| Objective 1.Objective to establish a stable cells line of human non-small cell lung cancer(A549/GR)with gefitinib resistance,and to provide a good drug resistance cell model for the study of the molecular mechanism of acquired resistance to gefitinib in non-small cell lung cancer;2.To investigate the relationship between insulin like growth factor 1 receptor(IGF-1R)and its downstream signaling molecule-phosphorylated protein kinase B1(p-AKT1)and the acquired resistance of gefitinib in A549 cells;3.Overexpression of miR-497 in gefitinib-resistant cells,to investigate the effects of miR-497 on IGF-1R signaling pathway in acquired resistance to gefitinib in non small cell lung cancer cells.Methods 1.Culture the A549 parental cells line of non small cell lung cancer in vitro sterile environment,and establish gefitinib resistant non-small cell lung cancer cells line A549/GR by the method of gradually increasing dosage of low concentration drugs;2.The morphological changes of A549/GR cells were observed in natural growth condition or HE staining satatus under the inverted microscope,and MTT assay was used to determine the cell viability of A549 and A549/GR cells line with different concentrations of drugs,and to determine the resistance of A549/GR cells to gefitinib;3.The expression of IGF-1R protein and p-AKT1 in A549 and drug resistant A549/GR cells was detected by Western Blot;4.The drug resistant cells line A549/GR was used as the model to be overexpressed miR-497 by the method of RiboFECTTMCP mediated miR-497 mimic to transient transfection,and the transfection effect was observed by the fluorescence microscope;5.MTT experiment was used to test the changes of resistance in the resistant cells after the transfection ofmiR-497;6.Western blot was used to detect the expression of IGF-1R protein and its downstream signaling molecule p-AKT1 in drug-resistant cells after the transfection.Results 1.The gefitinib resistant A549/GR cells could grow stably in the medium containing 40?mol/L concentration of gefitinib after the induction of gefitinib for 10 months;2.Both the A549 cells and A549/GR cells were adherent cells,and their size of cells were diferent;unlike the morphology of A549 cells which were mainly polygonal,the A549/GR cells were fusiform and its nuclear fission phenomenon was more obvious;3.The half inhibitory concentration(IC50)of A549 cels line was(8.90±0.48)?mol/L,and the A549/GR cells line was(51.96±2.39)?mol/L(p<0.01)which was detected by MTT,and the resistance index was 5.84;4.The results of Western blot showed that the expression of IGF-1R in A549/GR cells was1.36 times higher than that of A549 cells(p<0.05),while the expression of p-AKT1 was about 1.37 times higher(p<0.05);5.After the miR-497 mimic was transfected into A549/GR cells line,fluorescence microscope showed that the transfection effect was good(as Figure 4);6.The IC50 of A549/GR-miR497-mimic cells line whice was transfected with miR-497 mimic was(33.76±0.97)?mol/L,and it was 0.65 times lower than the control group(p<0.01);7.The expression of IGF-1R and p-AKT1 protein in A549/GR-miR497-mimic cells line with high expression of miR-497 was lower than that in negative control group(p<0.05).Conclusions 1.A stable gefitinib resistant non-small cell lung cancer A549/GR cells line was successfully established,and the morphological changes of the cell was consistent with the drug resistance;2.In the gefitinib resistant cells line A549/GR,the expression of IGF-1R protein and its downstream signal molecule p-AKT1 was more than the parental cell,and it's speculated that the activation of IGF-1R and its downstream AKT1 signaling pathway can promote the non-small cell lung cancer cells to acquired the gefibinib resistance;3.After the high expression of miR-497 in gefitinib-resistant cells line A549/GR,the drug resistance of the resistant cells to gefitinib was decreased,and it was speculated that mi R-497 could improve the sensitivity of A549/GR cells to epithelial growth factor receptor tyrosine kinase inhibitor(EGFR-TKIs)drugs;4.After the high expression of miR-497 in resistant cells line A549/GR,the expression of IGF-1R and p-AKT1 was lower than the controlgroup,and it's infered that miR-497 can inhibit the expression of IGF-1R and the activity of the downstream AKT1 signal pathway,futher,improve the sensitiveness of non-small cell lung cancer(NSCLC)cells to EGFR-TKIs. |
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