雄激素受体下调circR7/miR7-5p/notch4信号通路抑制肝细胞癌的血管拟态作用

In most cases, invasion and metastasis leads to recurrence of hepatocellular carcinoma (HCC). Angiogenesis is necessary for the invasion and metastasis, and vascular mimicry (VM) is a major cause of treatment failure from antiangiogenesis drugs. We had verdicted androgen receptor (AR) is negatively related to the VM in HCC, reducing AR raises the formation of VM,then leads to invasion and metastasis of HCC. So we speculated that the AR can inhibit invasion and metastasis of HCC via downregulating the formation of VM.In this syudy, first, we will establish VM model of HCC, HCC invasion and metastasis models in vitro through the knockout or gene expression of AR, examine the effects of AR on inhibiting HCC invasion and metastasis via down-regulating vascular mimicry. Second, reveal its mechanism on AR inhibiting the form of VM via down-regulating the level of HCC circRNA7 which promoter bonded by AR, then raising the expression of miRNA7-5p, thereby inhibiting notch4 expression, which 3’UTR is combined by miRNA7-5p, with a small interference RNA, luciferase reporter, ChIP, western blot, Q-PCR and PCR assays. Finally, set up nude mouse primary HCC model with knock-outing or over expression AR, to further verify the effects of AR on inhibiting HCC invasion and metastasis via down-regulating vascular mimicry and its mechanism. The study will reveal part of the mechanism on invasion and metastasis of HCC, and provide the basis to look for its new targets for HCC therapy and to screen new drugs to defeat HCC.

浸润转移导致肝细胞癌（HCC）的复发。血管生成是浸润转移的必要条件，血管拟态（VM）是抗血管药治疗失败的主要原因。课题组发现，HCC的雄激素受体（AR）与VM负相关，降低的AR上调VM形成，导致HCC侵袭转移。推测AR能够通过下调VM抑制HCC侵袭转移。本研究建立HCC的VM模型及侵袭转移模型，通过敲除或过表达AR基因，考察AR通过下调VM抑制HCC的侵袭转移作用；用小干扰RNA、荧光素酶报告基因、ChIP、western blot、Q-PCR等技术，揭示其机制与AR结合circR7 启动子下调HCC的circR7水平，从而上调miR7-5p的水平，miR7-5p与notch4 的3’UTR结合进而抑制其表达有关；再建立敲除或过表达AR原发性HCC裸鼠模型，进而验证AR通过下调VM抑制HCC侵袭转移的作用及机制。该课题将探明HCC侵袭转移的部分机制、为寻找其治疗的新靶点及筛选新药提供依据。

浸润转移是HCC的复发的主要原因。血管生成是浸润转移的必要条件，血管拟态（VM）是抗血管药治疗失败的原因。雄激素受体（AR）与HCC的VM负相关。本研究考察AR下调VM抑制HCC的侵袭转移作用及机制，揭示其机制与AR结合circR7 启动子下调circR7，上调miR7-5p，miR7-5p与notch4的3’UTR结合抑制其表达有关。.结果表明：.1、低转移组细胞MHCC97L、HepG2、Hep3B的AR显著高于中转移组MHCC97M、HA22T、SK-hep1，中转移组AR显著高于高转移组MHCC97H、SMCC7721、HCCC-9810。.2、低转移细胞MV形成数显著低于中转移组株，中转移组显著低于高转移组。.3、AR抑制了HCC细胞VM的生成及侵袭； .1）siAR显著增加了低、中转移组VM 的生成及侵袭；.2）oeAR显著抑制了中高转移组VM 的生成及侵袭；..4、AR抑制notch4 的表达，shnotch-4抑制了siAR增加的 notch4表达；.5、shnotch-4抑制了siAR增加的VM的形成；.6、oeAR 明显增加SK细胞miRNA7-5p, 而siAR 明显抑制其表达。.7、oeAR 明显抑制SK细胞circRNA7的表达, 而siAR明显增加其表达。.8、ShmiRNA7-5p 能够恢复oeAR 降低的VM及notch-4表达；.9、miR7-5p直接靶向notch4 的3'UTR抑制其表达.10、circRNA7抑制剂可恢复miRNA7-5p的表达、VM的形成以及notch-4的表达 .11、AR直接靶向circRNA7启动子，抑制其表达。.12、AR 下调HCC裸鼠VM生成抑制其侵袭转移的作用. 与PWPI组比较，oeAR组肿瘤大小显著降低，转移灶、VM、notch4表达减少；与PLKO组比较；siAR组肿瘤大小显著增大，转移灶、VM、notch4表达增加。PWPI组肝病理组织紊乱，胞核增大，oeAR组紊乱改善，胞核减小；与PLKO对照组比较，siAR组HE病理组织紊乱，胞核增大。.该课题明确了AR抑制HCC VM生成抑制其侵袭转移作用及机制；为寻找HCC治疗的新靶点及筛选新药提供依据。