内含miRNA122和miRNA224海绵体肝细胞癌特异靶向病毒样颗粒及其伴随检测的研究

The incidence of hepatocellular carcinoma (HCC) is high in China, and the effect of current treatment is limited. Studies have highlighted that dysregulated microRNA (miRNA) in the tissue of HCC play critical roles in HCC progression, and regulating target miRNA may be a new field worth studying on HCC treatment. Based on the positive results on HCC by delivering miRNA122 with MS2 virus-like particles (VLPs) in our previous work, in this research, we decide to upregulate miRNA122 and downregulate miRNA224 simultaneously using the methods such as prokaryotic expression and phage surface display to prepare VLPs packaging pre-miR122-miR224 sponge-pre-miR122 with HCC specific cell-penetrating peptide on the surface, and then discuss the therapeutic effects in vitro and in vivo as well as on cell and animal level comparing with VLPs only pakaging pre-miRNA122 or miRNA224 sponge in order to provide new idea for the treatment of HCC. Besides, at the time of treatment with VLPs on HCC mouse model, the levels of miR122 and miR224 and tumor size are measured, and the correlations between miRNAs level and tumor size are analyzed to adjust the dosage of VLPs and further to determine whether the level of miRNA could be used as a companion detection biomarker in the targeted therapy using VLPs to improve the accuracy of treatment.

肝细胞癌在我国发病率高，目前的治疗手段效果有限，研究提示miRNA表达失调与其发生发展密切相关，调控靶miRNA水平可能是肝癌治疗值得探讨的新领域。在我们前期通过病毒样颗粒递送miR122对肝细胞癌有良好治疗效果的基础上，本研究拟从同时上调miR122和下调miR224两个方面，采用原核表达、噬菌体展示等技术制备表面展示肝癌细胞特异性穿透肽、同时内含pre-miR122-miR224 sponge-pre-miR122序列的重组病毒样颗粒，通过体外、体内实验在细胞和动物水平验证其肝癌抑制作用，并与只含miR122前体或miR224海绵体的效果进行比较，以期为肝癌治疗提供新思路；此外，我们在对肝癌小鼠模型给药治疗时，同时监测肿瘤组织、血清中两种miRNA水平和肿瘤大小变化，分析二者相关性以调整治疗方案，探讨miRNA水平是否可作为上述特异靶向病毒样颗粒使用的伴随检测标志物，提高治疗精准性。

非编码RNA可通过调控基因表达影响肿瘤的恶性表型，其中miRNAs在肝癌发生发展中的作用已有广泛研究。根据肝癌miRNAs表达谱，同时靶向2种不同方向失调的miRNAs，可以增强靶向单一miRNAs的抑癌效果。本研究选取抑癌性miR-122的前体序列和促癌性miR-21的分子海绵（sponge），借助MS2病毒样颗粒（VLP）和EGFR特异性配体GE11小肽，将目标RNA片段递送进入肝癌细胞，从递送效果、细胞增殖、迁移、侵袭和凋亡等方面探究其治疗效果。本研究在5种肝癌细胞系中验证，结果显示GE11-VLP能将目标RNA有效递送进入肝癌细胞，在除Hep3B外的4种肝细胞中miR-122前体可显著提高成熟miR-122的胞内水平；miR-21 sponge可竞争性抑制miR-21，从而上调靶基因mRNA和蛋白质的表达水平；细胞功能实验结果显示，单独递送miR-21 sponge可以有效抑制多种肝癌细胞的增殖、迁移和侵袭，促进细胞凋亡；协同递送miR-21 sponge和miR-122前体可增强对肝癌细胞恶性表型的抑制效果。该microRNA递送策略同时调节肝癌细胞内2种不同方向失调的miRNAs，为肝癌的靶向治疗提供新的思路。.环状RNA（CircRNA）是近年新发现的共价闭环内源性非编码RNA，可通过与miRNAs相互作用，影响肿瘤发生发展。CircMTO1在肝细胞癌（HCC）中表达下调，抑制HCC的进展。然而，潜在的信号通路仍是未知的。生信预测以及相关实验验证了circMTO1和miR-541-5p之间的相互作用。CCK-8、transwell和凋亡实验显示miR-541-5p促进HCC的发展。生信预测以及相关实验发现ZIC1是miR-541-5p的下游靶基因。体外和体内rescue实验研究circMTO1、miR-541-5p和ZIC1的调控机制。结果表明，CircMTO1沉默后，ZIC1下游调节的Wnt/β-catenin信号通路标志物和间质标志物的表达上调，而上皮标记物表达下调。沉默miR-541-5p具有相反的作用，且可逆转circMTO1沉默对ZIC1下游调节因子的调节因子的调控作用。综上，circMTO1通过circMTO1/miR-541-5p/ZIC1轴调控Wnt/β-catenin信号通路和上皮-间质转化抑制HCC进展，使其成为一个新的治疗靶点。