下调PD-L1 表达对吉非替尼耐药性非小细胞肺癌细胞的影响

Effects of down-regulating PD-L1 expression on gefitinib-resistant non-small cell lung cancer cells

摘要:

目的：研究下调程序性细胞死亡配体1（programmed cell death ligand 1，PD-L1）表达对吉非替尼耐药性（gefitinib-resistant，GR）非小细胞肺癌细胞的作用及机制。方法：通过RT-qPCR和Western blot检测各非小细胞肺癌细胞程序性细胞死亡1（programmed cell death 1，PD1）和PD-L1 mRNA和其蛋白表达水平；获得GR细胞H1703/GR，CCK-8检测细胞活性，Western blot检测PD1和PD-L1 蛋白表达；转染sh-PD-L1进入H1703/GR细胞，CCK-8检测细胞活性，流式细胞术检测细胞凋亡，Western blot检测PI3K/Akt/mTOR通路相关蛋白的表达；加入PI3K/Akt/mTOR通路激活剂IGF-1，CCK-8检测细胞活性，流式细胞术检测细胞凋亡。结果：与人永生化肺上皮细胞株16HBE相比，非小细胞肺癌细胞株GLC82、A549、PC9、SK-MES-1、H1703、L78、H460、H661中PD1和PD-L1 mRNA及蛋白表达均上调（P<0.05、P<0.01），选取H1703细胞进行后续实验；与H1703细胞相比，H1703/GR细胞活性升高，PD1和PD-L1蛋白表达均上调（P<0.01）；与GR组相比，GR+sh-PD-L1组H1703/GR细胞活性明显降低，细胞凋亡率明显增加，p-Akt/Akt和p-mTOR/mTOR蛋白表达明显下调（P<0.01）；与GR+sh-PD-L1组相比，GR+sh-PD-L1+IGF-1组H1703/GR细胞活性明显升高，细胞凋亡率明显减少（P<0.01）。结论：下调PD-L1 表达可能通过抑制PI3K/Akt/mTOR通路激活，增强非小细胞肺癌对吉非替尼的敏感性。

Abstract:

Objective：To investigate the effect and mechanism of down-regulation of programmed cell death ligand 1（PD-L1） expression on gefitinib-resistant（GR） non-small cell lung cancer cells. Methods：The expression levels of programmed cell death 1 （PD1） and PD-L1 mRNA and protein in non-small cell lung cancer cells were detected by RT-qPCR and Western blot. GR cells H1703/GR were obtained. Cell activity was detected by CCK-8. Western blot was used to detect PD1 and PD-L1 protein expression. Sh-PD-L1 was transfected into H1703/GR cells，cell activity was detected by CCK-8，apoptosis was detected by flow cytometry，and PI3K/Akt/mTOR pathway-related protein expression was detected by Western blot. PI3K/Akt/mTOR pathway activator IGF-1 was added，cell activity was detected by CCK-8 and apoptosis was detected by flow cytometry. Results：Compared with human immortalized lung epithelial cell line 16HBE，the expressions of PD1 and PD-L1 mRNA and protein in non-small cell lung cancer cell lines GLC82，A549，PC9，SK-MES-1，H1703，L78，H460，and H661 were all up-regulated（P<0.05，P<0.01），and H1703 cells were selected for subsequent experiments. Compared with H1703 cells，H1703/GR cells increased activity，and both PD1 and PD-L1 protein expressions were up-regulated（P<0.01）. Compared with GR group，GR+sh-PD-L1 group significantly reduced H1703/GR cell viability，increased apoptotic rate and decreased p-Akt/Akt and p-mTOR/mTOR protein expression（P<0.01）. Compared with GR+sh-PD-L1 group，the activity of H1703/GR cells in GR+sh-PD-L1+IGF-1 group was significantly increased and the apoptosis rate was significantly reduced（P<0.01）. Conclusion：Down-regulating PD-L1 expression enhances the sensitivity of non-small cell lung cancer to gefitinib by inhibiting activation of the PI3K/Akt/mTOR pathway.