开发 miR-92a 递释系统用于基于抗血管生成的癌症治疗。

Development of a miR-92a delivery system for anti-angiogenesis-based cancer therapy.

机构信息

Department of Medical Biochemistry, University of Shizuoka Graduate School of Pharmaceutical Sciences, Shizuoka, Japan.

出版信息

J Gene Med. 2013 Jan;15(1):20-7. doi: 10.1002/jgm.2690.

DOI:10.1002/jgm.2690

PMID:23239404

Abstract

BACKGROUND

RNA interference has received much attention as a novel therapeutic strategy. MicroRNA (miRNA) appears to be promising as a novel nucleic-acid medicine because it is able to suppress a series of protein expression that relates to a specific event such as angiogenesis. In the present study, we used dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) as a delivery system for miR-92a, one of the miRNAs regulating angiogenesis, and attempted to deliver miR-92a to angiogenic endothelial cells for the development of cancer therapy by anti-angiogenesis.

METHODS

Cholesterol-grafted miR-92a (miR-92a-C) was bound to TEPA-PCL, and the ratio of nitrogen of TEPA-PCL to phosphorus of miR-92a-C (N/P ratio) was optimized. This complex was transfected into human umbilical vein endothelial cells (HUVECs), and the intracellular localization of miR-92a-C was observed under a confocal laser-scanning microscope by the use of fluorescein isothiocyanate-labeled miR-92a-C. After transfection of HUVECs with miR-92a-C/TEPA-PCL, the expression of miR-92a-target proteins (e.g. integrin α5, mitogen-activated protein kinase kinase 4, sphingosine-1-phosphate receptor 1) was examined by western blotting, and a tube formation assay was performed.

RESULTS

The complex of miR-92a-C with TEPA-PCL was formed and miR-92a-C remained stable with TEPA-PCL at the N/P ratio of 10. After transfection of HUVECs with miR-92a-C complex, miR-92a-C spread into the whole cytoplasm of the cells without any change of cellular morphology, and the expression of several proteins encoded by miR-92a-target genes was suppressed. Furthermore, the capability of forming capillary tubes was impaired in complex-treated HUVECs.

CONCLUSIONS

We have developed a miR-92a delivery system into angiogenic endothelial cells by the use of TEPA-PCL. These results suggest that miR-92a-C/TEPA-PCL is promising for the treatment of tumors via the suppression of angiogenesis.

摘要

背景

RNA 干扰作为一种新的治疗策略受到了广泛关注。miRNA（microRNA）似乎是一种很有前途的新型核酸药物，因为它能够抑制与特定事件（如血管生成）相关的一系列蛋白表达。在本研究中，我们使用磷酸二丁酯-四乙烯五胺基聚阳离子脂质体（TEPA-PCL）作为miR-92a 的递送系统，miR-92a 是调节血管生成的 miRNA 之一，我们试图将 miR-92a 递送到血管生成内皮细胞，通过抗血管生成来开发癌症治疗方法。

方法

胆固醇接枝 miR-92a（miR-92a-C）与 TEPA-PCL 结合，并优化氮与 miR-92a-C 的磷的比例（N/P 比）。将该复合物转染入人脐静脉内皮细胞（HUVECs）中，通过使用异硫氰酸荧光素标记的 miR-92a-C 在共聚焦激光扫描显微镜下观察 miR-92a-C 的细胞内定位。用 miR-92a-C/TEPA-PCL 转染 HUVECs 后，通过 Western blot 检测 miR-92a 靶蛋白（如整合素α5、丝裂原活化蛋白激酶激酶 4、鞘氨醇-1-磷酸受体 1）的表达，并进行管形成试验。

结果

miR-92a-C 与 TEPA-PCL 的复合物形成，并且在 N/P 比为 10 时 miR-92a-C 与 TEPA-PCL 保持稳定。用 miR-92a-C 复合物转染 HUVECs 后，miR-92a-C 扩散到细胞的整个细胞质中，而细胞形态没有任何变化，并且几个由 miR-92a 靶基因编码的蛋白的表达受到抑制。此外，在复合物处理的 HUVECs 中形成毛细血管的能力受损。