Baker B F, Lot S S, Condon T P, Cheng-Flournoy S, Lesnik E A, Sasmor H M, Bennett C F
Isis Pharmaceuticals, Inc., Carlsbad, California 92008, USA.
J Biol Chem. 1997 May 2;272(18):11994-2000. doi: 10.1074/jbc.272.18.11994.
Little is known about the mechanisms that account for inhibition of gene expression by antisense oligonucleotides at the level of molecular cell biology. For this purpose, we have selected potent 2'-O-(2-methoxy)ethyl antisense oligonucleotides (IC50 = 2 and 6 nM) that target the 5' cap region of the human intercellular adhesion molecule 1 (ICAM-1) transcript to determine their effects upon individual processes of mRNA metabolism in HUVECs. Given the functions of the 5' cap structure throughout mRNA metabolism, antisense oligonucleotides that target the 5' cap region of a target transcript have the potential to modulate one or more metabolic stages of the message inside the cell. In this study we found that inhibition of protein expression by these RNase H independent antisense oligonucleotides was not due to effects on splicing or transport of the ICAM-1 transcript, but due instead to selective interference with the formation of the 80 S translation initiation complex. Interestingly, these antisense oligonucleotides also caused an increase in ICAM-1 mRNA abundance in the cytoplasm. These results imply that ICAM-1 mRNA turnover is coupled in part to translation.
在分子细胞生物学水平上，关于反义寡核苷酸抑制基因表达的机制我们所知甚少。为此，我们选择了靶向人类细胞间黏附分子1（ICAM-1）转录本5'帽区的强效2'-O-（2-甲氧基）乙基反义寡核苷酸（IC50 = 2和6 nM），以确定它们对人脐静脉内皮细胞（HUVECs）中mRNA代谢各个过程的影响。鉴于5'帽结构在整个mRNA代谢中的功能，靶向目标转录本5'帽区的反义寡核苷酸有可能调节细胞内信使的一个或多个代谢阶段。在本研究中，我们发现这些不依赖核糖核酸酶H的反义寡核苷酸对蛋白质表达的抑制并非由于对ICAM-1转录本的剪接或转运产生影响，而是由于对80 S翻译起始复合物形成的选择性干扰。有趣的是，这些反义寡核苷酸还导致细胞质中ICAM-1 mRNA丰度增加。这些结果表明，ICAM-1 mRNA的周转部分与翻译相关。
