Overexpression Of NRF3 Reduces The Sensitivity Of Lung Adenocarcinoma Cells To Gefitinib And Its Mechanism

Objective:To investigate the expression level of NRF3 in lung adenocarcinoma and the effect of overexpression of NRF3 on the sensitivity of lung adenocarcinoma cells to gefitinib.Methods:The expression levels of NRF3 in lung adenocarcinoma tissues and adjacent tissues were predicted by bioinformatics methods.The expression of NRF3 in lung adenocarcinoma tissues and adjacent tissues was detected by immunohistochemistry.NRF3 overexpression cell lines were established by virus transfection of PC9 and A549 cells;The expression of NRF3 in NRF3 overexpression cells was verified by qPCR and Western blot.CCK-8 assay and EdU cell proliferation assay were used to detect the effects of different concentrations of gefitinib(0,4,8,16,32μg/ml)on the proliferation of PC9,lapNRF3-PC9,A549,lapNRF3-A549 cells.Flow cytometry was used to detect the effects of different gefitinib concentrations(0,4,8,16 μg/ml)on the cell cycle of PC9,lapNRF3-PC9,A549,and lapNRF3-A549 cells.The mRNA expression levels in PC9 and lapNRF3-PC9 cells were analyzed by transcriptome sequencing.Western blot was used to detect the effects of different gefitinib concentrations(0,4,8,and 16 μg/ml)on cyclinB1,CDK1,MLK1(MAP3K9),and p-JNK1/2 protein levels in PC9,lapNRF3-PC9,A549,and lapNRF3-A549 cells.Results:Bioinformatics analysis showed that the mRNA NRF3 expression level in lung adenocarcinoma tissues was higher than that in adjacent tissues.The immunohistochemical detection showed that the expression level of NRF3 protein in lung adenocarcinoma tissues was higher than that in adjacent tissues.CCK-8 assay showed that after PC9,lapNRF3PC9,A549 and lapNRF3-A549 cells were treated with different concentrations of Gefitinib,the proliferation inhibitory effect of different Gefitinib on NRF3 overexpression cells was reduced compared with the control group.EDU cell proliferation assay showed that the number of proliferating cells in PC9,LAPNRF3-PC9,A549 and LAPNRF3-A549 cells treated with different concentrations of gefinitinib was higher than that in the control group.The cell cycle of PC9,lapNRF3-PC9,A549NRF3-PC9,A549 and lapNRF3-A549 cells were detected by flow cytometry.The results showed that compared with the control group,the percentage of cells in S phase of NRF3 overexpression cells increased,and the percentage of cells in G2/M phase decreased.Through transcriptome sequencing analysis,it was found that compared with PC9 cells,the MLK1(MAP3K9)expression in NRF3 overexpression cells was up-regulated.At the same time,the expression of cell cycle-related proteins in PC9,lapNRF3-PC9,A549,lapNRF3-A549 cells treated with different concentrations of Gefitinib was detected by Western blot.The results showed that the expression of cyclinB1 and CDK1 in PC9 and A549 cells increased,and the expression of cyclinB1 and CDK1 in NRF3 overexpression cells decreased.The mechanism was further explored by Western blot,and it was found that the expression levels of MLK1 and p-JNK proteins in NRF3 overexpression cells were increased.Conclusion:Compared with adjacent tissues,NRF3 was up-regulated in lung adenocarcinoma tissues.Up-regulation of NRF3 expression could increase the percentage of S-phase cells and decrease the percentage of G2/M-phase cells in lung adenocarcinoma cells treated with different concentrations of Gefitinib,resulting in increased cell proliferation.Overexpression of NRF3 may regulate cell cycle-related proteins through MAPK/JNK signaling pathway,thereby reducing the sensitivity of lung adenocarcinoma to Gefitinib.