| Objective: In order to discuss the effects of FOXM1 for patients with EGFR mutation of advanced lung adenocarcinoma using EGFR-TKI.The expression of FOXM1 through the detection of patients with corresponding pathological tissues and analyze its correlation with clinicopathological parameters of patients,also tostudy the FOXM1 expression of EGFR mutation in lung adenocarcinoma cell lines and their effect on cell biological function,and tostudy the mutation of EGFR cells to erlotinib sensitivity and related mechanism.The aim is to lay the foundation for FOXM1 as a molecular marker for targeted treatment of EGFR mutant lung adenocarcinoma,and also provide a theoretical basis for the clinical treatment of EGFR-TKI resistance.Method: 1)140 cases of paraffin specimens were collected from patients with NSCLC in Xinjiang Medical University Affiliated Tumor Hospital from 2011 to 2014 confirmed by histopathology into this study.All the patients have EGFR mutation.The ARMS was used to detect the patients EGFR mutation.We tested the level of FOXM1 mRNA and protein insome patients,each tissue sample was detected by immunohistochemistry.We analyzed the relationship between the expression of FOXM1 mRNA and protein.2)We check the level of FOXM1 mRNA and protein in HCC827 and H1650 cells by fluorescence quantitative PCR and western blot.These two cells have same EGFR mutation.MTT assay was used to detect the effects of different concentrations of erlotinib on EGFR mutant cell line HCC827 and H1650.HCC827 cells were divided into HCC827group(conventional culture without any treatment)and HCC827 + Erlotinibgroup(adding 100μM erlotinib);H1650 cells were divided into H1650 group(conventional culture without any treatment)and H1650 +Erlotinib(100μM erlotinib was added).Transwell chamber was used to detect the invasive ability of each group.Cell migration was performed by cell scratch test.3)FOXM1overexpressed lentiviral vectors were constructed and transfected into 293 T cells to pack the virus.The virus was collected and infected with HCC827 and H1650 cells to obtain FOXM1 over expressing HCC827 and H1650 cell lines.The siRNA-FOXM1 plasmid was designed and constructed,and then transfected into human lung cancer EGFR mutant cell lines HCC827 and H1650,respectively,to obtain FOXM1 expression to inhibit HCC827 and HCC827 cell lines.The level of FOXM1 m RNA and protein was checked by fluorescence quantitative PCR and western blot.MTT assay was used to detect the proliferation activity of the cells.The invasive ability of the cells was detected by Transwell chamber method.Cell migration ability was measured by cell scratches.Results:1)The expression level of FOXM1 and mRNA in normal adjacent tissues was significantly lower than that in advanced lung adenocarcinoma tissues(P<0.05).Among140 cases of advanced adenocarcinoma of lung,the positive expression was 58 cases,and the negative expression was 82 cases.The positive rate was 41.43%.The expression of FOXM1 protein was independent of age,sex,clinical stage,smoking history and PS score(P > 0.05)in those patients,but was related to the degree of tumor differentiation and lymph node metastasis in advanced lung adenocarcinoma patients.The expression of PFS in patients with negative FOXM1 protein expression was significantly longer than that in patients with positive FOXM1 protein expression(P<0.05).In overall survival,patients with negative FOXM1 protein expression was significantly longer than in patients with positive FOXM1 protein expression(P<0.05).Univariate analysis showed that the degree of tumor differentiation,stage,lymph node metastasis and expression of FOXM1 were the prognostic factors of advanced lung adenocarcinoma(P < 0.05).The patient’s age,sex,smoking,PS score,and surgery were not associated with prognosis(P >0.05).Multivariate COX model was used forsurvival analysis.The results showed that the degree of tumor differentiation,lymph node metastasis and the expression of FOXM1 had an impact onthe prognosis of patients with advanced lung adenocarcinoma(P<0.05).2)Compared with H1650 cells,the expression level of FOXM1 mRNA in HCC827 cells was significantly decreased(P <0.05).Compared with H1650 cells,the expression level of FOXM1 protein in HCC827 cells was significantly decreased(P<0.05).With the increase of the concentration of erlotinib,the proliferation inhibition rate of HCC827 and HCC1650 cells increased gradually.Compared with H1650 group,the proliferation inhibition rate of HCC827 cells was significantly increased(P < 0.05).The number ofinvasive cells in H1650 group was no statistical difference with that in HCC827group(P>0.05).Compared with HCC827 group,the number of invasive cells in HCC827+Erlotinibgroup was significantly lower than that in HCC827group(P<0.05).Compared with H1650 group,the number of invasive cells in H1650+Erlotinib group was significantly decreased(P<0.05).Compared with H1650 + Erlotinibgroup,the number of invasive cells in HCC827 + Erlotinibgroup was significantly decreased(P<0.05).The Compared with H1650 group,the migration distance of HCC827 was no statistical difference.