The patients underwent computed tomography scan covering target lesions 4-6 weeks after the initiation of EGFR-TKIs treatment and then every 8 weeks or when indicated by symptoms. (N=150) who received EGFR-TKIs (gefitinib or erlotinib) as second- or third-line therapy from September 2008 to December 2012 were included in the study. In 66 NSCLC patients with wild-type mutation, the trend remained the same but not statistically significant. Overall, these findings indicated that Galanthamine miR-200c might be a predictive biomarker for sensitivity to EGFR-TKIs in advanced NSCLC patients with wild-type fusion may experience unprecedented success on treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) or anaplastic lymphoma kinase (ALK) inhibitor [2-4]. Unfortunately, only minority patients have these driver mutations. About 3-5% of NSCLC patients harbor ALK-rearrangement [5-7], and about 30-40% East Asian patients harbor mutation (wild type (could be the main cause of acquired resistance to EGFR-TKIs in H1975. MiR-200c could upregulate the expression of E-cadherin and trigger MET in H1975, but cannot reverse the resistance to gefitinib owing to existence. Inversely, blocking miR-200c expression of PC9 caused resistance of gefitinib compared with parental and NC cells (Figure ?(Figure5D5D). Open in a Galanthamine separate window Figure 5 Low expression of miR-200c contributes to gefitinib drug resistance(A-C) Effects of LV-hsa-miR-200c on gefitinib sensitivity in A549, H1975 and H1299 cells. (D) Effects of miR-200c inhibiter on gefitinib sensitivity in PC9 cells. Data are mean Galanthamine SD from 3 independent experiments. EGFR-TKIs resistance induced by miR-200c downexpression was mediated through PI3K/AKT and MEK/ERK pathway PI3K/AKT and MEK/ERK signal Galanthamine pathways are the main downstream pathways of EGFR. To explore the downstream mechanisms of miR-200c mediated in EGFR-TKIs resistance, we detected pAKT and pERK expression before and after Mouse monoclonal to COX4I1 LV-hsa-mir-200c infecting A549, H1299 and H1975. We found that pAKT and pERK were repressed when miR-200c was upregulated (Figure 6A-C). Moreover, phosphorylation of AKT and ERK were markedly activated after silencing miR-200c expression in PC9 by miR-200c inhibitor (Figure ?(Figure6D6D). Open in a separate window Figure 6 PI3K/AKT and MEK/ERK are two important signal pathways regulated by miR-200c(A-C) Western blot analysis of AKT, pAKT, ERK and pERK levels in A549, H1975 and H1299 cells after infected with LV-hsa-miR-200c or NC. (D) Western blotanalysis of AKT, pAKT, ERK and pERK levels in PC9 cells after transfected with miR-200c inhibitor or NC. GAPDH is included as a loading control. Results are representative of at least three independent experiments. Patient characteristics A total of 150 patients with advanced NSCLC were included into this study. The median age was 59 years (range, 30-81 years). The proportions of male patients, ever smokers and patients with adenocarcinoma accounted for 56.0%, 27.3% and 67.3%, respectively. Patients received a median of two prior chemotherapy regimens (range, 1-2 regimens). MiR-200c expression levels were detected in all of the 150 patients. Mutations of were successfully performed in 139 patients, including 73 patients with activated mutation and 66 with wild type, while the other 11 failed the test owing to poor quality of DNA. The expression of miR-200c was significantly lower in mutation status and miR-200c expression level were the main factors identified as predicting the disease control to EGFR-TKIs treatment. The ORR and DCR were 57.1% and 84.5% in the patients with unknown and activated mutation subgroup respectively, which is significantly higher than 6.1% (P 0.0001) and 39.4% (P 0.0001) in patients with wild-type status in the whole population (12.0m [95%CI: 7.37-16.63m] vs. 5.00m [95%CI: 1.82-8.18m], P=0.009, Figure ?Figure7).7). Univariate analysis showed lower risk of progression in patients of female, never smoker, ECOG PS1, age65, activated mutation and high level of miR-200c expression (Table ?(Table2).2). In multivariate analysis, mutations [Hazard ratio(HR): 0.29, 95%CI: 0.19-0.45, P 0.0001], high level of miR-200c expression (HR: 0.55, 95%CI: 0.36C0.84, P=0.006) and ECOG PS 1 (HR: 0.41, 95%CI: 0.21-0.80, P =0.009) remained independent predictors of PFS (Table ?(Table22). Open in a separate window Figure 7 Kaplan-Meier curves Galanthamine showing the probability of progression free survival for patients with NSCLC, according to miR-200c expression level and stratified according to EGFR mutation status. Table 2 Survival analysis in the whole population activated mutation and high level of miR-200c expression were the main predictors for longer OS in univariate analysis..