| Objective:To investigate the effect of autophagy on drug sensitivity of gefitini-resistant lung adenocarcinoma PC9/GR and programmed death ligand-1(PD-L1).Methods:1.Culture of PC9 parental cells and acquired drug resistant PC9/GR cells and detection of autophagy related indicators: PC9 and PC9/GR cells were cultured,and q RT-PCR and Western Blot were used to detect the expression levels of autophagy related indicators in the two kinds of cells.2.The gefitinib IC50 of PC9 and PC9/GR cells and the cell proliferation of each group were detected by design group: CCK-8 was used to detect the proliferation inhibition of gefitinib on PC9 and PC9/GR cells at different concentrations(0,0.04,0.2,1,5,25,50,100 μmol/L),and the corresponding IC50 was calculated.PC9/GR grouping experiment was divided into the following six groups: Control group,chloroquine(CQ)(50μmol/L)group,3-methyladenine(3-MA)(5mmol/L)group,Gefitinib group(1μmol/L),Gefitinib+3-MA group,and Gefitinib+CQ group.The cell proliferation of the six groups was detected by CCK-8 method.3.Western Blot was used to detect the expression of basal PD-L1 in PC9 cells and PC9/GR cells,and the changes of autophagy level and PD-L1 content in PC9/GR group treated with 3-MA(5mmol/L)for 24 h.4.Experiment groups were designed to detect the changes of PD-L1 after 3-MA inhibition of PC9/GR autophagy level by different concentrations and different time.PC9/GR concentration groups were divided into Control group,3-MA(2.5μmol/L)group,3-MA(5mmol/L)group,and 3-MA(10mmol/L)group.PC9/GR was divided into Control group,3-MA(1h)group,3-MA(4h)group,3-MA(8h)group,3-MA(24h)group and 3-MA(48h)group for time.Proteins were extracted from cells in the above groups,and autophagy level and PD-L1 expression were detected.5.Experimental grouping was designed to detect the expression of PD-L1 after different drugs regulated the autophagy level of PC9/GR: PC9/GR was grouped into Control group,3-MA(5μmol/L)group,CQ(50μmol/L)group and Rapamycin(RAP)(100 N mol/L)group.The above groups were collected to extract proteins and detect the autophagy level and the expression of PD-L1.6.Detection of PD-L1 changes after upregulating the autophagy level of PC9 parental sensitive cells: PC9 cells were collected,RAP(100N mol/L)was applied to PC9 cells for 24 h,and proteins were extracted to detect the autophagy level and PD-L1 expression.Results:1.Compared with gefitinib-sensitive PC9 parental cells,autophagy-related protein LC3II/LCI was highly expressed in gefitinib-resistant PC9/GR cells(P<0.001).2.CCK8 results showed that the IC50 of PC9/GR cells to gefitinib was higher than that of PC9 cells to gefitinib(P<0.001);Compared with Control group and Gefitinib group(1μmol/L),the inhibitory rate of cell proliferation in Gefitinib+3-MA group and Gefitinib+CQ group was significantly increased(P<0.01);3.Compared with PC9 cells,the expression of PD-L1 was significantly increased in PC9/GR(P<0.05),PD-L1 expression was decreased after 3-MA inhibited PC9/GR autophagy(P<0.05);4.Western blot results showed that the expression of PC9/GR PD-L1 after 3-MA treatment at different concentrations and at different times decreased to different degrees compared with the Control group(P<0.05),and 3-MA(5mmol/L)group and3-MA(24h)group were the most obvious reduction compared with Control group;5.Compared with Control group,the inhibition of PC9/GR autophagy in 3-MA(5μmol/L)group and CQ(50μmol/L)group decreased PD-L1 compared with Control group(P<0.05);After PC9/GR autophagy was induced in RAP(100N mol/L)group,PD-L1 increased compared with Control group(P<0.05);6.Compared with the Control group,after PC9 autophagy was induced by RAP(100N mol/L),PD-L1 was increased compared with the Control group(P<0.05).Conclusion:Inhibition of autophagy in vitro partially restored the sensitivity of lung adenocarcinoma resistant cells to gefitinib,a process in which PD-L1 can be regulated by autophagy,suggesting that PD-L1 may be involved in the process of reversing gefitinib resistance by inhibiting autophagy. |
