| Objective:Exploring the resistance of EGFR wild-type lung adenocarcinoma drug-resistant cell line A549/GR to gefitinib,and through the positive and negative regulation of autophagy key gene Beclin-1 to affect autophagy,to explore the effect of autophagy on the drug resistance and stemness of lung adenocarcinoma drug-resistant cell line A549/GR,and the relationship between gefitinib resistance,autophagy,and tumor cell stemness in lung adenocarcinoma cells.Methods:1.The sensitivity difference between stable drug-resistant cell line A549/GR and common cell line A549 to gefitinib was detected by CCK8 drug sensitivity assay,and the proliferation abilitity of A549/GR and A549 cells was detected by CCK8 cell proliferation assay.2.The expression of autophagy-related proteins such as Beclin-1,LC3-II/ LC3-I and p62 in stable drug-resistant cell line A549/GR and normal cell line A549 treated with 5μg/ml gefitinib was detected by WB assay,and the changes of autophagosome number of A549 and A549/GR were detected by TEM.3.Clone formation and migration of A549/GR and A549 cells treated with 5μg/ml gefitinib were detected by clonae formation and transwell migration assay.WB assay was used to detect the expression of stemness related proteins(Nanog,Bmi-1,CD44)and drug resistance related proteins(ABCG2)in both groups after 5μg/ml gefitinib treatment.4.Transfection of drug-resistant cell lines A549/GR and A549 by lentivirus.knocking down Beclin-1 cell lines(A549/GR +sh-Beclin-1)and control cell lines(A549/GR + shNC)were constructed.Overexpressing Beclin-1 cell line(A549 + oe-Beclin-1)and control cell line(A549 + oe-NC)were constructed.TEM,CCK8 drug sensitivity assay,Clone formation,Transwell migration assay,and WB assay were used to detect the changes of autophagy level,drug resistance,proliferation and migration ability,expression of stemness-related proteins of the above constructed cell lines in the presence of 5μg/ml gefitinib.Results:1.CCK8 drug sensitivity assay showed that the resistance of stable drug-resistant cell line A549/GR to gefitinib was higher than that of its parent cell line A549(P < 0.05);CCK8cell proliferation assay showed that A549/GR cells had higher proliferation ability than A549 cells after gefitinib treatment(P < 0.05).2.The expression of autophagy-related proteins in gefitinib-treated stable drug-resistant cell line A549/GR and normal cell line A549 treated with 5μg/ml gefitinib was detected by WB.and the results showed that the expression of Beclin-1,LC3-II/ LC3-I proteins in A549/GR cells were higher than that in A549 cells,The protein expression of p62 was lower than that of A549 cell.The number of autophagosomes in A549/GR cells was significantly higher than that in A549 cells(P < 0.05).3.The ability of clone and migration of A549/GR and A549 cells treated with 5μg/ml gefitinib were detected by clonogenic assay and Transwel assay.The results showed that the number of colonies and migration cells of A549/GR was significantly higher than that of A549.WB showed that the levels of stemness-related protein Nanog,Bmi-1,CD44 and drug-resistant protein ABCG2 in A549/GR cells were higher than those in A549,with significant differences.(P < 0.05).4.Compared with A549/GR + sh-NC group,the expression of autophagy-related proteins Beclin-l,LC3-II/LC3-I decreased,the expression of p62 increased,and the number of autophagosomes decreased significantly in A549/GR + sh-Beclin-1 group,the resistance to gefitinib decreased,the number of clones formed and the number of migrating cells decreased,At the same time,the expressions of stemness and drug resistance-related proteins Nanog,Bmi-l,CD44 and ABCG2 were significantly decreased;Compared with the A549 + oe-NC group,the A549 + oe-Beclin l group had increased autophagy-related protein Beclin-l,LC3-II/LC3-I expression,decreased p62 expression,and increased autophagosome number.Gefitinib The drug resistance increased,the number of clones formed and the number of migrating cells increased,and the expression of stemness and drug resistance-related proteins Nanog,Bmi-l,CD44,and ABCG2 increased(P < 0.05).Conclusion:1.EGFR wild-type lung adenocarcinoma drug-resistant cell line A549/GR has high levels of autophagy and tumor cell stemness,The autophagy key gene Beclin-1 can regulate autophagy and then affect the stemness and gefitinib resistance in lung adenocarcinoma cell line A549/GR;2.Autophagy and stemness of tumor cells may be related to gefitinib resistance of EGFR wild-type lung adenocarcinoma drug-resistant cell line A549/GR。... |
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