Research On Regulating Activity Of Telomerse By EGF Through Up Regulating Expression Of RFPL3 Protein In NSCLC Cells Via MEK Signaling Pathway

Objective:1.To research the influence of stimulation of epidermal growth factor(EGF)of different concentrations at different time on RFPL3 and h TERT protein in non-small-cell lung cancer cells(A549,H1299),and screen out the optimum concentration and time of stimulation;2.To research whether the change of expression of RFPL3 and h TERT protein in non-small-cell lung cancer cells(A549,H1299)caused by EGF stimulation is related to MEK signaling transduction pathway;3.To research the relation of RFPL3 protein with h TERT protein and related protein expression change in MEK signaling pathway;4.The experiments above are hopeful to provide possible new target spot for the molecular targeted treatment of advanced lung cancer.Methods: 1.With A549 and H1299 cells as experimental subject and added EGF in different concentrations to stimulate 24 h and 48h;detected the expressions of RFPL3 and h TERT proteins from m RNA and protein level based on RT-PCR as well as Western blot technology;2.Applied MTT method to detect the cell viability of A549 and H1299 cells when sitimulated by EGF,and adopted flow cytometry to detect cell apoptosis;3.Added EGFR inhibitor(AG1478 and Erlotinib)to pretreat cells for 4h and then added EGF to stimulate for 48h;set control group and detected expressions of RFPL3 and h TERT proteins via Western blot;4.Pretreated cells for 2h by using MEK signal pathway specificity inhibitor(PD98059);then added EGF to stimulate for 48h;set control group and detected expressions of RFPL3,h TERT,general ERK and p-ERK proteins via Western blot;5.Transfected RFPL3 overexpression plasmid in A549 and H1299 cells;detected expressions of RFPL3,h TERT and related proteins of MEK signal pathway 48 h later.Result: 1.EGF could upregulate the expressions of RFPL3 and h TERT proteins with presenting time and concentration dependence within certain concentration scope;20ng/ml and 48 h separately were the optimum concentration and time;2.EGF could promote proliferation of non-small cell lung cancer cells;withincertain concentration scope(< 20ng/ml),cell viability increased and cell apoptosis decreased with the increase of EGF concentration;3.After adding EGFR inhibitor(AG1478 and Erlotinib),the expressions of RFPL3 and h TERT proteins in cells A549 and H1299 lowered,which further indicating that EGF could upregulate the expressions of RFPL3 and h TERT proteins;4.After adding MEK signaling pathway specificity inhibitor(PD98059),the expression of total ERK protein was unchanged,the expression of p-ERK protein in cells A549 and H1299 lowered,MEK signal pathway was activated;Expression of RFPL3 and h TERT protein also decreased,indicating the function of EGF up-regulating expression of RFPL3 and h TERT protein in A549 and H1299 cells may be closely related to MEK signaling pathway;5.After successfully transfecting RFPL3 expression plasmid,the expression of h TERT protein increased,and expression of related protein(Ras,Raf,ERK,p-ERK protein)for MEK signaling pathway increased.Conclusion:EGF combines with EGFR in lung cancer cells,activates MEK signaling pathway,increases the expression of upstream molecules(Ras,Raf)of MEK signaling pathway,then increasing expression of ERK,regulatory tumor protein RFPL3,promotes transcription and expression of h TERT,increasing activity of telomerase,promoting infinite proliferous of tumor cells.