pHLIP酸性穿膜肽和RGD肽共修饰miR-210反义核酸的抗肝癌作用及机制研究

Though it has been proved that microRNAs plays crucial role in cancer，but its therapeutic application is still rare. Our previous studies showed that miR-210 is a oncogene in HBV/HCV relative HCC, but its function and application has not been reported now. Hence, a new pHLIP- RGD-anti-miR-210 AMO (antisense oligonucleotide) peptide will be synthesised with anti-angiogenesis pepetide-RGD, pHLIP ( pH Low Insertion peptide ) and miR-210 AMO according the latest medical research achievement and the theory of tumor microenviroment and angiogenesis, so that to investigate new HCC gene therapy and a novel treatment of inhibiting HCC metastasis and recurrence. Firstly, pHLIP- RGD-anti-miR-210 AMO peptide will be synthesis and its relative therapeutic efficacy, the toxicity and side effect, tumor-targeting and penetrating function, angiogenesis, HBV DNA and HBsAg expression and immunology mechanism will be assessed and studied. In all, it’s important to perform this study, thus providing new insight and methods for exploring RNA relative delivery and treatment in HCC.

microRNA与癌症发生发展的关系已被证实，但其在癌症治疗中的应用较少报道。本课题根据最新医学研究成果，针对肿瘤微环境设计智能纳米载体，结合我们前期筛选出的HBV/HCV肝癌密切相关的miR-210，构建智能纳米药物载体pHILP酸性穿膜肽和RGD肽共修饰miRNA-210反义核酸，探讨肝癌靶向治疗及抑制肝癌转移与复发的新方法。首先化学合成pHILP-RGD-miR-210反义核酸，分别从分子、细胞、组织以及实验动物水平研究pHILP-RGD-miR-210反义核酸对肝癌细胞的靶向性、穿透性、杀伤性、抗肿瘤血管生成和对肿瘤微环境的影响；探讨pHILP-RGD-miR-210反义核酸靶向治疗肝癌的效果及对HBV的抑制与清除作用，分析其免疫机制。本研究有望解决RNA治疗过程中药物分子输送的瓶颈问题, 为RNA干扰在肝癌治疗中的应用奠定基础。

本项目以索拉菲尼耐药与乏氧微环境调控为切入点，深入探讨了乏氧诱导的miR-210-5P及其靶基因线粒体膜蛋白ATAD3A对肝癌细胞索拉菲尼治疗敏感性及其他恶性生物学行为的调控作用、分子机制和意义，取得多项有意义的结果：（1）乏氧可诱导肝癌细胞索拉菲尼耐药，乏氧处理可降低肝癌细胞线粒体膜蛋白ATAD3A的表达。（2）ATAD3A在肝癌组织及肝癌细胞系中呈低表达，其表达与肝癌患者肿瘤大小及组织病理分级呈明显的负相关；肝癌组织中ATAD3A的表达是判断肝癌患者预后的重要指标，高表达ATAD3A的肝癌患者其预后越好。（3）ATAD3A低表达能促进肝癌恶性生物学进展：使用敲低及过表达ATAD3A的LM3及Huh7细胞，通过细胞增殖实验、克隆形成实验、细胞周期、周期相关蛋白检测及体内成瘤实验发现ATAD3A低表达具有促进肝癌细胞增殖加快、克隆形成能力增强、细胞周期进展及体内成瘤能力增强的作用。（4）ATAD3A低表达与肝癌细胞的乏氧状态密切相关，与HIF1α的表达呈负相关，乏氧诱导的miR-210-5P能直接靶向ATAD3A的3’UTR区。（4）miR-210-5P类似物能够部分回复ATAD3A对肝癌索拉菲尼耐药、增殖及细胞周期进展的影响等肝癌细胞恶性生物学行为的影响，而miR-210-5P的抑制剂无此作用。由此进一步证实ATAD3A是miR-210-5P的重要功能性靶基因。（5）肝癌细胞ATAD3A低表达参与激活mTOR信号通路的活化，并且其作用受到miR-210-5P的调节，miR-210-5P抑制ATAD3A表达激活下游mTOR及ERK信号通路。研究结果为丰富索拉菲尼耐药与乏氧的关系提供更多的理论依据，关键耐药介导分子的寻找为肝癌患者治疗索拉菲尼耐药提供更多的靶点和思路。