负载miRNA-221-shRNA的双配体修饰壳聚糖纳米颗粒靶向抑制肝细胞癌侵袭转移的作用及机制

Invasion and metastasis mainly limit to improve the prognosis of hepatocellular carcinoma (HCC) and epithelial-mescenchymal transition (EMT) is the essential approach to tumor invasion and metastasis. Therefore, blocking the EMT process is the significant treatment to improve the long-term survival of HCC. However, current targeting drug-carrier and molecular target are not satisfactory. In previous study, we successfully found active targeting ligands and discovered that miRNA-221 downregulated adiponectin receptor 1 (ADIPOR1) to mediate EMT of HCC. Therefore, we hypothesize that upregulation of ADIPOR1, mediated by dual targeting ligand-modified nanoparticles (NPs), perhaps is an efficacious targeting drug for HCC.. In this research, we will attempt to fabricate the dual-ligand modified chitosan NPs loaded with miRNA-221 shRNA to upregulate the level of ADIPOR1 and incubate hepatoma cells with the NPs, then investigate the targeting efficacy to hepatoma cells, and explore the inhibition and molecular mechanism of EMT, invasion and metastasis by the NPs in vitro. Simultaneity, mouse liver cancer model with high metastasis will be prepared and treated with the NPs through intravenous injection. Then we will detect the biodistribution and pharmacokinetics in vivo, and investigate the inhibition and mechanism of liver cancer growth and metastasis through targeted intervention miRNA-221. The research aim at seeking for a novel effective targeting drug treatment method for HCC.

侵袭转移是提高肝细胞癌预后的主要瓶颈，而上皮间质转化(EMT)是肿瘤侵袭转移的关键步骤，故阻断EMT是提高肝癌长期生存的重要手段。但是，目前的靶向药物载体和分子靶点均不理想。在前期研究中，我们成功筛选出肝癌细胞主动靶向配体，并发现miRNA-221下调ADIPOR1介导肝癌EMT过程。为此，我们设想：以ADIPOR1为靶点，用双靶向纳米载体为运载工具，上调其表达可能是一种有效的肝癌靶向药物治疗方案。 . 本项目拟制备负载miRNA-221 shRNA的双靶向壳聚糖纳米药物以上调ADIPOR1表达，干预体外培养的肝癌细胞，检测该纳米药物的靶向性，并探讨对EMT和侵袭转移能力的影响及分子机制；建立高转移肝癌裸鼠模型，静脉给予纳米颗粒，以检测其在体内组织分布及药代动力学，并探讨靶向干预miRNA-221对肝癌生长和转移的抑制作用及机制。本项目旨在探索一种有效的肝癌靶向药物治疗方案。

无法根治性切除和高转移潜能的肝细胞癌切除患者，针对侵袭转移的全身药物治疗有着举足轻重的作用。纳米药物技术是近年来新兴的肿瘤治疗策略，可通过纳米载体系统将药物或基因靶向性运载至肿瘤细胞内，进而提高其在肿瘤细胞内的浓度并降低副反应，以达到靶向治疗目的。本项目中，我们合成了一种新型肝癌双靶向壳聚糖纳米药物载体，并发现miRNA-221是肝癌治疗的一个理想分子靶点。在此基础上，进一步制备了负载miRNA-221-shRNA双靶向壳聚糖纳米载体系统，并处理人肝癌细胞，以检测该纳米药物载体的肝癌细胞靶向性，并探讨该纳米载体系统抑制miRNA-221对肝癌细胞生物学行为的影响及分子机制。同时，建立小鼠肝癌移植瘤模型，尾静脉注射纳米载体系统，检测靶向干预miRNA-221对肝癌上皮间质转化和侵袭转移的抑制作用及分子机制。研究结果表明：（1）通过下调肝癌细胞miRNA-221的表达，可显著抑制肝癌细胞的增殖能力，促进凋亡，并可遏制其上皮间质转化和侵袭转移的能力。（2）通过下调肝癌细胞miRNA-221表达后ADIPOR1表达上调，进而抑制了JAK/STAT3信号通路，证实了抑制上皮间质转化和侵袭转移的分子机制。（3）制备了负载miRNA-221-shRNA的双靶向壳聚糖纳米载体系统，体外细胞实验和体内肿瘤模型证实该纳米载体系统更高效靶向抑制miRNA-221/ADIPOR1/JAK/STAT3信号通路，抑制肝癌上皮间质转化和侵袭转移。本项目证实了该纳米靶向载体系统可高效干预miRNA-221对肝癌生长、上皮间质转化和侵袭转移的抑制作用，为肝癌的靶向治疗提供了一种新的手段。