MLL促进肝癌的靶基因鉴定及其表观遗传学特性的研究

MLL is a critical oncogene on inducing Leukemia, but its biological functions in other tumors are still unclear. We have shown that MLL is involved in the activation of Yap1 transcription and the induction of hepatocellular carcinoma (HCC) promoted by menin, Its mechanism maybe depend on the H3K4 positive histone methylation, but the exact regulation mechanisms and histone modification patterns need to be further determined. This study utilizes HCC model induced by DEN in MLL gene tissue-specific knockout mice, in order to confirm that MLL promotes the key biological functions of the malignant phenotype in HCC, and evaluate its pathological characteristics. Based on the analysis of the chromatin histone methylation which has been obtained from data integration, using ChIP and other key technologies, the target genes network regulated by MLL will be screened and identified in HCC. The advantage of MLL on regulating histone methylation modification of target genes transcription and its partners of histone methyltransferase complex will be further clarified. We will also investigate the potential application prospect of the small molecular MLL target compounds on the treatment of HCC. This study will be helpful on revealing the epigenetic mechanism of the positive histone methylation modification on promoting hepatocarcinogenesis. It will provide a new therapeutic target on the treatment of HCC.

MLL是诱发白血病的关键原癌基因，但在其他肿瘤中的生物学作用尚不清楚。我们发现，MLL参与menin促进的Yap1基因转录激活及肝癌发生，其机制可能依赖于H3K4等正性组蛋白甲基化修饰，但精确的调控机制及组蛋白修饰规律有待进一步深入阐明。本项目利用MLL基因组织特异性敲除等小鼠建立DEN诱导的原发性肝癌疾病模型，证实MLL促进肝癌恶性表型的关键生物学功能，评价病理学特点；从已获得的染色质组蛋白甲基化等组学数据的整合分析入手，利用ChIP等关键技术深入筛选和鉴定肝癌中受MLL调控的靶基因网络，明确MLL调控关键靶基因转录的优势组蛋白甲基化修饰规律、鉴定组蛋白甲基转移酶复合物组成；探讨以MLL调控的正性组蛋白甲基化修饰为靶点的小分子化合物在肝癌治疗中的潜在应用前景。这将有助于揭示正性组蛋白甲基化异常修饰促进肝癌发生的表观遗传学机制，为肝癌防治提供崭新的靶点。

肝细胞癌是世界第四大癌症致死原因，具有侵袭力强、易转移、预后差、死亡率高等特点，因此阐明肝细胞癌发生相关的关键途径和机制以及研发新的高效靶向制剂尤为重要。已有研究表明组蛋白修饰的异常与肝细胞癌的发生密切相关，MLL复合物为组蛋白甲基转移酶复合物，可以诱导基因启动子区域发生H3K4me3、H3K79me2等组蛋白甲基化修饰，在多种恶性肿瘤中发挥重要的生物学作用，但其对肝细胞癌的作用尚未见报道。.本课题首先从小分子抑制剂MI-2和EPZ004777对肝癌细胞恶性表型的影响入手，深入探究MLL复合物介导的正性组蛋白甲基化修饰是否参与肝细胞癌原癌基因c-Myc的转录调控；其次，明确了靶向 BET蛋白小分子抑制剂I-BET151对肝细胞癌增殖等恶性表型的影响，阐明其调控肝癌关键原癌基因Yap1转录的表观遗传学机制；在此基础上，我们又深入探讨了缺氧应激调控的H3K4me3组蛋白甲基化修饰规律及其在肝细胞癌恶性进程中的生物学作用；设计、合成了一系列小分子化合物，并对其抗肝细胞癌活性进行了筛选和鉴定。.结果表明Menin-MLL调控的H3K4me3及MLL-Dot1L调控的H3K79me2是小分子抑制剂MI-2和EPZ004777抑制c-Myc转录的重要表观遗传学机制，联合应用MI-2和EPZ004777可协同抑制肝癌细胞恶性增殖和c-Myc的转录；I-BET151可明显降低Yap1启动子H3K79me2正性组蛋白甲基化修饰水平，H3K79me2甲基转移酶DoT1L的功能沉默可明显抑制肝癌细胞的增殖及Yap1的转录；此外，缺氧应激可通过诱导H3K4甲基转移酶MLL的表达显著上调肝细胞癌恶性增殖表型相关基因HOXB9启动子区域H3K4me3的修饰水平，促进HOXB9的转录，进而促进了VEGF及TGFβ1等缺氧微环境因子的表达；设计合成的一系列4-苯氧基苯甲酰胺类小分子化合物可明显抑制肝细胞癌的恶性增殖表型。.综上所述，Menin-MLL、MLL-Dot1L介导的H3K4me3、H3K79me2正性组蛋白甲基化修饰是调控c-Myc、Yap1、HOXB9等关键原癌基因转录促进肝癌进程的重要分子事件。本研究阐明了MLL调控肝癌关键原癌基因转录的表观遗传学机制，探讨了联合应用的协同治疗意义，拓展了MLL相关蛋白小分子抑制剂在抗肝癌领域上的认识，为肝细胞癌的防治提供了崭新的靶点。