MicroRNA-224 Modulates Gefitinib Drug Resistance Of NSCLC Cells By Targeting Bim

Background:Lung cancer is the highest mortality rate of cancer in China,of which about 85%of non-small cell lung cancer.NSCLC patients are with poor prognosis,and most of them are already in the advanced stage which can not be surgical resection.Therefore,platinum-containing combination of chemotherapy is the main treatment of advanced NSCLC,but the efficiency is still not enough.In recent years,it has been found that epidermal growth factor receptor tyrosinase inhibitors,represented by gefitinib,play an important role in the treatment of advancedEGFR-sensitive mutant NSCLC.But the occurrence of acquired resistance are limiting the efficacy.MicroRNA is a non-coding small RNA that acts as a oncogene or tumor suppressor gene in tumors by degrading the target gene mRNA or inhibiting its translation.With the further study of miRNA,it has also played a crucial role in tumor resistance.Studies have shown that miR-224 is not only involved in methotrexate resistant in colon cancer,but also associated with the radiosensitivity of glioblastoma.But there is no research report about the relationship between miR-224 and gefitinib.Objective:The aim of this study is to evaluate the interaction between miR-224 and gefitinib resistance in NSCLC tissues and cells.Methods:(1)Gefitinib-resistant phenotypes in PC9/GR cells were identified by MTT assay.(2)Real-time quantitative PCR(qRT-PCR)assays was performed to detect the expression of miR-224 in NSCLC gefitinib tissues and cells.(3)MiR-224 inhibitors was synthesized and introduced to cells.The expression of miR-224 in transfected cells was detected by qRT-PCR.MTT and clone formation assays were used to evaluate the viability of PC9/GR cells transfected with miR-224 inhibitor.The half maximal inhibitory concentration(IC50)values of gefitinib were detected by MTT assay.(4)Compared with the control group,inhibition of miR-224 in PC9/GR cells introduced increased apoptosis and proportion of G0-G1 cell cycle.(5)In vivo experiments confirmed that inhibition of miR-224 resensitized PC9/GR cells to gefitinib.(6)Bioinformatis analysis showed that there were Bim binding sites in miR-224 sequence.Luciferase reporter assay,qRT-PCR and Western Blot assay confirmed that Bim is the target gene of miR-224.Results:(1)The IC50 of PC9/GR cells to gefitinib was higher than that of PC9 cells.(2)miR-224 was relatively highly expressed in the post-resistant tissue compared with the pre-drug-resistant tissue.Compared with the parental cell PC9,miR-224 was relatively highly expressed in the PC9/GR.(3)MTT assay and clone formation assay showed that inhibition of miR-224 expression could attenuate cell proliferation and enhance the sensitivity of PC9/GR cells to gefitinib.(4)Flow cytometry detection of cell cycle and apoptosis found that inhibition of miR-224 expression can block the cell cycle in the G0-G1 phase,and can promote apoptosis.(5)Inhibition of miR-224 expression can enhance the sensitivity of cells to gefitinib at the in vivo level.(6)Bioinformatics found that Bim was the downstream target gene of miR-224 and was verified by luciferase reporter assay.Meanwhile,qRT-PCR and Western Blot confirmed that Bim was the downstream target gene of miR-224.Conclusion:In this paper,a series of in vivo and in vivo experiments confirmed that inhibition of miR-224 can reverse the NSCLC cells PC9/GR on gefitinib resistance by regulating the downstream target gene Bim.