鸦胆子调控外泌体miRNA抑制非小细胞肺癌侵袭转移的药效物质基础及机制研究

Traditional Chinese Medicine Fructus Bruceae is the dry fruit of simaroubaceae plant Brucea javanica (L.) Merr. It's now recorded by Chinese Pharmacopoeia of 2015 version. Fructus Bruceae can be applied to treat non-small cell lung cancer (NSCLC), which is serious threat to humankind. However, the effective substances and acting mechanisms are not clearly elucidated. In our previous study, bruceine D was found to inhibit invasion of NSCLC A549 cells via regulation of exosome miRNA. This project will isolate exosome by ultra high speed centrifugation, identify exosome and sequence exosome-included miRNA, compare the difference of exosome miRNA in human serum between NSCLC patients and normal persons, forecast the downstream gene of miRNA. Furthermore, isolation of active compounds or active compounds group will be under guidance of activity tracking expressed as regulating exosome miRNA. Thereafter, the influence of active compounds or active compounds group on exosome miRNA target group and the downstream gene expression will be investigated. Subsequently, the impact of exosome on invasion and metastasis of NSCLC in cell or animal model will be clarified. Finally, the structure-activity relationship and active compounds group will be summarized. Our research will expound the effective substances and acting mechanisms of Fructus Bruceae inhibiting NSCLC invasion and metastasis. Moreover, the results will provide foundation for developing drugs against NSCLC.

中药鸦胆子是苦木科植物鸦胆子Brucea javanica (L.) Merr的干燥成熟果实，收载于《中国药典》2015版。非小细胞肺癌(NSCLC)严重威胁人类健康，鸦胆子可用于治疗NSCLC，但药效物质基础及作用机制不清楚。前期研究中，我们发现鸦胆子素D可调控NSCLC细胞A549外泌体miRNA而抑制NSCLC侵袭转移。本项目拟以超速离心提取外泌体、鉴定外泌体及miRNA测序、比较NSCLC患者与正常人血清外泌体miRNA差异、预测miRNA下游基因；以调控外泌体miRNA为活性指标，活性追踪指导鸦胆子活性成分（群）的分离纯化，研究活性成分（群）对外泌体miRNA靶点群及下游基因影响，考察外泌体在细胞与动物模型中对NSCLC侵袭转移的作用，总结构效关系与活性成分（群）。本项目将阐明鸦胆子调控外泌体miRNA抑制NSCLC侵袭转移的药效物质基础及机制，对研究NSCLC药物具有重要意义。

传统中药鸦胆子是苦木科植物鸦胆子Brucea javanica (L.) Merr的干燥成熟果实，收载于《中国药典》2015版。非小细胞肺癌（NSCLC）严重威胁人类健康，鸦胆子可用于治疗NSCLC，但药效物质基础及作用机制不清楚。我们的研究发现：（1）鸦胆子苦醇可抑制肺癌PC-9细胞的增殖、迁移和侵袭，其抗癌作用可能与EGFR、β-catenin、Akt和STAT3下调有关。（2）鸦胆子素D显著降低肺癌干细胞（A549细胞分选得到）中相关信号因子β-catenin、FZ-2、Cyclin D1、Survivin的mRNA表达水平。（3）鸦胆子素D可抑制肺癌H1299细胞生长，并下调Snail、Twist、MMP7、Integrin β4、αv的表达。（4）研究发现非小细胞肺癌A549细胞产生的外泌体能促进自身的侵袭转移，A549外泌体中miR-34c-3p的表达明显低于BEAS-2B的。双荧光报告实验表明Integrin α2β1是miR-34c-3p的直接靶点，免疫共沉淀实验表明Integrin α2与β1以二聚体形式起作用。细胞实验、体内实验结果均表明Integrin α2β1的高表达可促进非小细胞肺癌的侵袭转移，Integrin α2β1在临床样本处于高表达状态。（5）鸦胆子素D可调控非小细胞肺癌外泌体的miRNA表达，从而抑制非小细胞肺癌外泌体的促侵袭迁移作用。本项目发表18篇文章，获得2项科技奖励，培养硕士研究生5名。我们的研究为阐明NSCLC侵袭转移的机制、阐明鸦胆子抗NSCLC的药效物质及作用机制提供支持。