HOTAIR与miR-214-3p的表达及相互作用介导澳洲茄边碱增强EGFR-TKI对抑制肺癌的分子机制探讨

Lung cancer is currently one of the most common malignant tumors in the world with low 5-year survival rate. The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) acquired drug resistance is the important and key reason for the failure of the targeted therapy. Therefore, search and find other promising nonsurgical modalities, especially in the case of sensitization and reversing drug resistance, is one of the hot research spot in the lung cancer targeted therapy. Our cell based in vitro and animal experimental results have showed that solamargine demonstrated inhibitory effect on lung cancer cell growth, and even greater response in the combination treatment of solamargine and EGFR-TKI gefitinib. However, the molecular mechanism underlying this, especially for sensitization and inversing the resistance of targeted therapy of lung cancer still remain to be elucidated. This project will examine the following two areas: 1) use of modern molecular biology related technologies and methods including genetic and proteomics,lung cancer cells in vitro to understand the molecular mechanism by which solamargine enhances the sensitization of target therapy 2) To observe the enhancing effect of solamargine on tumor targeting therapy. Our research is focused on the molecular mechanisms underlying the solamargine enhancing sensitization reverse resistance of EGFR-TKI, including the signal transduction, genes and lncRNA, miRNA expression, regulation and cross-talks among non-coding RNA, proteins, and multiple target responses. This study from the perspective of medical treatment, and strive to explore the therapeutic effect of solamargine on enhancing the response of EGFR-TKI in order to improve the life quality and prolong survival of patients with lung cancer.

肺癌是目前世界上最常见的恶性肿瘤之一。EGFR-TKI失敏感以及获得性耐药是靶向药物治疗肺癌失败的重要原因。因此,寻找有希望的增敏以及逆转耐药方面，是目前肺癌靶向治疗研究热点。我们体外以及动物实验结果发现澳洲茄边碱能有效抑制肺癌细胞的生长。联合靶向药物效果更佳。然而,其分子作用机制特别是对肺癌靶向治疗的增敏方面还了解甚少。本项目将从两方面开展研究：1）利用现代分子生物学相关技术和方法包括基因结合非编码RNA等技术手段，采用人EGFR-TKI敏感以及不敏感肺癌细胞体外培养研究澳洲茄边碱对肺癌靶向治疗增敏分子作用机制。2）在动物体内观察澳洲茄边碱对肿瘤靶向治疗增效作用效果。我们的研究重点是澳洲茄边碱对靶向治疗的增敏以及可能逆转耐药方面的分子机制，包括信号传递，靶基因，非编码RNA( lncRNA 和 miRNA) 表达，以及它们之间的相互调控，作用和可能的‘交叉对话’，多靶效应等。

肺癌是全球癌症死亡的首要原因，生存率极低。针对EGFR突变的NSCLC患者，分子靶向药物表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）已成为临床用药首选。EGFR-TKIs获得性耐药和低敏感性是靶向药物治疗失败的重要原因。因此,探索可增强EGFR-TKIs抗癌疗效的药物或方法已成为目前肺癌靶向治疗的研究热点。.本研究创新性的发现澳洲茄边碱（SM）联合EGFR-TKIs（如吉非替尼Gefitinib和厄洛替尼Erlotinib）具有协同抑制肺癌生长的作用。深入研究后，我们揭示了SM单用以及联合EGFR-TKIs协同抗癌的分子靶点和调控机制。首先，SM单用或联合Gefitinib显著抑制长链非编码RNA HOTAIR的表达，同时上调miR-214-3p的表达，进而下调PDPK1（磷酸肌醇依赖性蛋白激酶1)基因启动子活性和蛋白表达水平，最终抑制肺癌生长。其次，Stat3，Sp1和FOXO3a共同介导SM联合Gefitinib对IGFBP1蛋白表达和功能的激活，从而协同抑制肺癌进展。再次，在长链非编码RNA-MALAT1的参与下，Sp1作为miR-141-3p的直接靶点介导SM联合Gefitinib对IGFBP1蛋白表达的调控。更重要的是，SM联合Gefitinib对上述靶点均具有显著的协同调控作用。总之，本项目成功揭示了SM联合EGFR-TKIs基于HOTAIR/miR-214-3p/PDPK1，Stat3/Sp1-FOXO3a/IGFBP1和MALAT1/miR-141-3p/Sp1/IGFBP1信号通路抑制肺癌生长的分子机制。通过体外和体内实验双重验证SM联合Gefitinib的协同抗癌活性和多靶效应。.另外，在本课题经费和技术思路的支持下，我们进行了相应的拓展性研究。结果发现消积饮复方和中药单体小檗碱也可显著增加EGFR-TKIs吉非替尼对肺癌的抑制作用。其中消积饮复方通过调控HOATIR和Sp1的表达，进而减少细胞前列腺素E受体蛋白4（EP4），最终增强Gefitinib对肺癌的抑制作用。小檗碱通过下调HOTAIR表达，调控HOTAIR与miR-34a-5p的相互作用，共同影响肿瘤EMT过程，最终增强Gefitinib对肺癌的抑制效应。由此可见，本课题中HOTAIR和Sp1可能是介导肺癌进展以及EGFR-TKIs抗癌增敏机制的潜在靶点，值得更加深入的研究。