EGF修饰的靶向性载miR-7脂质体的构建及其抗卵巢癌作用研究

Gene therapy provides great opportunities for the treatment of cancer．miRNAs play an important role in the regulation of tumor progression, invasion and metastasis. Our study found that low expression of miR-7 in ovarian cancer tissues, especially in metastatic tissues, except that EGFR protein expression levels were significantly increased. In further research, we also found that miR-7 can significantly regulate EGFR expression and EMT in ovarian cancer cells, which significantly inhibited the cell proliferation and invasion. However, miRNAs are poor in vivo stability, short half-life, low transfected efficiency, so the key of application miRNAs molecule is selecting an appropriate carrier to reach and enter the tumor cells for the purpose of RNAi. In this study, we intend to prepare EGF-modified cationic liposomes containing miR-7, through in vitro and in vivo experiments to clarify the physicochemical properties of the liposome and the target for ovarian cancer. We also plan to clarify the effect of the liposomes in inhibiting ovarian cancer cell growth and invasion or migration, and their associated mechanisms; to study the antitumor effect and safety of the liposomes containing miR-7 in nude mice of ovarian cancer. The obtained research results would form the basis for the future application of miR-7 therapy in the clinic.

基因治疗为癌症的治疗提供了良好的前景。miRNA 在调控肿瘤的发生发展、侵袭和转移方面起到重要作用。我们的研究发现miR-7在卵巢癌组织尤其是转移灶组织低表达，而EGFR的蛋白表达水平显著升高。进一步的研究还发现转染miR-7后，能够明显调控卵巢癌细胞的EGFR表达，从而显著抑制卵巢癌细胞的增殖和侵袭能力。但miRNA具有不稳定性、半衰期短、细胞转染效率低等缺点，故应用miRNA分子的关键是选择合适的载体使其到达肿瘤部分并进入细胞，达到RNAi的目的。本课题拟通过制备EGF修饰的靶向性载miR-7脂质体、 体外细胞和裸鼠皮下/原位移植卵巢癌模型体内实验来明确该脂质体的理化性质和对卵巢癌细胞的靶向情况；阐明该脂质体对卵巢癌细胞生长和侵袭迁移的影响及其相关机制；研究该载miR-7脂质体对荷卵巢癌裸鼠的靶向治疗作用及其有关机制，并进行安全性评价，为今后的临床应用提供理论和临床前研究基础。

本研究项目原拟通过制备靶向性载miR-7纳米载药递送系统、 体外细胞和裸鼠移植卵巢癌模型体内实验来明确该纳米粒的理化性质和对卵巢癌细胞的靶向情况；阐明纳米粒对卵巢癌细胞生长的影响及其相关机制；研究该载miR-7 纳米粒对荷卵巢癌裸鼠的靶向治疗作用及其有关机制，并进行安全性评价。为此，我们反复研究，最终选择了一种毒性较小的材料来制备共载紫杉醇及miR-7的mPEG-PLGA-PLL纳米粒，通过体外细胞和裸鼠皮下移植卵巢癌模型体内实验，明确了该纳米粒的理化性质和对卵巢癌的抑制作用及其相关机制。通过三年工作，已经成功地证明了以下科学事实：第一：成功制备的P/MNPs粒径位于100nm左右，能保护RNA不被降解，并且缓慢释放紫杉醇及miR-7，具有药物缓释效果；第二：P/MNPs易于被细胞摄取，高效将miR-7转入细胞，抑制由于PTX而被激活的与耐药相关的EGFR/ERK通路，增加肿瘤细胞对PTX的敏感性，增强PTX诱导肿瘤细胞凋亡的能力；第三：P/MNPs在体内实验中能通过EPR效应靶向肿瘤部位，增加肿瘤组织miR-7表达，抑制EGFR表达，促进肿瘤细胞凋亡，增强化疗药物抑瘤效果，并且无明显的毒副作用。我们的研究首次成功制备了一种安全的共载PTX及miR-7的纳米载药递送系统，抑制化疗药物引起的耐药相关细胞通路改变，从而增加化疗效果，在卵巢癌治疗中具有潜在的应用价值。