| Background The lung cancer is the main cause of cancer related death,accounting for over 1/4 of all diagnosed cancers,accounting for over 1/4 of all diagnosed cancers.Due to the late diagnosis and multiple metastases,most of the patients lost the chance of complete resection,and the five-year survival rate is 15%.NSCLC is the most common group of lung cancer(about 80%).Activation of RTKs(such as EGFR gene)are the most common type of driving mutations in NSCLC.TKIs,targeting EGFR,is widely used as first-line drugs in EGFR mutant NSCLC.However,resistance to EGFR-TKIs is inevitably happened within one year,which greatly limits the long-term efficacy of these drugs.FGL1 is a specific mitotic factor in hepatocytes.The expression of FGL1 is up-regulated in NSCLC reported recently.Therefore,this paper aims to probe the change of FGL1 before and after resistance to gefitinib PC9 cells(NSCLC cells with EGFR mutation),and to explore the potential relation between FGL1 level and the sensitivity of PC9/GR cells to gefitinib,so as to provide some experimental basis of improving the sensitivity of PC9/GR cells to gefitinib and offering more useful therapy for NSCLC patients.Objective To study the level of FGL1 expressed in PC9 cells and PC9/GR cells,and to explore the mechanism of FGL1 effecting the migration and invasion of PC9/GR cells.Method(1)The sensitivity of NSCLC cells(PC9 and PC9/GR cells with or without transfecting si RNA)to gefitinib and IC50 were measured by CCK-8;(2)Western blot and q RT-PCR were used to explore the expression of FGL1 protein and m RNA in PC9 cells and PC9/GR cells;(3)The FGL1 gene of PC9/GR cells was silenced by transfecting si RNA,and the interference effect was determined by q RT-PCR and Western blot;(4)The ability of cell migration and invasion was detected by scratch assay and Transwell chamber assay;(5)The expression of Zeb1,Vimentin and E-cadherin were detected by Western blot.Results(1)CCK-8 showed that the survival rate of PC9 cells and PC9/GR cells decreased as dependence of gefitinib concentration,the inhibition of gefitinib on PC9 cells was stronger than that of PC9/GR cells(P < 0.01);The IC50 values of PC9 cells((1.513± 1.296)umol /L)is evidently lowered than PC9/GR cells((19.179± 1.091)umol /L)(P < 0.01).(2)Western blot and q RT-PCR showed that the level of protein and m RNA of FGL1 in PC9/GR cells was significantly higher than that in PC9 cells.(3)Western blot and q RT-PCR showed that the level of FGL1 protein((0.579 ± 0.005)vs(0.593 ± 0.039)vs(0.495 ± 0.180),(P < 0.05))and FGL1 m RNA((0.135 ± 0.026)vs(0.152 ± 0.054)vs(0.217 ± 0.052),(P < 0.05))decreased significantly compared to group of Con.(4)After decreasing the level of FGL1 in PC9/GR cells,the CCK-8 assay showed that gefitinib could inhibit the proliferation of PC9/GR cells in group of Con,NC and Fi in the form of concentration dependence,and the effect on Fi group were more abvious than Con group and NC group(P < 0.01);IC50 in Fi group,Con group and NC group were(1.967 ± 1.147)umol/L,(19.179 ± 1.091)umol/L and(20.7543 ± 2.7348)umol/L respectively(P < 0.01).(5)Scratch assay showed that PC9/GR cells migrated to the edge of scratch significantly less and scratch distance more wide than those in Fi group compared with Con group(P < 0.01),and compared with G group,group of Fi and Con,that of G+Fi group were more significantly(P < 0.01,P < 0.01,P < 0.01).There was no significant difference between group G and group con(P > 0.01).(6)As traswell assay showed,compared with Con group,the number of cells migrated to the bottom of ependyma in group Fi and G decreased obviously(P < 0.05,P < 0.05,respectively);Compared with Fi group,G group and Con group,that of G+Fi group decreased more significantly in(P < 0.05,P < 0.05,P < 0.01respectively).(7)As invasive test showed,compared with group Con,gefitinib could inhibited the invasion of PC9/GR cells((P < 0.05),and knockdown of FGL1 alone or cotreatment with gefitinib significantly decreased the number of cells invading the basement of ependyma(P < 0.05,P < 0.05,respectively).(8)Western blot showed that the expression of Zeb1 and Vimentin decreased and E-cadherin increased in Fi group compared with Con,NC and G;And the level of Zeb1 and Vimentin decreased and E-cadherin increased more significantly in G+Fi group(P < 0.01);The expression of FGL1 protein increased significantly in G group compared with Con group(P < 0.01).Conclusion FGL1 expression in PC9/GR cells increased significantly,which may be related to the resistance of PC9 cells to gefitinib;interference with FGL1 expression can increase the sensitivity of PC9/GR cells to gefitinib,and inhibit the invasion and migration of PC9/GR cells by inhibiting the EMT process. |
PDF Full Text Request