LncRNAHOTAIR与雄激素受体AR协同调控HIF-2α转录促进肾细胞癌血管生成和耐药

Targeting drugs brought revolutionary developments of the treatment of late stage renal cancer; however, most patients will be non-sensitive and even resistant to them. HIF-2α is a key gene in the promoting of renal cancer development and resistance to targeting drugs, while the transcriptional modulation of it is still not clear. Our results demonstrated that AR promotes growth of renal cancer cells by activating HIF-2α transcription. Furthermore, we found that LncRNA HOTAIR participates in the transcriptional modulation of HIF-2α by AR. Therefore, we hypothesized that HOTAIR may play an important role in the binding of AR and HIF-2α promoter. In the present project, we will detect whether HOTAIR carry AR into the promoter region of HIF-2α as a “scaffold” through RIP assay and other methods. In addition, we will identify whether HOTAIR and AR promotes renal cancer angiogenesis and resistance to targeting drugs via HIF-2α through tumor cell- vascular endothelial cell interaction assay, xenografts in nude mice and other experiments, and whether knockdown f HOTAIR and suppression of AR activity by bicalutamide and enzalutamide will increase the response of renal cancer cells to targeting drugs. This project will demonstrate new mechanism of HIF-2α transcriptional modulation, and provide pre-clinical evidence and new targets for the utilization of multiple targeting strategy to improve the treatment of renal cancer.

靶向药物为晚期肾癌治疗带来了革命性进展，但多数患者终将发生耐药甚至抵抗。HIF-2α是肾癌进展和靶向耐药的关键基因，但是它的转录调控机制尚不清楚。我们的研究证实：AR可通过激活HIF-2α转录促进肾癌细胞生长。进一步研究发现：LncRNA HOTAIR参与了AR对HIF-2α的转录调控。我们推测：HOTAIR在AR与HIF-2α启动子结合过程中可能扮演着重要角色。本课题拟通过RNA结合蛋白免疫沉淀等方法检测HOTAIR是否发挥“脚手架”作用将AR携带到HIF-2α启动子区域促进其转录；通过检测肿瘤细胞-血管内皮细胞相互作用及裸鼠体内实验等验明HOTAIR与AR是否通过HIF-2α促进肾癌血管生成与靶向药物抵抗，并进一步明确敲除HOTAIR、用药物抑制AR活性是否增加肾癌细胞对靶向药物的敏感性。本课题有望明确调控HIF-2α转录的新机制，为多靶点策略提高肾癌靶向治疗效果提供理论依据和新靶点。

肿瘤血管生成是肾细胞癌（RCC）的重要特征，也是RCC靶向治疗的重要靶点。已有报道和我们的肿瘤数据库分析都发现lncRNA HOTAIR在RCC中高表达，并且其高表达与患者的不良预后相关；但是，HOTAIR是否能促进RCC肿瘤血管生成及其潜在机制尚不清楚。据报道HOTAIR能介导多种激素受体的促癌功能，而我们之前的研究发现雄激素受体（AR）促进RCC生长与侵袭。因此，我们在RCC细胞中检测HOTAIR和AR是否相互调节，发现HOTAIR和AR形成了正反馈环，促进彼此的表达。有趣的是，我们还通过生物信息学分析发现，在RCC中HOTAIR与Hedgehog通路（尤其是GLI2）相关。Hedgehog-GLI信号通路在细胞癌变时被重新激活以促进组织生长、血管生成。我们发现在AR表达阳性RCC细胞中HOTAIR促进GLI2表达，而在AR表达阴性RCC细胞中HOTAIR不影响GLI2表达；进一步通过RNA纯化—染色质分离实验（ChIRP）发现HOTAIR能与AR相互作用，而且AR敲低后HOTAIR与GLI2启动子区域结合减少；通过荧光素酶报告基因实验发现，HOTAIR与AR共同敲除后，GLI2启动子转录活性降低程度高于二者的单独敲除。这些结果表明HOTAIR与AR协同结合GLI2启动子并增加其转录活性。功能分析证实了HOTAIR-AR轴促进GLI2的表达，并发现HOTAIR和AR协同增强了VEGFA，PDGFA和肿瘤干细胞转录因子等GLI2下游基因的表达；而且，体外实验和动物异种移植肿瘤实验都证实了HOTAIR和AR协同促进了RCC细胞肿瘤血管生成和肿瘤干性。本课题发现了lncRNA和AR协同驱动Hedgehog通路促进RCC中肿瘤血管生成的新机制；这一通路独立于肾癌中的VHL/HIF促血管生成通路，HOTAIR和GLI2可能是抑制肾癌血管生成的新靶点。此外，我们还鉴定了AR在GLI2启动子区域的结合序列，发现GLI2是AR的靶基因。总体上，本课题对于理解肾癌进展的分子机制和研发新的肾癌的治疗方法有重要意义。