以转铁蛋白偶联纳米金为载体递送miR-375治疗肝癌的实验研究

Primary liver cancer, mainly hepatocellular carcinoma (HCC), is a worldwide malignancy resulted in serious threat to human health but lack of effective therapeutic medications. Therefore, further studies of molecular mechanisms and therapeutic targets underlying this disease are urgently needed, and development of effective targeted therapeutic interventions is an important direction for liver cancer research. In previous study, we found that miR-375 functioned as an important tumor suppressor in liver cancer；the expression of miR-375 was significantly reduced or absent in HCC tissues and cell lines，and gradually reduced in liver tissues of the pathological process of "chronic hepatitis - cirrhosis - liver cancer". Furthermore, miR-375 can decrease cell proliferation, migration/invasion, and also induce G1 arrest and apoptosis by negative regulation of AEG-1, and overexpression of miR-375 can significantly reduced the malignant phenotype of liver cancer cells in vitro and in vivo. In past study, we also found that miR-375 inhibited autophagy by targeting ATG7 and impaird viability and growth of liver cancer cells under hypoxic conditions in culture and in mice. Hence, we believe that miR-375 is an ideal target for liver cancer treatment. In recent years, there has been an unprecedented expansion in the field of nanomedicine with the development of new nanoparticles for the treatment of cancer. Nanomaterials, especially gold nanoparticles (AuNPs) have unique physico-chemical properties for drug delivery, such as ultra small size, large surface area to mass ratio, and high surface reactivity, biocompatibility and ease of surface functionalization. Transferrin conjugated nanoparticle is an attractive formulation as a vehicle for specific cellular uptake and targeted drug delivery for liver cancer. In this study, we will design and prepare a transferrin conjugated miR-375 gold nanoparticles (TF-miR-375-AuNPs), which facilitate the efficient delivery of miR-375 to liver cancer cells. Moreover, we will detect the stabilty, molecular mechanisms, efficacy and safety of TF-miR-375-AuNPs to treat liver cancer both in vitro and in vivo, so as to provide scientific experimental basis for miR-375 replacement therapy for liver cancer.

针对分子发病机制开发靶向药物是目前肝癌治疗研究的重要方向。我们前期研究发现：miR-375是肝癌中的一个重要抑癌分子，其表达在肝癌组织和细胞株中显著下调；miR-375通过负调控AEG-1而抑制肝癌细胞的增殖、迁移侵袭，并诱导细胞凋亡；过表达miR-375则可在体内外显著减弱肝癌细胞的恶性表型。我们还发现，在缺氧条件下miR-375通过靶向自噬关键分子ATG7抑制自噬，从而抑制肝癌细胞的活性和生长。因此，miR-375是肝癌治疗的一个重要靶点。近年来，纳米金由于其有独特的理化性质，作为药物载体在癌症治疗领域发展迅猛，而转铁蛋白被证实可显著提高纳米金的主动肝癌靶向性。本研究中，我们将设计制备一种新型的转铁蛋白介导靶向的纳米金miR-375偶联物，通过体内外研究考察其稳定性、递送效率、抑癌机制及其治疗肝癌的有效性和安全性，为基于miR-375治疗肝癌的临床应用提供科学的实验依据。

针对分子发病机制开发靶向药物是目前肝癌治疗研究的重要方向。我们的前期研究发现miR-375是肝癌中的一个重要抑癌分子和干预靶点。本研究中：①我们成功制备了一种纳米金miR-375偶联物，证实其具有较好的稳定性、良好的肝癌细胞摄取率以及较高的miR-375携带效率，体外细胞实验证实其能显著抑制肝癌细胞的恶性表型并对miR-375靶基因的表达有显著抑制作用，体内动物实验揭示其具有一定的肝脏和肝癌组织靶向性、对重要脏器的毒性较低，并能在鼠皮下肝癌移植瘤和Akt/Ras诱导的原位肝癌模型上显著抑制肝癌细胞的生长。②探索了应用阳离子脂质体纳米共递送miR-375和阿霉素对肝癌细胞体内外的作用和机制。发现脂质体共递送的miR-375 和阿霉素能够被肝癌细胞及耐药肝癌细胞高效摄取，发挥显著的抑制肝癌细胞恶性表型的作用，能同时作用于miR-375 和阿霉素各自的靶基因，并且，miR-375通过负调控靶基因AEG-1作用于MDR-1(p-gp)的表达而减轻肝癌细胞的耐药性。③优化了纳米递送给药材料，进一步探索了应用阳离子脂质体包被的碳酸钙纳米材料共递送miR-375和阿霉素，并检测了其对肝癌细胞（尤其是耐药肝癌细胞株）体内外的抗肿瘤作用和分子机制，特别就其脏器组织分布特点、药代动力学、心脏毒性、在Akt/Ras诱导的小鼠原位肝癌模型上的抗癌效果进行了细致研究。本课题可为microRNA分子的干预研究提供递送工具和方法，为肝癌的干预治疗提供新的思路与策略。