Antisense treatment directed against mutated Ki-ras in human colorectal adenocarcinoma
Antisense treatment directed against mutated Ki-ras in human colorectal adenocarcinoma
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DOI：
10.1136/gut.48.2.230
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发表时间：
2001-02-01
期刊：
影响因子：
24.5
通讯作者：
Clarke, PA
中科院分区：
文献类型：
--
作者：
Andreyev, HJN;Ross, PJ;Clarke, PA
背景-Kirsten ras (Ki-ras) 突变在胃肠道癌症中很常见，而一种密码子 12 突变（甘氨酸到缬氨酸）在结直肠癌中尤其具有侵袭性。目的-研究该缬氨酸点突变是否可以用反义寡核苷酸靶向，并确定任何反义/mRNA 相互作用的功效。方法-在无细胞系统中针对目标和对照 Ki-ras RNA 以及针对目标和对照筛选 29 种反义寡核苷酸结果-寡核苷酸的活性和特异性各不相同。各个寡核苷酸的结果在无细胞模型和使用两种不同摄取启动子的细胞培养物中是一致的。尽管无法检测到单次处理后 Ki-ras mRNA 和蛋白质表达的变化，但只有一种寡核苷酸在无细胞系统中对目标 Ki-ras mRNA 的切割具有特异性，并且在细胞培养物中表现出特异性。无细胞系统中的实验表明，寡核苷酸相对难以接近点突变。 Ki-ras RNA 分子上远离点突变的其他位点可以更有效地靶向。结论-用反义寡核苷酸成功靶向临床相关的 Ki-ras 点突变是困难的，因为突变位点处的 RNA 结构，并且与 Ki-ras mRNA 上的其他位点相比效率较低。
Background-Kirsten ras (Ki-ras) mutations are common in gastrointestinal cancer and one codon 12 mutation, glycine to valine, is particularly aggressive in colorectal cancer.Aims-To investigate if this valine point mutation could be targeted with antisense oligonucleotides and to determine the efficacy of any antisense/mRNA interaction.Methods-Twenty nine antisense oligonucleotides were screened against target and control Ki-ras RNA in a cell free system and against target and control cell lines in culture.Results-The activity and specificity of the oligonucleotides varied. Results for the individual oligonucleotides were consistent in a cell free model and in cell culture using two different uptake promoters. Only one oligonucleotide was specific in its cleavage of target Ki-ras mRNA in the cell free system and appeared specific in cell culture, although changes in Ki-ras mRNA and protein expression following a single treatment could not be detected. Experiments in the cell free system showed that the point mutation is relatively inaccessible to oligonucleotides. Other sites on the Ki-ras RNA molecule, away from the point mutation, can be targeted more effectively.Conclusions-Successful targeting of the clinically relevant Ki-ras point mutation with antisense oligonucleotides is difficult because of RNA structure at the mutated site and is inefficient compared with other sites on the Ki-ras mRNA.