15-氧代绣线菊内酯在肾癌中对Wnt信号通路的作用机制研究

Renal cell carcinoma(RCC) is one of the most common malignant tumors of urology, but there are lack of effective method to therapy the advanced metastatic RCC. Our previous research and other's studies show that Wnt signling plays an important role in RCC pathogenesis, it may be an effective way to therapy advanced metastatic RCC through designing small molecule inhibiter of Wnt signaling. Our collaborating laboratory have synthesised and screened a new Wnt molecule inhibiter named 15-oxospiramilactone, it can inhibit Wnt signaling. A preliminary study found, 15-oxospiramilactone inhibits the growth of RCC and the activity of Wnt signaling reporter Top-flash, induces cell apoptosis. The small molecule effects at the Wnt signaling transcriptional complex level, but the specific role of the target is not clear. Studies of a member of the project team show that SET8 is a key factor of the Wnt transcriptional complex, and it plays an important role in RCC. So we hypothesis that 15-oxospiramilactone may inhibit the activity of Wnt signaling to suppress the growth and metastasis of RCC, and it maybe through inhibiting SET8 involved in the formation of the transcription complex. This project intends to use Flow cytometry, Co-IP, ChIP, Nude mice tumor-bearing and lung metastasis model technologies to study the effect of malignant phenotype of RCC by the small molecule in vitro and in vivo, such as cell proliferation, migration, invasion, cell cycle and apoptosis, and study the The specific target of the small molecule. The next, we use primary cell cultures technology to verify the effect of 5-oxospiramilactone in RCC, provide plausible therapy methods for advanced metastatic renal cancer.

肾癌发病的分子机制不清楚，晚期肾癌缺乏有效治疗手段。文献和我们前期研究表明Wnt信号通路与肾癌密切相关，设计抑制Wnt的小分子是肾癌治疗研究新思路。我们合作的实验室新合成筛选出小分子15-氧代绣线菊内酯，其能够特异性抑制Wnt信号通路。我们前期研究表明：肾癌中该小分子药物能够抑制肾癌细胞生长，诱导细胞凋亡，抑制Wnt报告基因活性，该小分子作用于Wnt转录复合物水平。但该小分子具体作用靶点不清楚。我们研究还表明：SET8是Wnt转录复合物上的关键因子，在肾癌中具有重要作用。由此推测15-氧代绣线菊内酯可能通过抑制SET8参与转录复合物形成来抑制Wnt信号通路，进而抑制肾癌。本项目拟通过流式细胞检测、Co-IP、ChIP、裸鼠皮下荷瘤及肺转移模型等技术研究该小分子对肾癌恶性表型的影响及其对Wnt的机具体调控机制，进行肾癌原代细胞培养验证，探讨肾癌发病机制，为肾癌分子靶向治疗新方向提供理论依据。

肾癌发病的分子机制不清楚，晚期肾癌缺乏有效治疗手段，Wnt信号通路在肾癌的发生、发展中具有重要作用。本项目在体外细胞水平研究发现小分子15-氧代绣线菊内酯能够抑制肾癌786－0及ACHN细胞的增殖、迁移，诱导细胞凋亡的发生，阻滞细胞周期于G2/M期。本项目进一步研究发现，在肾癌786－0及ACHN细胞中，小分子15-氧代绣线菊内酯能够抑制Wnt信号通路报告基因活性，在mRNA和 /或蛋白水平显著抑制Axin2、LEF1、NKD1、Cyclin D1和Survivin等Wnt靶基因以及Cdc25和Cdc2等细胞周期相关基因的表达，小分子15-氧代绣线菊内酯不影响Wnt信号通路中的关键蛋白b-cateinin的入核，但是影响b-cateinin与TCF4相互结合，综上说明小分子15-氧代绣线菊内酯能够能过抑制b-cateinin与TCF4相互结合，进而抑制Wnt信号通路，最终抑制肾癌细胞的生长。本项目组还发现SET8在肾癌中高表达，肿瘤分期分级越高，SET8异常表达越明显，且与Wnt信号通路中的关键因子b-cateinin正相关，通过下调SET8在肾癌中表达，能够通过抑制LEF1/TCF4转录复合物的形成来抑制Wnt信号通路，进而抑制肾癌细胞的生长。