法尼酯X受体促进腺苷A2AR受体表达的分子机制及抗肝癌的作用

Farnesoid X receptor(FXR) is overexpressed in normal liver tissue, which plays a key role in inhibiting tumorigenesis, whereas defect of FXR gene is relevant to hepatocellular carcinoma(HCC). Our previous experiments showed that FXR could upregulated obviously adenosine A2A receptor(A2AR) expression significantly in HCC cells. Further study demonstrated that there might have potential FXR binding sites in A2AR promoter region. Here, we propose a hypothesis that FXR can induce A2AR expression in transcriptional level, and further explore the mechanism and significant of FXR-A2AR pathway in hepatocarcinogenesis. Our proposal is aim to investigate the molecular mechanism of A2AR expression upregulated by FXR, which clarify the possible mechanism including activating FXR via its ligands binding, forming FXR-RXRα heterodime , binding to A2AR promoter region and enhancing A2AR expression by using real-time PCR, Western blot, gene engineering technology, dominant negative(D/N) strategy , terminal sequence analysis and reduction assay, Luciferase reporter gene assay, promoter site-directed mutation, electrophoretic mobility shift assay (EMSA) , chromatin immunoprecipitation (ChIP) , RNA interference(RNAi) and animal experiments. Our study will not only determine whether FXR bind to sites of A2AR promoter, but also explore further significant and mechanism of FXR-A2AR pathway involved in HCC. Therefore, the completion of the project successfully will provide a novel target for HCC therapy.

法尼酯X受体（FXR）在正常肝脏中高表达，且具有抗肿瘤的功能，其功能缺陷与肝细胞肝癌（HCC）的发生有关。前期研究结果显示：在肝癌细胞中FXR能显著上调腺苷A2A受体（A2AR）的表达；进一步研究发现A2AR启动子区域可能存在与FXR结合的潜在位点。在这里我们假设，FXR能从转录水平调控A2AR表达，并能在抑制HCC发生的过程中发挥作用。本研究拟采用real-time PCR、Western blot、分子克隆、显性失活、末端序列续减分析、荧光素报告基因分析法、基因突变、凝胶迁移阻滞实验、染色质免疫沉淀、RNA干扰及动物实验等方法，探讨FXR活化后形成的FXR-RXRα异源二聚体是否可与A2AR启动子区域关键位点相结合，促进A2AR的表达，以期明确FXR调控A2AR表达的新机制，并进一步解析FXR-A2AR通路的抗HCC作用。因此，该项目成功实施可为肝癌的靶向治疗提供新型靶点。

本研究的结果包括以下两方面：（1）初步解析、鉴定人FXR在A2AR基因的启动子区域的结合位点，（2）在人肝癌细胞水平解析FXR-A2AR通路在抗HCC发生的作用。. 在第一方面，对于寻找、解析、鉴定人FXR在A2AR基因启动子区域的结合位点方面得到重要的实验数据，发现和A2AR启动子区域结合的两个FXR序列，为探讨FXR在转录水平上活化A2AR表达的分子机制奠定了实验依据和基础。. 在第二方面，对于探讨人肝癌细胞中FXR-A2AR通路抗HCC发生的作用方面，建立了人肝癌细胞SMMC-7721 FXR、A2AR表达缺失的细胞株，经鉴定后从细胞水平检测了FXR-A2AR通路的抗HCC发生发展的作用。并进一步采用FXR配体干预、予以A2AR抑制剂刺激等手段，验证FXR-A2AR通路具有抗HCC作用，为下一步动物实验验证奠定了基础。