proteins.Inhibition of PD-L1 expression may enhance the sensitivity of Hep3B cells to cisplatin.