EGFR-TKIs对EGFR突变的非小细胞肺癌有良好的临床疗效，但耐药的产生严重限制了肺癌患者临床获益，对此目前缺乏有效的防治手段。研究发现miRNA在诱导EGFR-TKIs耐药方面有着重要作用，调控miRNA网络已成为防治EGFR-TKIs耐药最具潜力的治疗策略。我们在前期研究中证实，蟾毒灵可通过抑制EGFR和cMET通路并激活凋亡级联逆转EGFR-TKIs耐药。应用miRNA微阵列芯片技术，我们发现蟾毒灵对引起耐药的miR-221有显著的调控作用。基于以上基础，本项目拟应用基因过表达、基因敲减、双荧光素酶报告基因实验、免疫荧光、核酸锁定原位杂交、qRT-PCR、免疫印迹等技术，通过体外、体内实验从miRNA→mRNA→蛋白→细胞→整体等水平,研究miRNA-221诱导EGFR-TKIs耐药的机制及蟾毒灵逆转耐药的作用及机制，以期为研发抗肿瘤中药及防治靶向药物耐药提供理论依据和新思路。

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs) have shown promising therapeutic efficacy in non-small cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR)-activating mutation. However, the inevitable recurrence resulting from acquired resistance has limited the clinical improvement in therapy outcomes. By now, there is no therapy to prevent this resistance. Many studies demonstrate that microRNA plays an important role in tumor progression and drug sensitivity; thus, miRNAs could serve as critical and potential therapeutic targets for EGFR-TKIs resistance. We previously reported that bufalin could reverse EGFR-TKIs resistance by inhibition of EGFR and cMET pathways. We then used miRNA microarray analysis to identify the miRNAs regulated by bufalin in PC-9GR cells, and found that bufalin regulated miR-221 expression levels, which could induce EGFR-TKIs resistance. Based on these results, we will reveal the mechanisms of miR-221 induces EGFR-TKIs resistance and bufalin reverses EGFR-TKIs resistance in vitro and in vivo by over-expression or knockdown some genes in lung cancer cells, qRT-PCR, Luciferase reporter assay, Western blotting, immunofluorescence and Locked Nucleic Acid In situ Hybridization. Our study will provide a novel strategy for development of new antitumor drugs and reversion of targeted drug resistance.