DNA甲基化/羟甲基化介导肝癌CD133+细胞促侵袭转移的机制研究

A highly distorted epigenome (including aberrant DNA methylation and histone modification patterns) is now accepted to be a general feature of many cancers. 5-hydroxymethylcytosine (5-hmC), is oxidized from 5-methylcytosine (5-mC) by the TET (ten-eleven translocation) family, and is proposed to be a new epigenetic mark that constitutes the first step in an active pathway for DNA demethylation. Cancer stem cells (CSCs) are associated with increased aggressiveness and adverse prognosis in carcinomas, which is suggested to contribute to tumorigenesis. CD133-expressing cancer cells are not only responsible for tumor initiation or progression but also have stem-cell-like properties. The "plasticity" regulatory factors of DNA methylation play an important role in metastasis and invasion via CD133+ cancer cells. Based on the previous studies, we first screen specific tumor-related genes or methylation factors (Dnmts, MBDs,Tets) by which induced CD133+ cells metastasis and invasion in hepatocellular carcinoma (HCC) metastasis tissue samples. After obtained HCC cell lines, we will filter out methylation factors in CD133+ cells with metastasis and invasion properties. The specific factors siRNA or expression vector are constructed, then CD133+ cells are transfected with control vectors and positive vectors. CD133+ cells with vectors are injected into the nude mice and established intrahepatic metastasis and extrahepatic metastasis model (MHCC-97H cell line), then tumor formation is monitored and the tumor volume is measured weekly and calculated. The 5-mC/5-hmC, methylation factors and CD133 expression will be to analyze with metastasis and invasion properties. The results will be advantageous for future therapeutic treatments to target the CSCs in HCC.

紊乱的表观遗传基因组已经成为许多肿瘤的共同特征。DNA羟甲基化胞嘧啶是由Tets蛋白家族氧化甲基化胞嘧啶而来的，已经成为新的表观遗传学分子标记。肝癌肿瘤干细胞CD133+细胞具有更强的促转移能力和侵袭特性，阐明DNA甲基化/羟甲基化介导肝癌CD133+细胞促肿瘤侵袭转移的甲基化分子机制意义重大。本课题拟在前期研究基础上，首先从肝癌转移组织标本中筛选介导CD133+细胞转移的特异性甲基化修饰因子（Dnmts, MBDs,Tets）；其次建立肝癌转移细胞株，分选CD133+细胞，筛选具有CD133+细胞转移特性的甲基化调控分子；利用CD133+细胞构建甲基化修饰因子稳定表达或siRNA质粒转移细胞株，同时建立裸鼠肝癌肝内转移模型和MHCC-97H细胞株肝外转移模型，观察转移进程中5-mC/5-hmC、甲基化修饰改变、肝内肝外转移特性及可能的治疗作用。预期为肝癌干细胞靶点治疗提供新的理据。

在细胞再生和生理活性方面，表观遗传学调控机制维持基因表达和染色质结构方面起重要作用，如DNA甲基化和组蛋白修饰等。5-mC是肿瘤中一种重要的表观遗传修饰，它可以被相关的酶催化生成5-hmC。5-hmC修饰已经发现在胚胎干细胞和神经细胞中大量出现。我们的研究探讨人肝癌组织中CD133(+) 细胞和CD133(-)细胞中5-hmC的修饰频率变化，发现CD133(-)细胞中5-mC含量高于CD133(+)细胞，但CD133(-)细胞中5-hmC含量低于CD133(+)细胞，同时5-hmC与肿瘤转移密切相关。.DNA 甲基化转移酶家族成员(Dnmt1, Dnmt3a,和Dnmt3b)和Tets家族成员(Tet1-3)，在肿瘤组织中维持和建立DNA甲基化进程中发挥重要作用。MDB家族成员(MBD1, MBD2, MBD3, 和MBD4)具有特异性结合核转录因子的功能，在转录表达中起调控效应。异常的DNMTs，Tets和MBDs表达成为肿瘤组织中的普遍现象。我们发现，CD133(+)细胞Dnmts mRNA表达低于CD133(-)细胞，二组细胞间差异显著。且MBD1, MBD3, 和MBD4 mRNA表达也出现类似现象，但Tets mRNA 在CD133(+)细胞表达高于CD133(-)细胞。. PADI4是一个转录共调控因子，催化Ca2+依赖的特异性精氨酸转化成胱氨酸，一种可逆性的表观遗传修饰效应。PADI4蛋白广泛分布于各组织，其表达与许多病理过程相关。化疗仍然是晚期肝癌患者术后的首选治疗方法，本研究关注于PADI4在肝癌化疗耐药中的作用机制。我们观察到PADI4 mRNA高表达出现在肝癌患者术后TACE治疗的患者中，同时体内和体外实验证实，PADI4过表达的肝癌细胞系发生化疗耐药。研究同时发现，PADI4过表达的肝癌细胞系经历自噬，这可能是导致癌细胞耐药而产生的一种自我保护机制。体内外实验证实，自噬抑制剂能有效地恢复肝癌细胞的化疗敏感性。研究结果表明，PADI4蛋白可能通过诱导肝癌细胞自噬现象而致化疗耐药。以上结果提示，DNA甲基化相关性修饰因子在肝癌CD133(+)细胞中扮演重要角色。