The Significant Of EGFR D896N Mutation In Non-small Cell Lung Cancer

Background Lung cancer,especially non-small cell lung cancer,is an important factor causing the death of patients around the world.In recent years,epidermal growth factor tyrosine kinase inhibitor(EGFR-TKI)has been considered an effective treatment for non-small cell lung cancer.The drug,targeting the epidermal growth factor receptor(EGFR)which is a multi-domain transmembrane tyrosine kinase receptor,consists of the extracellular coordination region,the transmembrane region and the tyrosine kinase region with the regulation of the terminal carboxyl group in the cell.Activation of EGFR can activate a variety of downstream signaling pathways,which plays an important role in the proliferation,differentiation and angiogenesis of cells.EGFR mutations are associated with the development of a variety of tumors.Studies show that about 40% of NSCLC patients carry positive EGFR mutations.The most commonly known EGFR-positive mutations are the 19 exon deletion mutations and the 21 exon L858 R mutations.The first generation of EGFR-TKI such as gefitinib and erlotinib work through binding to EGFR tyrosine kinase as well as blocking EGFR activation and downstream signaling.Although a substantial proportion of EGFR-positive patients with NSCLC have a good response to targeted therapy,not all EGFR kinase mutations are drivesor drug-sensitive.D896 N,a rare EGFR mutation,was detected clinically in lymphoid tissue from a patient with non-small cell lung cancer.D896 N is a point mutation at codon 896 on EGFR which resulting in asparagine being substituted for aspartic acid.According to the sequence of EGFR gene on NCBI,this site is located in the tyrosine kinase domain of EGFR.After the mutation was measured,the patientwas treated with gefitinib.The lymph nodes were contracted after treatment.There have been no relevant report on this site so far.The meaning of this mutation is unknown.The study on it could be valuable and of guiding significance in future clinical practice.For those reasons,we explored the significance of EGFR mutation by constructing a cell line carrying the mutation and observing the changes of the cell line in the growth,proliferation and EGFR protein expression.Objective To construct a recombinant plasmid carrying D896 N mutant EGFR gene and investigate its expression in Ba / F3 cells as well as its sensitivity to gefitinib.Methods The recombinant plasmid MSCV-Tel-EGFR D896 N was constructed by PCR and cotransfected with two kinds of helper plasmids,MLV and VSVG,to produce lentivirus.The recombinant plasmids were transfected into BA / F3 cells by polybrene.After puromycin screening,Western blotting was used to detect the expression of EGFR and p-EGFR.Theproliferation cells were observed under the microscope after removing IL-3.After Ba/F3-tel-EGFR L858 R cells and Ba/F3-Tel-EGFR D896 Ncells were treated with different concentrationsof gefitinib,western blotting was used to d compare the sensitivity of the two cells to gefitinib.Results The recombinant plasmids were confirmed by restriction enzyme digestion and sequencing.Western blottingshowed that the expressions of EGFR and p-EGFR significantly increased in Ba / F3 cells transfected with IL-3.Abnormal proliferation was also observed in cells.Besides,the expression of p-EGFR significantly decreased in both Ba/F3-tel-EGFR L858 R cells and Ba/F3-Tel-EGFR D896NcellsConclusion The Ba / F3 cell line expressing D896 N mutant EGFR was successfully constructed.It was preliminarily determined that this site was not a driver gene but had some sensitivity to gefitinib.