MeCP2/HDAC1募集DNMT3A表观调控AR转录促进肝癌细胞的迁移侵袭

Methyl-CpG-binding protein 2 (MeCP2) is a kind of key epigenetic regulator. The role and the molecular mechanisms of MeCP2 in migration and invasion of cancer cells remain unknown. Our previous results showed that MeCP2、HDAC1 and DNMT3A expressions were remarkably upregulated in hepatocellular carcinoma tissues, and the significant inverse correlations were identified between the androgen receptor (AR) expression and them. 5’-Aza-dC and TSA promoted the AR expression in hepatocellular carcinoma cells. In addition, bioinformatic analysis showed that MeCP2 and DNMT3A could bind to the promoter region of AR, and silencing of MeCP2 inhibited the migration and invasion of hepatocellular carcinoma cells. These findings suggest that MeCP2/HDAC1 complex promotes the migration and invasion of hepatocellular carcinoma cells via epigenetic regulation of AR by recruiting DNMT3A. In the present study, firstly, the expressions, correlations and clinical significances of MeCP2, HDAC1, DNMT3A and AR in hepatocellular carcinoma are analyzed by using immunoblotting, immunohistochemical and quantitive real-time PCR analysis. Then, BSP、ChIP、Co-IP, cellular and molecular biological technique are applied to explore the epigenetic regulation mechanism of MeCP2/HDAC1 complex on the AR transcription in hepatocellular carcinoma by recruiting DNMT3A. To use slow virus pack LV-sh-MeCP2, LV-sh-HDAC1 and LV-sh-DNMT3A expression vector, next, we will transfect hepatocellular carcinoma cell line, construct permanent cell line, establish bearing cancer hairless mouse model, examine the metaptosis of tumor through small animal living body imaging system, which will authenticate the effects of MeCP2 on the migration and invasion of hepatocellular carcinoma cells and the molecular mechanism in vitro and in vivo. These results will provide a novel cognition for clarifying the regulatory mechanism of MeCP2 on the migration and invasion of hepatocellular carcinoma cells.

甲基化结合蛋白MeCP2是表观调控关键分子，其对肿瘤细胞迁移侵袭的调控机制尚未阐明。前期研究表明，肝癌组织中MeCP2、HDAC1和DNMT3A高表达且与雄激素受体AR表达负相关，5’-Aza-dC和TSA处理肝癌细胞引起AR表达上调，生物信息学分析显示MeCP2、DNMT3A在AR启动子区存在结合位点，沉默MeCP2可抑制肝癌细胞的迁移侵袭，提示MeCP2/HDAC1募集DNMT3A表观调控AR转录促进肝癌细胞的迁移侵袭。本研究拟通过Western Blot、qRT-PCR等检测肝癌中MeCP2、HDAC1、DNMT3A、AR的表达、相关性及临床意义；利用BSP、ChIP、Co-IP等探索MeCP2/HDAC1募集DNMT3A对AR转录的表观调控机制；建立荷瘤小鼠模型等体内体外实验分析MeCP2对肝癌细胞迁移侵袭的影响及分子机制，为阐明MeCP2在肝癌细胞迁移侵袭中的调控机制提供新认识。

肝癌是世界范围内高发病率高死亡率的肿瘤之一。在全世界范围内，肝癌患者死亡数占癌症相关死亡数的第四位，在发病率方面排名第六（4.7%），死亡率排名第三（8.3%），男性发病率和死亡率远远高于女性。前期研究表明，肝癌组织中MeCP2、HDAC1和DNMT3A高表达且与雄激素受体AR表达负相关，5’-Aza-dC和TSA处理肝癌细胞引起AR表达上调，生物信息学分析显示MeCP2、DNMT3A在AR启动子区存在结合位点，沉默MeCP2可抑制肝癌细胞的迁移侵袭。课题基本按照原有计划进行，课题从细胞水平、动物水平和临床标本等不同层次解析了MeCP2调控肝癌生长的作用机制。MeCP2表达在HCC组织和细胞系中显著上调，高MeCP2水平与较差的总生存率、肿瘤大小和TNM晚期密切相关；体内体外实验表明，过表达MeCP2能够促进肝癌的侵袭和转移；MeCP2通过调控HB-EGF/EGFR通路加强HOXD3基因的甲基化和表达参与肝癌的侵袭和转移，YY1招募HDAC1通过HOXD3相关的ITGA2/ERK1/2细胞信号通路直接调控肝癌细胞的进展；沉默MeCP2抑制HCC细胞增殖，阻止G1-S细胞周期转变，并诱导细胞凋亡；MeCP2能够招募CREB1正向调控RPTOR和PIK3CD的转录过程，激活mTOR信号通路，发挥促进肝癌细胞的增殖，抑制细胞凋亡作用，有可能成为HCC治疗的靶向新疗法；AR转录调控MBL2的表达参与了HCC的发生和性别二态性的形成，为揭示肝癌性别二态性提供新的理论基础。课题在执行期间共发表论文13篇，其中SCI 13篇；参加国内学会会议9人次、省内学术会议15人次；培养硕士研究生5人（其中2人考取博士研究生）；入选省级以上人才计划3人次，荣获省级科研成果奖2项，孵化获批省部级以上科研项目6项，肿瘤细胞分子与表观调控研究创新团队获批陕西省重点科研创新团队，新增科研平台3个。