SMP30表观遗传学对肝癌细胞生物学特性影响机制研究

Hepatocellular carcinoma (HCC) is first killer of malignant tumor in Guangxi province. The occurrence and development of HCC is relative to abnormal expression of various proteins and their interactions.SMP30 is one of new tumor-associated antigen found in our laboratory.SMP30 had a low expression in HCC tissue with a high expression in adjacent tissues of HCC. However,the regulating mechanism of its expression is not clear. We found that epigenetic regulation,such as DNA methylation and micoRNA, was the mainly way for the gene silence of SMP30. Bioinformatics predict that there is a relationship of protein-protein interaction between SMP30 and the three proteins of AFP, PON1 and PBLD.These three proteins are closely relevant to liver cancer and the regulation of MAPK signaling pathway. We propose that the protein-protein interaction between SMP30 and its relating proteins involving in the development of HCC by the role of signaling pathways. This project aims to study the effect of epigenetic regulation of DNA methylation and microRNA on SMP30 expression levels by the model of cell in vitro and nude mice in vivo, to research the coordination effect of methylation and microRNA. We are making further research the role of overexpressing and silencing SMP30 gene on cell biological behavior and eluciate the relationship of HCC occurrence, development and protein interaction of SMP30 with the change of protein level in MAPK signal pathway.

肝细胞癌(HCC)是广西死亡率最高的恶性肿瘤。肝癌发生发展涉及多种蛋白异常表达及相互作用。SMP30 是本课题组发现的新肝癌抗原，在肝癌组织低表达且与肝癌发生发展相关，但机制尚不清楚。我们前期发现DNA甲基化、micoRNA表观遗传调控是SMP30基因沉默的主要方式，生物信息学分析表明SMP30与AFP、PON1、PBLD三个蛋白存在互作关系，而这三个蛋白又与肝癌关系密切并参与MAPK信号通路调节，推测SMP30与这些蛋白相互作用并通过该信号通路调节肝癌发生发展。本课题拟在体外细胞模型和体内裸鼠移植瘤模型上研究SMP30 DNA甲基化及microRNA表观遗传调控对SMP30表达水平影响及其二者的协同作用，观察高表达和沉默SMP30基因对肝癌细胞生物学行为影响，分析验证SMP30与之互作蛋白关系，通过互作基因共表达研究相关MAPK信号通路蛋白的变化，以阐明SMP30在肝癌发生发展中的作用。

SMP30 是本课题组发现的新肝癌抗原，其在肝癌组织低表达且与肝癌发生发展相关，但其潜在临床意义和机制尚不清楚。本项目将研究SMP30低表达与临床肝癌患者预后的关系，探讨表观遗传学（DNA 甲基化、miRNA）对 SMP30 表达及其细胞生物学行为的影响及其机制。结果：①发现SMP30在正常肝脏中优先表达，但在肝细胞癌组织中的表达水平明显降低，其表达水平显著地与肝细胞癌患者肿瘤大小和TNM分期相关，是HCC患者总体生存率降低的独立风险因素。②发现SMP30基因表达下调与其启动子DNA的高甲基化密切相关，焦磷酸测序发现肝细胞癌中SMP30基因CpG岛中4个CpG位点存在甲基化修饰，加入DNA甲基化转移酶抑制剂DAC恢复SMP30基因的表达；发现TFAP2A转录因子通过与SMP30基因启动子区甲基化的CpG序列（-1到-122bp）结合进而抑制SMP30表达。③发现SMP30基因表达下调也与miR-382的调节相关，分析验证发现SMP30是miR-382作用靶点，miR-382在肝癌临床样本中表达高于癌旁组织；在肝癌细胞97L中转染miR-382使细胞中的SMP30表达降低，并促进肝癌细胞的侵袭与迁移。④体外、体内实验均表明SMP30过表达可抑制肿瘤转移，将不同迁移能力的亚细胞株进行转录组、蛋白组的检测及其关联分析验证，发现存在六个差异表达基因，分别为COL6A3、FN1、LAMB3、MMP9、MMP14、MMP15；此外发现，高转移能力的细胞株中钙离子浓度明显升高。⑤发现SMP30与肿瘤转移相关蛋白ROCK1间存在互作关系，并调节MLC蛋白影响细胞骨架结构形成，从而促进细胞迁移；同时，发现SMP30通过Wnt/β-catenin-MMP9信号通路抑制肝癌细胞的侵袭与迁移。本项目阐明了SMP30可作为肝癌临床预后的预测因子，证实SMP30是一个肿瘤抑制因子，为其今后临床应用打下基础。