Notch3信号通路对透明细胞性肾细胞癌生物学行为调控的机制研究

Renal cell carcinoma (RCC) constitutes the majority of urological neoplasms, which is insensitive to chemotherapy and presents a poor clinical outcome. Exploration of the molecular mechanisms will help unveil more effective therapeutic targets and is an important topic currently being investigated by many groups around the world. Previous study has demonstrated that up-regulation of the Notch3 signaling pathway plays a key role in the pathogenesis of many tumors development and progress. Our results show Notch3 is up-regulated in clear cell RCC (CCRCC) as well. Meanwhile, silencing of the Notch3 suppresses tumor cell proliferation. This indicates Notch3 may play an important role in CCRCCs. By performing a ChIP-on-chip, we were also able to elucidate series of other downstream target genes such as TIAM1，which has been implicated to involve in the metastasis of many cancers.In this project,we will further investigate the mechanism of Notch3 in CCRCCs, by ways of over-expressing its intracellular domain in both cell lines and orthotopic-transplant nude mice model via retrovirus. We will also validate TIAM1 as a de novo downstream of Notch3 pathway by series of experiments such as Expression correlation,EMSA, Promoter Luciferase Assay etc. Taken together, we expect to discover the mechanism of Notch3's regulation in the biological behavior of CCRCCs, which may help to broaden our view for either the diagnoses or therapeutic approaches for this disease.

肾癌是泌尿系统常见肿瘤，对药物治疗不敏感且预后不良。探索其发生发展的分子机制，寻找更有效的分子诊疗靶点一直是该领域的研究热点。Notch3信号通路过度激活是多种恶性肿瘤形成发展中重大的分子事件。在前期工作中我们发现Notch3在透明细胞性肾细胞癌（CCRCC）中高表达，对其沉默可显著抑制肿瘤生长，表明其重要性。我们还通过染色质免疫沉淀芯片发现参与多种肿瘤侵袭转移的TIAM1可能是Notch3下游靶基因之一。本课题中，我们将以探索Notch3在CCRCCs中的作用机制为目标，利用逆转录病毒载体在细胞和裸鼠肾原位移植瘤模型中高表达Notch3胞内活性段来研究该通路对CCRCCs发生发展的生物学作用。同时运用表达相关性分析、EMSA、启动子荧光素酶表达实验等论证TIAM1为Notch3下游靶基因。从而阐明Notch3信号通路对CCRCCs生物学行为调控的分子机制，为诊疗提供新思路。

得益于本课题的资助，我课题组成功地对Notch3信号通路在肾透明细胞癌中作用机制进行了初步探索。发现在将近半数的中晚期肾癌组织中Notch3表达下降；进一步研究发现原发肿瘤相对于的远处转移灶其表达呈现上升的趋势。通过在多种肾癌细胞系中运用慢病毒表达载体沉默或过表达Notch3蛋白后，可显著影响肿瘤细胞侵袭和转移能力。通过蛋白免疫印迹实验，我们在多种肾癌细胞系中证实Notch3信号参与上皮间质转化 ( EMT ) 。据此我们推测Notch3失活可能导致EMT而参与肾癌的侵袭和转移。通过对肾癌细胞用药物抑制Notch3信号通路后，可以观察到广泛参与多种恶性肿瘤EMT过程的ARTN蛋白表达亦上调。综上，我们得出Notch3失活可能参与肾透明细胞癌的侵袭和转移过程，其具体机制可能通过上调ARTN蛋白而实现。为进一步深入探索Notch3在肾癌中的分子机制奠定了可靠的基础。