MiR-483-5p通过靶基因CacyBP调控Wnt信号通路关键分子维持双表型肝细胞癌双向分化的机制研究

We previously found that dual-phenotype hepatocellular carcinoma (DPHCC) can express both hepatocellular biomarkers(AFP/Hep Par-1) and biliary biomarkers(CA19-9/CK19), and showed the characteristics of hepatic progenitor cells (HPCs) with highly postoperative recurrent risk; HPCs were activited with highly proliferation activity by the activation of Wnt signaling pathway.By microRNAs screening assay, we firstly found that the expression of miR-483-5p was significantly increased in the postoperative recurrent HCC,ite target gene,CacyBP was also related to the activity of Wnt pathway.The above results reasonably leading to the hypothesis that miR-483-5p, through its target gene of CacyBP, regulates the activity of Wnt/β-catenin pathway, which may present the molecular mechanism underlying the regulation of DPHCC's bidirectional differentiation through miR-483-5p-CacyBP-Wnt/β-catenin signaling pathway axis. Therefore, in the present study, our aims are: (a). to clarify that CacyBP suppression by miR-483-5p accounts for Wnt/ β-catenin hyperactivation,and the miR-483-5p- CacyBP-Wnt/β-catenin-dependent pathway might enhance CK19 expression and the invasive ability of DPHCC cells; (b).to confirm the correlation among the expressions of key moleculars of Wnt/β-catenin pathway, miR-483-5p and its target gene CacyBP in DPHCC cells and DPHCC tissues; (c).and to establish the molecular phenotype by analyzing the association of the above molecular expressions with tumor bioloical behaviours and clinicopathological features, including long-term prognoses in 200 patients with DPHCC. This project holds great promises to firstly discover special miR483-5p-CacyBP-Wnt/β-catenin signaling cascade for regulating the dual genetic phenotypes of DPHCC,clarifies the molecular mechanism in regulating the double- phenotype characteristics and biological behaviours of DPHCC, provides the molecular targets with potential diagnostic and therapeutic values, and explicits the significance of DPHCC submolecular classification in determining the new individualized therapeutic strategy.

我们前期研究发现：双表型肝细胞癌(DPHCC)表达胆管CK19分子、具有肝祖细胞特征和高复发风险、肝祖细胞的增殖受Wnt通路调控、从术后复发肝癌筛选出miR-483-5p高表达，预测靶基因CacyBP与Wnt信号通路活性相关，这些结果为探讨miRNA-靶基因-Wnt通路轴调控DPHCC双向分化的分子机制提供了新思路。为此，本课题拟通过调控DPHCC细胞miR-483-5p和CacyBP的表达，明确对Wnt通路关键分子的调控路径及对CK19表型及侵袭能力的影响；检测100例DPHCC中miR-483-5p、CacyBP和Wnt通路关联分子的表达特点，并与预后和生物学特性的相关性进行统计学分析。本研究有望率先提出DPHCC类型相关miRNA-靶基因-Wnt信号通路轴、阐明其调控双表型特性与双重生物学行为的分子机制、提出有潜在诊治价值的分子靶标、明确DPHCC亚型对制订个体化诊疗策略的指导意义。

本项目利用高通量miRNA芯片筛选、结合生物信息学分析及经典的分子生物学技术验证，筛选鉴定出在双表型肝细胞癌（DPHCC）中表达显著降低的微小核糖核酸miR-101-3p，并在48对DPHCC癌及癌旁石蜡组织中进行大样本验证；在多种肝癌细胞系中外转miR-101-3p的mimics后证实了CK19表达水平的降低，我们发现miR-101-3p可以通过靶向核转录因子POU2F1（ OCT1）的3’-UTR区下调OCT1的表达，而CK19也同时出现相应的表达降低，这一现象在一系列正常肝细胞和肝癌细胞系中得到了验证；ChIP实验证实 OCT1与CK19两者间有相互结合作用，且过表达或干扰OCT1可以调控CK19的表达；利用216例肝癌临床样本、临床病理信息和患者预后资料，我们证实了相较于 CK19-的肝癌病例， CK19+的DPHCC患者的无瘤生存期及总生存期更短，miR-101-3p 在CK19+肝癌病人组织中表达更低，而OCT1则与miR-101-3p的表达情况成反比；分析发现两者表达水平与患者预后相关，低表达miR-101-3p及高表达OCT1的病人生存期更短。.功能研究方面，目前我们已经构建及包装完成了针对miR-101-3p及OCT1的高低表达腺病毒及慢病毒并成功构建了DPHCC人源肿瘤异种移植（PDX）小鼠模型，下一步计划是利用这些载体工具研究miR-101-3p及OCT1对肝癌细胞恶性表型的影响及其用于肝癌治疗的可行性，着重关注两者对肿瘤细胞由肝细胞表型向胆管细胞表型转化过程中起到的作用，拟检测贴壁与悬浮培养的肝癌细胞系/原代肝癌细胞中miR-101-3p、OCT1与干性相关基因、胆系相关基因等的表达变化；最终通过3D培养动态观察两者对肝癌细胞系/原代肝癌细胞及裸鼠肝原位种植瘤的组织形态学影响。.目前已有结果提示miR-101-3p不但可能作为肝癌患者预后判定的生物标志物，还可能作为靶向OCT1及CK19治疗肝癌的重要工具，具有较好的科学技术价值和应用前景。后续细胞及动物实验正在进行中，并计划拟从新鲜肝癌组织建立一个高表达CK19的DPHCC细胞系，为治疗靶点验证提供研究工具，并申报国家发明专利。项目执行期间，培养研究生1名，课题组发表标注受本课题资助的论文8篇，目前正在对本项目的创新点进行总结，准备投稿有国际影响力的高质量杂志。