ZEB2基因促进肾细胞癌侵袭转移的分子机制研究

Our previous studies found that ZEB2 expression in RCC(renal cell carcinoma) tissues was closely correlated with the prognosis of RCC patients. We also found that over expression of ZEB2 promote RCC cells migration and invasion in vitro, but the mechanism is still unclear. In our project, we would firstly detect the migration and invasion ability of the ZEB2 overexpression and knockdown RCC cells in vitro. ZEB2 overexpression and knockdown RCC cells were then injected intravenously through the tail vein into each BALB/c_nu mouse to gain the lung metastasis animal model, we used an in vivo ImagineTechnology to observe the metastatic ability of the tumor cells in vivo. Secondly, western blotting, immunofluorescence and immunohistochemistry were used to detect the expression activity relationships between ZEB2 and the mesenchymal transition (EMT) markers in cell and tissues levels. Then, the PCR gene chips and other techniques were conducted to screen the potential related signaling pathways by which ZEB2 induced EMT，such as the TGF-β/Smad signalling pathway, Wnt/β-catenin signalling pathway, TNFα/NF-κB signalling pathway, etc. Furthermore, co-immunoprecipitation, chromatin immunoprecipitation assay and luciferase reporter assay were administered to confirm the signalling pathway by which ZEB2 regulates EMT. We expect to clarify that ZEB2 promotes RCC invasion and metastasis as well as the molecular mechanism by which ZEB2 induction of EMT. And our researches would provide foundation for in-depth understanding of the pathogenesis of RCC and the potential therapeutic targets for RCC.

我们前期研究发现ZEB2基因表达与肾细胞癌患者预后相关, 且ZEB2基因能促进肾癌细胞株体外的迁移侵袭,但其机制尚不清楚。本项目首先检测ZEB2基因过表达和敲除后肾癌细胞株体外迁移侵袭能力的变化,并通过裸鼠尾静脉注射肿瘤细胞肺转移模型利用活体成像技术观察肿瘤细胞体内转移能力的变化。其次, 应用western blotting、免疫荧光，免疫组化等技术分别在细胞和组织水平检测ZEB2与上皮间质转化(EMT)标记物表达活性之间的关系。然后,通过PCR基因芯片等筛查ZEB2诱导EMT可能相关的信号通路(TGF-β/Smad、Wnt/β-catenin、TNF-α/NF-κB等)。进一步通过免疫共沉淀、染色质免疫共沉淀和荧光素酶报告等实验确定ZEB2调控EMT的信号通路。期望能阐明ZEB2促进肾癌侵袭转移、诱导EMT的分子调控机制，为深入理解肾癌的发病机制及治疗靶点的选择提供实验基础。

我们前期研究发现ZEB2基因表达与肾细胞癌患者预后相关, 且ZEB2基因能促进肾癌细胞株体外的迁移侵袭，但其机制尚不清楚。本项目旨在阐明ZEB2促进肾癌侵袭转移、诱导EMT的分子调控机制，为深入理解肾癌的发病机制及治疗靶点的选择提供实验基础。我们通过体外细胞实验、体内动物实验、生物信息学方法证实了在肾癌细胞中ZEB2受到miR-30a-5p的负调控，外源性过表达miR-30a-5p可通过下调ZEB2抑制肾癌细胞EMT，从而抑制肾癌细胞的体外侵袭与体内转移。我们还进一步通过回复实验反向论证了ZEB2受miR-30a-5p调控进而调控EMT，同时长链非编码RNA DLEU2可通过竞争性内源RNA(ceRNA)机制调控miR-30a-5p从而进一步调控ZEB2。我们的研究成果阐明了miR-30a-5p可通过下调ZEB2抑制肾癌细胞EMT，从而抑制肾癌细胞的体外侵袭与体内转移的分子机制。该项目按计划按时完成。已发表结题论文10篇，均为SCI论文。