Jere Dhananjay, Kim Ji-Eun, Arote Rohidas, Jiang Hu-Lin, Kim You-Kyoung, Choi Yun Jaie, Yun Cheol-Heui, Cho Myung-Haing, Cho Chong-Su
Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.
Biomaterials. 2009 Mar;30(8):1635-47. doi: 10.1016/j.biomaterials.2008.12.005. Epub 2008 Dec 20.
Efforts directed in ameliorating silencing studies with shRNA, siRNA and ssiRNA (siRNA with sticky overhangs) are faltered mainly due to the lack of efficient carrier system. In the present study, we developed reductable polyspermine (RPS) carrier composed of multiple spermine units with disulfide linkages for gene expression and silencing studies. In gene expression studies, EGFP expression was found to be almost 4 folds higher and 20 folds safer with RPS carrier than with PEI25K. Moreover, on systemic administration, RPS exhibited significantly high EGFP expression in mice lungs. Similarly in gene silencing studies, EGFP silencing achieved was nearly 1.5 times superior with RPS carrier than PEI25K. Also, RPS delivered Akt1 shRNA (shAkt), siRNA (siAkt) and ssiRNA (ssiAkt) efficiently silenced oncoprotein Akt1 and thereby decreased A549 cell survival. The degrees of cell survival, proliferation and metastasis were differed with the nature of siRNA treatment. Further study at different time intervals revealed that ssiAkt treatment, although superior to sh/siAkt, was highly transient while, shAkt treatment was uniform and prolong. These finding demonstrate the potential use of RPS carrier in gene expression and silencing studies, and significance of the nature of siRNA employed in cancer study.
旨在改善使用短发夹RNA（shRNA）、小干扰RNA（siRNA）和粘性末端小干扰RNA（ssiRNA，带有粘性突出端的siRNA）进行基因沉默研究的努力主要因缺乏高效载体系统而受阻。在本研究中，我们开发了一种由多个带有二硫键的精胺单元组成的可还原多精胺（RPS）载体，用于基因表达和沉默研究。在基因表达研究中，发现与聚乙烯亚胺25K（PEI25K）相比，RPS载体介导的增强绿色荧光蛋白（EGFP）表达量几乎高4倍，安全性高20倍。此外，经全身给药后，RPS在小鼠肺部表现出显著较高的EGFP表达。同样，在基因沉默研究中，RPS载体实现的EGFP沉默效果比PEI25K高出近1.5倍。而且，RPS递送的Akt1短发夹RNA（shAkt）、小干扰RNA（siAkt）和粘性末端小干扰RNA（ssiAkt）有效沉默了癌蛋白Akt1，从而降低了A549细胞的存活率。细胞存活、增殖和转移的程度因小干扰RNA处理的性质而异。在不同时间间隔的进一步研究表明，ssiAkt处理虽然优于sh/siAkt，但具有高度短暂性，而shAkt处理则较为均匀且持久。这些发现证明了RPS载体在基因表达和沉默研究中的潜在用途，以及在癌症研究中所使用的小干扰RNA性质的重要性。
