oncomiR驱动肝癌干细胞特性的功能及机制研究

Cancer stem cells (CSCs) are considered to be the root cause of tumor initiation, recurrence, drug resistance and metastasis. MicroRNAs are important factors for regulating the self-renewal and differentiation of tumor stem cell. Thus, it is valuable for research on the roles of microRNAs in maglinant characters of cancer stem cell and its regulation law. Using our previous model of liver cancer stem cell, Hep-11 and Hep-12 cells (most of Hep-12 cells with cancer stem cell properties, on the contrast Hep-11 with no tumorigentic ability), we analysis of miRNA expression profile in Hep-12 cells. Then, we establish all the different miRNA over-expression cell lines. By soft agar clone formation test and mice tumorigenic experimental, we screen out 6 miRNAs (miR-512, miR-516b, miR-375, miR-7, etc) that have a regulatory role in malignant characteristics of liver cancer stem cell. These miRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Further studies of miR-512 oncomiR suggest that it is significantly up-regulated in liver cancer tissues. This study intends to explore the role of miR-512 oncomiR in regulating the malignant characteristics of liver cancer stem cells and its potential molecular mechanism. Hopefully, the study will help us to understand the roles and mechanisms of miR-512 oncomiR in the regulation of liver cancer stem cells, and to provide new target molecules for the development of prognosis and therapy of liver cancer.

肿瘤干细胞是肿瘤发生发展、耐药和复发转移的根源，miRNAs是调控肿瘤干细胞自我更新和分化的重要因子。因此研究miRNAs对肿瘤干细胞生物学特性的调控作用具有理论和应用价值。本课题组利用肝癌干细胞-非干细胞之间的miRNA表达谱，筛选在肝癌干细胞恶性生物学行为调控中发挥重要功能的oncomiR。结果发现miR-512，miR-516b等在肝癌干细胞中高表达，体外及体内实验表明miR-512等oncomiR促进肝癌干细胞的自我更新和成瘤能力，提出miR-512等oncomiR驱动肝癌干细胞的恶性生物学特征转化的工作假说。本研究将证实上述oncomiR驱动肝癌干细胞的恶性生物学特征转化的作用；确定肝癌干性相关oncomiR在肝癌发生发展中的功能意义及分子机制；明确miR-512等oncomiR及其靶基因与肝癌临床预后的关系，发现可用于肝癌干细胞特性诊断的分子标志物及药物治疗的分子靶标。

肿瘤干细胞是肿瘤发生发展、耐药和复发转移的根源，miRNAs是调控肿瘤干细胞自我更新和分化的重要因子。因此研究miRNAs对肿瘤干细胞生物学特性的调控作用具有理论和应用价值。本课题组创建基于肝癌肿瘤起始细胞的新型表面标志物--电压依赖性钙通道α2δ1（亚型 5）亚基研究体系。其中，Hep11和Hep12细胞对为研究肝癌复发耐药等干性特征提供了特有的细胞模型；而α2δ1抗体为筛选肝癌及其他癌肿肿瘤干细胞提供了基础。该课题也在此研究基础上建立与肝癌干细胞特性相关 miRNAs及其靶基因的预警调控网络。首先，利用原发和复发肝癌的原代培养细胞系为比较肝癌干细胞-非干细胞之间的miRNA表达谱，检测到miR-512-3p 等oncomiR在相对富集肝癌干细胞中明显上调，而且高表达 miR-512-3p等oncomiR能明显促进肝癌细胞的克隆形成能力及小鼠成瘤能力，证实miR-512-3p等oncomiR驱动肝癌干细胞的恶性生物学特征转化功能。通过RT-qPCR检测本科室收集的肝癌患者临床标本中miR-512-3p的表达量，发现miR-512-3p在肝癌组织中高表达；进一步通过分析TCGA肝癌数据库分析，发现miR-512-3p在肝癌组织中高表达并且与肝癌预后成负相关，进而明确了miR-512-3p与肝癌细胞干性特征的相关性。通过分子生物学软件预测miR-512-3p可能的靶基因--UBE2G2,后续通过双荧光素报告实验发现miR-512-3p直接结合在UBE2G2 3‘UTR区域；然后Western blot实验证实其为miR-512-3p靶基因，接着我们进一步通过UBE2G2银染质谱实验，寻找到其相互作用的E3连接酶CUL1；通过免疫共沉淀实验证实UBE2G2的作用底物A-RAF，从而阐明miR-512-3p通过下游靶基因UBE2G2泛素化调控A-RAF/p-ERK通路，进而影响肝癌的发生发展。