Notch-1/AP-1/miR-451信号轴参与人肺腺癌细胞化疗耐药表型形成的分子机制研究

Chemoresistance is the most significant problem of the clinical efficacy of treatment in advanced lung adenocarcinoma so far.The mechanism involved in genetic change with multiple tumor drug resistance ralted protein, and it can regulate various physiological and pathological processes by complicated signaling axis network. On the previous basis of establishment of docetaxel-resistant lung adenocarcinoma cell lines, our group has proved that Notch-1 may be involved in the formation of lung adenocarcinoma resistant phenotype, consistent with other researches. We also found a negative regulatory role between Notch-1 and miR-451, however the exact mechanisim was unclear, further study was seem to be needed. Combined with the microRNA array results and bioinformatics analyses, AP-1, one of the downstream target of Notch-1, might be an important transcription factor which binding to the upstream promoter region of miR-451 and regulating it. The aim of this study is to investigate the molecular mechanisms of Notch-1/AP-1/miR-451 signaling axis in drug resistant lung adenocarcinoma.by luciferase reporter gene, gel retardation, chromatin immunoprecipitation, site-specific mutation. Meanwhile, we using molecular biology regulation and specific Notch signaling pathway inhibitors DAPT to verify as well. It will provide a novel target for reversing chemoresistance and clinical individualized treatment of lung adenocarcinoma.

肿瘤化疗耐药表型的形成涉及肿瘤耐药相关蛋白基因的遗传学及表观遗传学分子调控机制改变，是目前限制肺腺癌临床疗效的重要难题。在成功建立人肺腺癌耐多西他赛细胞模型的基础上，本课题组前期研究结果提示：Notch-1表达激活可能参与了肺癌腺化疗耐药表型的形成，且与miR-451表达之间存在负相关关系，但其确切的分子调控机制尚不清楚。生物信息学并结合microRNA芯片筛选结果提示：Notch-1、AP-1与miR-451三者之间存在可能的调控关系。本课题拟在前期研究基础上，采用荧光素酶报告基因、凝胶阻滞、免疫共沉淀、RNA干扰等实验方法从细胞和活体动物水平探讨Nocth-1通过转录因子AP-1调控miR-451及其下游靶基因MDR1表达从而参与肺腺癌化疗耐药表型形成的分子机制，为临床逆转肺腺癌化疗耐药提供新的分子靶标。

化学耐药的产生是限制晚期肺腺癌病人临床疗效的重要障碍，化疗耐药的机制错综复杂，涉及众多耐药相关基因的表达调控异常,还通过复杂的网络信号轴参与调节各种生理和病理过程。阐明化疗耐药相关机制、发现重要分子靶标的研究进程尚面临巨大的挑战。在多种生物体内Notch信号通路，尤其是其中重要的受体Notch-1，在决定细胞命运和联系细胞间信号通讯发挥了显著的作用,包括干细胞表型维持、调节肿瘤分化、增殖、凋亡和化疗耐药形成。Notch信号通路与微小RNA（miRNAs）之间亦存在交互调控作用。本研究旨在寻找参与人肺腺癌耐药表型形成的分子靶标，借助信号通路特异性抑制剂和基因转染等方式人为调控基因表达，体内外验证Notch信号通路上受体Notch-1对肺腺癌化疗耐药的影响，结合课题组前期针对miRNA在肺腺癌中已取得的研究进展，深入研究肺腺癌中Notch通路和miRNAs之间的交叉对话作用。继而探索microRNA-451在肺腺癌耐药中的功能，凭借生物信息学分析和应用分子生物学方法明确激活蛋白1为重要枢纽后，试阐明Notch-1/AP-1/miR-451/MDR-1信号轴参与调控化疗耐药表型形成的分子机制。研究结果表明，1..Notch-1在肺腺癌化疗耐药表型形成中发挥重要作用，抑制其表达能够促进肺腺癌化疗增敏，抑制增殖，增加凋亡和改变细胞周期分布水平。2..MiR-451与Notch-1负向调控，呈抑癌基因表现，与化疗增敏作用，增殖抑制，凋亡增加和细胞周期再分布密切相关。3..MDR-1为miR-451直接靶基因，AP-1与miR-451启动子区存在可能结合位点。Notch-1/AP-1/miR-451功能轴的调控异常促进肺腺癌化疗耐药表型的形成。4..本研究首次探讨了Notch-1/AP-1/miR-451信号轴调控异常在人肺腺癌化疗耐药中的重要作用，其为肺腺癌临床个体化治疗和逆转化疗耐药提供了新的依据和思路。