Petch Amelia K, Sohail Muhammad, Hughes Marcus D, Benter Ibrahim, Darling John, Southern Edwin M, Akhtar Saghir
Pharmaceutical Sciences Research Institute, Aston University, Aston Triangle, Birmingham B4 7ET, UK.
Biochem Pharmacol. 2003 Sep 1;66(5):819-30. doi: 10.1016/s0006-2952(03)00407-6.
Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.
扫描寡脱氧核苷酸（ODN）阵列似乎是预测有效反义试剂的很有前景的体外工具，但尚未见其在哺乳动物系统中的应用报道。在本研究中，我们评估了使用扫描ODN阵列来预测在人表皮样癌细胞系和原代人胶质瘤细胞中靶向人表皮生长因子受体（EGFR）mRNA的有效反义ODN。通过将放射性标记的EGFR转录本与2684个从单体到27聚体的反义序列的扫描阵列杂交，确定了人EGFR mRNA编码区前120nt的杂交可及性图谱。设计了两个与EGFR mRNA内可及序列互补的ODN，即AS1和AS2，并通过体外RNase H介导的切割试验进一步证实了它们与EGFR mRNA杂交的能力。硫代磷酸酯修饰的21聚体AS1和AS2 ODN作为阳离子脂质体递送时，可抑制已建立的人A431癌细胞系以及来自人类受试者的原代胶质瘤细胞的生长。相比之下，乱序对照和与EGFR mRNA中不可及位点互补的反义ODN AS3则无活性。蛋白质免疫印迹显示，AS1 ODN在纳摩尔范围内对A431细胞中EGFR蛋白表达具有剂量依赖性抑制作用。对用21聚体硫代磷酸酯AS1 ODN处理的A431细胞进行的基于微阵列的基因表达谱研究表明，下游信号分子被成功抑制，进一步证实了通过扫描ODN阵列技术设计反义ODN可有效抑制人癌细胞中EGFR的表达。
