基于PPARγ/CYP39A1信号通路的蛇床子素抗肝细胞肝癌分子机制研究

Our previous study has found that CYP39A1, a new CYP450 family member, was under-expressed in the HCC tissues, which was closely related to the degree of tumor differentiation as well as the prognosis of patients. The HCC cell proliferation was inhibited by over-expression of CYP39A1. Osthole, one of the active components of Traditional Chinese Medicine, can increase the promoter activity of CYP39A1 and decrease the proliferation of HCC cells. It is reported that transcription factor PPAR gamma (PPARγ) can be activated by osthole. Furthermore, PPARγ and CYP39A1 promoter have potential binding site through bioinformatic analysis. It is speculated that PPARγ may participate in the inhibititory process of osthole on HCC occurrence and progression by up-regulating the promoter activity and protein expression of CYP39A1. Therefore, in the light of the related research performed by our laboratory and the other investigators, the molecular biology methods including the assay of gene over-expression / silence and dual luciferase reporter gene assay, hepatoma bearing mice will be employed in this study. This project will explore the following mechanism from three aspects such as cells, animals and human samples. Firstly, whether PPARγ could bind with the promoter of CYP39A1 and increase its protein expression; Secondly, whether osthole can inhibit HCC occurrence and progression through increasing CYP39A1 protein expression mediated by activating PPARγ. From the above mentioned research, it will help to elucidate the molecular mechanism of the inhibititory effects of osthole on HCC, which will lay the foundation for osthole in the treatment of HCC.

本课题组前期发现细胞色素P450家族新成员CYP39A1在肝细胞肝癌（Hepatocellular carcinoma, HCC）组织中低表达，与肿瘤分化程度及预后相关，过表达CYP39A1后HCC细胞增殖被抑制。中药单体蛇床子素能上调CYP39A1启动子活性并抑制HCC细胞增殖。据报道转录因子PPARγ可被蛇床子素激活，生物信息学显示PPARγ和CYP39A1启动子区具有潜在结合位点，推测PPARγ可能通过上调CYP39A1启动子活性和表达参与蛇床子素抗HCC的过程。本项目拟在此基础上采用基因过表达/沉默、双荧光素酶报告基因及肝癌荷瘤模型等方法，在细胞、动物及人体样本水平，探讨①PPARγ是否可与CYP39A1启动子结合并上调CYP39A1蛋白表达；②蛇床子素是否通过PPARγ调控CYP39A1表达抑制HCC发生发展，藉此阐明蛇床子素抑制HCC的分子机制，为其治疗HCC提供新的研究基础。

背景与目的：CYP39A1作为细胞色素P450超家族的新成员，参与24羟基胆固醇的代谢，近年来该酶在肿瘤中发挥的作用逐渐被认识，但其在肝细胞肝癌(HCC)中的作用尚未明确。申请人前期在临床初步发现CYP39A1在HCC中表达降低，且与预后有关。预实验表明蛇床子素能抑制HCC细胞的增殖，生信分析提示PPARγ和CYP39A1启动子区具有潜在结合位点。本项目拟扩大临床样本探讨CYP39A1在HCC中的表达及临床意义，及蛇床子素是否通过PPARγ调控CYP39A1表达而抑制HCC发生发展。藉此阐明OST抑制HCC的分子机制，为其治疗HCC提供新的研究基础。 .材料与方法：采用生物信息学和免疫组化分析人CYP39A1在HCC中的表达和临床意义，并用Western blot研究人新鲜配对组织中CYP39A1的表达；构建Akt/c-met诱导的肝癌小鼠模型，探讨蛇床子素对HCC的抑制作用，及对CYP39A1和PPARγ表达调控作用；构建CYP39A1过表达质粒，并转染HepG2和SMMC7721细胞株，探讨CYP39A1对HCC的抑制作用及可能涉及的信号通路。采用PPARγ的抑制剂和激动剂来探讨PPARγ对CYP39A1的调控作用及蛇床子素是否通过PPARγ参与对CYP39A1的调控。.结果：CYP39A1在人HCC组织中表达降低，与肿瘤分化程度相关，且CYP39A1表达越低，生存越短。此外，CYP39A1低表达患者血清中总胆汁酸、总胆红素和直接胆红素水平均明显升高。小鼠体内研究发现蛇床子素能抑制HCC的生长，CYP39A1在模型鼠中呈现低表达，而在蛇床子素给药组，CYP39A1和PPARγ的表达均上调，并呈现剂量依赖性。进一步体外研究表明，过表达CYP39A1后，HepG2和SMMC-7721细胞活力受到抑制，p-NF-κB和Nrf2表达也受到抑制。另外，抑制PPARγ后，CYP39A1的表达也随之下降，反之，激活PPARγ后，CYP39A1的表达上调。同时，抑制PPARγ后，蛇床子素对CYP39A1的表达有回调作用。分子对接提示蛇床子素结合于PPARγ配体结合区域的口袋中。.结论：CYP39A1在人和小鼠HCC中表达均降低，并与HCC患者的肿瘤分化程度和预后相关。蛇床子素可通过激活PPARγ进而上调CYP39A1表达而抑制HCC的发生发展。