新型纳米载体介导靶向BTBD7基因治疗肝癌的实验研究

Liver cancer, primarily hepatocellular carcinoma (HCC), is the third leading cause of death from cancer worldwide. Surgical resection is the mainstay of HCC treatment. Although advances in surgical techniques have improved HCC clinical outcomes, the overall survival of patients with HCC remains unsatisfactory because of a high incidence of recurrence and metastasis after hepatic resection. Therefore understanding the mechanisms of HCC metastasis is critical to developing new therapies and improving clinical outcomes. Accumulated evidence has established that angiogenesis and epithelial-mesenchymal transition (EMT) is a critical step in the metastasis of HCC. BTB/POZ domain-containing protein 7 (BTBD7) regulates EMT- associated proteins implicated in HCC progression. In our previous studies, we show that BTBD7 mRNA and protein was highly expressed in HCC cells and tumor tissues, with such expression being associated with: enhanced cell motility, venous invasion, and poor prognosis. BTBD7 promoted HCC angiogenesis and metastasis in vitro and in vivo, but did not influence cell proliferation or colony formation. BTBD7 enhancement of HCC invasion and EMT phenotype occurred via activation of a RhoC-Rock2-FAK signaling pathway resulting in MMP-2/9 production and microvessel formation. Collectively, BTBD7 serves as a novel prognostic factor and a potential therapeutic target of HCC. To explore the efficiency of a noval gene-target therapy system in HCC treatment in vitro and in vivo, by using BTBD7 gene as the target. In the present project, a new fusion promoter (yCDglyTK) driving pcDNA3.1 (-)AV plasmid, which contained the alpha-fetoprotein (AFP) promoter, was constructed by molecular biologic method. The eukaryotic expression plasmid pcDNA3.1(-)AVGFP and pcDNA3.1 (-)AVsiRNA-BTBD7 were constructed by cloning and inserting the green fluorescent protein (GFP) and siRNA-BTBD7 sequence into pcDNA3.1(-) AV plasmid separately. Then these two plasmids were transfected into HCCLM3, SMMC-7721 and Hela cells by using nanoparticles of calcium phosphate. The transfection efficiencies were detected by GFP. Reversed transcript polymerase chain reaction (RT-PCR) and western-blot were used to evaluate the knockdown efficiency of siRNA-BTBD7. The growth curves, cell death and metastasis of HCCLM3 cells transfected with or without pcDNA3.1(-)AVsiRNA-BTBD7 were detected by MTT assay and flow cytometry assay, orthotropic tumor model, respectively. These present project indicated that this noval gene-target therapy system could specifically target HCC cells with high efficiency, providing a new gene therapy strategy for HCC.

肿瘤血管新生在肝细胞癌（HCC）发生和转移复发过程中极为重要。我们前期工作发现BTB/POZ结构域蛋白7（BTBD7）调控肝癌细胞发生上皮间质转化；并激活下游RhoC-ROCK2-FAK通路信号，导致肝癌细胞分泌较多的基质金属蛋白酶2/9(MMP-2/9)和血管内皮细胞生长因子（VEGF）。强烈提示，BTBD7表达具有调控HCC血管新生形成促进转移复发的重要功能，BTBD7是肝癌治疗潜在的重要干预靶点，显示出较好的应用前景。本课题拟构建以BTBD7基因为靶向的RNA干扰质粒与甲胎蛋白（AFP）启动元件调控融合自杀基因yCDglyTK相结合的治疗体系，并以转染效率高且无毒副作用的磷酸钙纳米颗粒为基因运载系统，将该基因治疗体系靶向导入肝癌细胞中，达到增强基因治疗靶向性及安全性的目的。通过研究该体系体外杀伤肝癌细胞和体内靶向抑瘤作用机理，研究结果将为临床肝癌的分子靶向治疗提供新思路和理论依据。

肿瘤微环境和血管新生在肝细胞癌（HCC）发生和转移复发过程中极为重要。对于肝癌转移复发机制的阐明将是临床有效防治复发转移的前提和关键。本项目根据肝癌新生血管和上皮间质转化的病理机理特点，围绕调控肝癌新生血管关键基因BTBD7促进上皮间质转化、激活下游RhoC-ROCK2-FAK通路信号、促使肝癌细胞分泌较多的MMP-2/9等问题，开展以下研究：①构建AFP增强子/启动子驱动融合自杀基因靶向BTBD7siRNA表达基因治疗体系，通过选择磷酸钙纳米颗粒作为基因运输载体将该基因治疗体系导入肝癌细胞；②考察该基因治疗体系杀伤肝癌细胞、抑制瘤内新生血管生成的作用及抗血管药物治疗的旁观者效应；③考察靶向BTBD7siRNA表达基因治疗体系治疗肝癌的作用及机理。分析发现采用的磷酸钙纳米粒由于未经聚乙二醇（PEG）修饰，体内易消除，siRNA 基因沉默的疗效会受到影响。通过Tween80 和PEG 6000优化，提高了BTBD7siRNA基因治疗体系复合物抑瘤效果。并获得新的重要研究发现，HCC细胞中的BTBD7表达上调激活CNPY2，通过磷酸化FAK/src-p130case复合物，促进HCC细胞Snail和纤连蛋白的活化，MMP-9的产生，从而增强HCC的侵袭转移能力。研究成果不但加深对肿瘤血管生成机理、肿瘤EMT机制以及肝癌转移复发分子机制的理解，而且为肝癌治疗拓展新的思路和理论依据。