(P>0.05).Compared with HCC827 group,the migration distance of HCC827+ Erlotinib group was significantly decreased(P<0.05).Compared with H1650 group,the migration distance of H1650 + Erlotinibgroup was significantly lower than that of H1650group(P<0.05)Compared with H1650 + Erlotinibgroup,the migration distance of HCC827 + Erlotinibgroup was significantly decreased(P<0.05).3)The expression of FOXM1 mRNA in HCC827 cells was significantly higher than that in HCC827-NCgroup(P<0.05)after transfection with FOXM1 lentivirus(HCC827-FOXM1)(P<0.05).The expression of FOXM1 mRNA in HCC827-siFOXM1 group was significantly lower than that in HCC827-siNCgroup(P < 0.05).The expression level of FOXM1 mRNA in H1650-FOXM1 group was significantly higher than that in H1650-NCgroup(P <0.05).After transfection with si RNA-FOXM1(H1650-siFOXM1group)The expression level of FOXM1 m RNA was significantly lower than that of H1650-siNCgroup(P<0.05).The expression of FOXM1 protein in HCC827 cells was significantly higher than that in HCC827-NCgroup(P < 0.05)after transfection with FOXM1 lentivirus(HCC827-FOXM1)cells.The expression of FOXM1 protein in HCC827-siFOXM1 group was significantly lower than that in HCC827-siNCgroup(P<0.05).The expression level of FOXM1 protein in H1650-FOXM1 group was significantly higher than that in H1650-NCgroup(P<0.05).After transfection ofsiRNA-FOXM1(H1650-siFOXM1group)The expression level of FOXM1 protein was significantly lower than that of H1650-siNCgroup(P<0.05).MTT assay showed that the proliferation inhibition rate of HCC827-FOXM1 group was significantly lower than that of HCC827-NCgroup(P<0.05).The proliferation inhibition rate of HCC827-si FOXM1 group was significantly higher than that of HCC827-siNCgroup(P < 0.05).The proliferation inhibition rate of H1650-FOXM1 group was significantly lower than that of H1650-NCgroup(P < 0.05).The proliferation inhibition rate of H1650-siFOXM1 group was significantly higher than that of H1650-siNCgroup(P<0.05).Transwell assay showed that the number of invasive cells in HCC827-FOXM1 group was significantly higher than that in HCC827-NCgroup(P < 0.05).The number of invasive cells in HCC827-siFOXM1 group was significantly lower than that in HCC827-siNCgroup(P < 0.05).The number of invasive cells in H1650-FOXM1 group was significantly higher than that in H1650-NCgroup(P <0.05).The number of H1650-si FOXM1 group was significantly lower than that of H1650-siNCgroup(P < 0.05).The migration distance of HCC827-FOXM1 group was significantly longer than that in HCC827-NCgroup(P<0.05).The migration distance of HCC827-siFOXM1 group was significantly shorter than that of HCC827-siNCgroup(P<0.05).The migration distance of H1650-FOXM1 group was significantly longer than that of H1650-NCgroup(P < 0.05).The migration distance of H1650-siFOXM1 group was significantly shorter than that of H1650-siNCgroup(P<0.05).Conclusion: 1)FOXM1 in the tissue of most advanced lung adenocarcinoma was highly expressed,and the degree of tumor differentiation,lymph node metastasisstatus;positive and negative expression in patients with PFS were 6 months and 10 months,the expression of FOXM1 protein,overallsurvival,the positive expression of FOXM1 protein and the expression of negative patients with advanced lung adenocarcinoma of the OS were 18 and 28 months.Single factor analysis showed that the expression of tissue differentiation,tumor stage,lymph node metastasis and FOXM1 are prognostic factors of patients with advanced lung adenocarcinoma,COX model multivariate survival analysis showed that the expression of FOXM1 and metastasis and prognosis of patients with advanced lung adenocarcinoma has affected the degree of differentiation and lymph tumor node.2)The expression level of FOXM1 in H1650 cells was significantly higher than that in HCC827 cells,and the invasion and migration ability of H1650 cells after treated with erlotinib was significantly higher than that of HCC827 cells.With the increase of the concentration of erlotinib,the proliferation inhibition rate of HCC827 and HCC1650 cells increased gradually.Compared with H1650 group,the proliferation inhibition rate of HCC827 cells was significantly increased(P < 0.05).3)The proliferation,invasion and metastasis of HCC827 and H1650 cells increased with the increase of FOXM1 expression level.The proliferation,invasion and metastasis of HCC827 and H1650 cells were significantly decreased when FOXM1 expression was inhibited. |
