Effect Of Silencing FGL1 On Sensitivity Of Gefitinib In Lung Adenocarcinoma Cells

Objective: FGL1 lentivirus vector was used to target the expression of silenced fibrin original protein 1(FGL1),and the effect of silenced fibrin original protein 1(FGL1)on the drug sensitivity and related biological behaviors of lung adenocarcinoma cells was observed.Methods:(1)Culture of Gefitinib sensitive cells PC9 and acquired drug-resistant cells PC9/GR and detection of the relative expression level of FGL1: PC9 and PC9/GR cells were cultured,and the relative expression level of FGL1 in the two strains was detected by Western Blot.(2)PC9/GR cells were infected with FGL1 lentiviral vector(Lv-FGL1)and FGL1 expression was detected: The Control group was set as PC9/GR cells without any treatment;PC9/GR-Lv FGL1 cells were infected with FGL1 lentiviral vector;PC9/GR-Lv NC cells were infected with empty vector;and FGL1 expression was detected by Western Blot.(3)Detecting the drug resistance in each group of PC9,PC9/GR,PC9/GR-Lv NC and PC9/GR-Lv FGL1: CCk-8 was used to detect the proliferation inhibition of Gefitinib at different concentrations(0,0.008,0.04,0.2,1,5,10,20,40 mol/L)on PC9,PC9/GR,PC9/GR-Lv NC and PC9/GR-Lv FGL1 groups and calculate the half inhibitory concentration(IC50).(4)Designed experimental grouping and detected the apoptosis rate and clone formation ability of each group: the following five groups were set for the experiment: Control group,Lv NC group,Gefitinib group,Lv FGL1 group and Gefitinib+Lv FGL1 group.(5)Expressions of apoptosis-related proteins in cells of each group were detected:expression levels of Bax,Bcl-2,Caspase-3 and Cleaved-Caspase-3 were detected by Western Blot.Results:(1)Compared with PC9 cells,FGL1 was highly expressed in gefitinib-resistant PC9/GR cells(P<0.01).(2)The infection efficiency of lentiviral vector infection in PC9/GR cells was more than80%,and Western Blot showed stable and low expression of FGL1 in PC9/GR-Lv FGL1cells(P<0.01).(3)PC9/GR cells showed significantly higher drug resistance than PC9 cells(P<0.001),and the Gefitinib IC50 of PC9/GR-Lv FGL1 cells was significantly lower than that of PC9/GR-Lv NC and PC9/GR cells(P<0.001).(4)Compared with the Control group,the clone formation rate of the Gefitinib group,Lv FGL1 group and the combination group all decreased to different degrees(P<0.05),and the cell apoptosis rate also increased to different degrees(P<0.05),with the most significant changes in the combination group.There was no significant difference in clone formation rate and apoptosis rate between Lv NC group and Control group(P>0.05).(5)Compared with the Control group,the expression of Bax,Bcl-2 and Caspase-3 in the Gefitinib group showed no significant difference,while the expression of Cleaved-Caspase-3 was up-regulated,with statistically significant difference(P<0.05).After FGL1 was silenced,Bcl-2 protein expression was significantly down-regulated and Bax protein expression was significantly up-regulated,and Gefitinib was combined with the most significant changes,with statistically significant differences(P<0.05).In addition,there was no significant difference in the expression of caspase-3 protein in each group,while Cleaved caspase-3 protein was significantly up-regulated in cells with low FGL1 expression,with statistically significant differences(P<0.05).There was no significant difference in protein expression between Lv NC group and Control group(P>0.05).Conclusion: Silencing FGL1 gene could improve the sensitivity of gefitinib-acquired resistant cells to gefitinib in lung adenocarcinoma,inhibit the proliferation and growth of resistant cells,and promote the apoptosis of resistant cells,which might be related to up-regulation of pro-apoptotic protein Bax,Cleaved-Caspase-3 and down-regulation of anti-apoptotic protein Bcl-2.