摘要
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qPCR、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，5 μmol/L）和顺铂+MK2206组（5 mg/L顺铂处理前1 h给予5 μmol/L MK2206），分别干预Hep3B细胞，Western blotting检测AKT、p-AKT和PD-L1的表达情况。siRNA-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mRNA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。siRNA-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。
Abstract
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, 5 μmol/L) and cisplatin+MK2206 group (5 μmol/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.
基金项目
安徽省高校自然科学研究重点项目(KJ2018ZD022)
蚌埠医学院研究生科研创新项目(Byycx22089)
蚌埠医学院科技项目(2022byzd096)
万方数据