| Background Lung cancer is the leading cause of cancer-related fatalities worldwide,and non-small cell lung cancer(NSCLC)is the main pathology type.As Molecular Biology and Genomics developed,EGFR has become the valid target of NSCLC.TKIs not only increased the overall survival rate,but also improved the living standard of the patients.However,acquired resistance to TKIs inevitably occurs.The underline mechanism includes: over-expression of c-met,T790 M mutation,over-expression of IGF-1R,et.What role does IGF-1R play in NSCLC ? It has not been clarified.Micro RNAs(mi RNAs or mi Rs)are a class of small non-coding RNAs,which are involved in tumor initiation and progression.MiR-223 is a tumor suppressor mi RNA that has been reported in various types of cancer,including lung cancer.In our previous study,a low expression level of miR 223 was observed in lung cancer.IGF-1R is one of the target of mi R-223.Then we hypothesized that if IGF-1R plays an important role in NSCLC.We explored the role and mechanism of IGF-1R in EGFR-TKIs resistant NSCLC.Objection To explored the drug resistant mechanism of IGF-1R and miR-223 in NSCLC both in the level of the cell and nude mice.Methods1.We used CCK-8 to detect the drug resistant index.2.We used lentiviral transfection to build PC-9/ER-mi R-223 and PC-9/ER-EV cells.3.We used flow cytometry(FCM)to purified the transfection cells.4.We used FCM to detect the apoptosis ratio of PC-9/ER-miR-223 and PC-9/ER-EV cells.5.We used q RT-PCR to detect the level of m RNA of IGF-1R.6.We detected the activation time and concentration of IGF-1.7.The IGF-1R/AKT/S6 signaling pathway was detected by Western blotting.8.We detect the volumes of tumors in nude mice.Results1.We subcultured the PC-9 cell line.2.We builded the lentiviral over-expression vector of mi R-223.3.We used FCM to purify the GFP positive cells: PC-9/ER-mi R-223 and PC-9/ER-EV.The transfection ratio was 95.6% and 97% respectively.4.The expression of mi R-223 in the miR-223 over-expression group was up-regulated approximately 16 fold compared with that in the EV group.5.MiR-223 enhanced cell apoptosis compared with the EV group following erlotinib treatment for 48 h as determined by flow cytometry.The apoptotic rates were 18.92±2.123 vs.11.13±1.127%.6.The sensitivity of the PC-9,PC-9/ER,mi R-223 and EV-infected PC-9/ER cells to erlotinib was determined by cell counting kit-8(CCK-8)assay.IC50 of groups PC-9,PC-9/ER,PC-9/ER-mi R-223 and PC-9/ER-EV: 0.25,5.16,1.19 and 5.29.7.The IGF-1R m RNA level of PC-9/ER cell was 2.85 fold compared with PC-9 cells.The IGF-1R protein level of PC-9/ER was 1.75 fold compared with PC-9 cells.8.The IGF-1R mRNA level of PC-9/ER-mi R-223 decreased 43% and the IGF-1R protein level of PC-9/ER-mi R-223 decreased 34%.9.MiR-223 inhibited the IGF-1R/AKT/S6 signaling pathway.The inhibition can be rescued by IGF-1.10.In vivo,mi R-223 improves the tumor response to erlotinib in tumor xenografts.Growth curves of tumors in 6 groups(n=3 mice per group;*P<0.05).Nude mice with high level of IGF-1 have poor prognosis.ConclusionsIGF-1R over-expressed in erlotinib resistant lung cancer cells(PC-9/ER).MiR-223 enhanced the sensitivity of NSCLC cells to erlotinib by targeting IGF-1R/AKT/S6 signaling pathway. |
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