中文摘要：

目的探讨目前常见的甲基化、锁核酸和肽核酸三种方式修饰的反义寡聚核苷酸对miR-125b的抑制作用,寻求一种高效的、特异性的miRNA表达沉默方式。方法分别合成甲基化、锁核酸和肽核酸三种方式修饰的反义寡聚核苷酸转染U87细胞,流式细胞仪分析转染效率,MTT评价细胞毒性,实时荧光定量PCR分析miR-125b表达水平。结果 48h甲基化、锁核酸、肽核酸修饰的反义寡聚核苷酸使用FugeneHD转染效率无明显差异。无转染试剂共培养甲基化、锁核酸和肽核酸修饰的反义寡聚核苷酸,肽核酸修饰的转染效率均显著高于甲基化、锁核酸修饰的反义寡聚核苷酸。使用FugeneHD转染的甲基化、锁核酸和肽核酸修饰的反义寡聚核苷酸三者之间无明显毒性差异。锁核酸和肽核酸修饰的反义寡聚核苷酸对miR-125b的抑制作用显著高于甲基化;肽核酸修饰的反义寡聚核苷酸抑制能力及持续性显著高于甲基化和锁核酸修饰。结论肽核酸修饰的反义寡聚核苷酸在抑制miRNA表达能力上具有显著的高效性、安全性和持续性。

英文摘要：

Objective To investigate the current common methylation, locked nucleic acid and peptide nucleic acid modified three ways antisense oligonucleotides on the impact of miR-125b inhibition effect,and seek an efficient, specific way of miRNA expression silence. Methods Methylation, locked nucleic acid and peptide nucleic acid modified three ways antisense oligonucleotides were respectively transfected into U87 cells. Transfeetion efficiency was analyzed by flow cytometry, cytotoxicity was evaluated by MTT assay, and miR-125b expression levels were analyzed by real-time quantitative PCR analysis. Results 48h after methylation, locked nucleic acid, peptide nucleic acid-modified antisense oligonucleotides transfeetion using Fugene HD, the transfeetion efficiency was no significant difference among them. Methylation, locked nucleic acid and peptide nucleic acid-modified antisense oligonucleotides were cultured without transfection reagent;peptide nucleic acid-modified transfection efficiency was significantly higher than methylation, locked nucleic acid-modified antisense oligonucleotides. There were no significant differences of toxicity among methylation,locked nucleic acid and peptide nucleic acid-modified antisense oligonucleotides transfection using Fugene HD. locked nucleic acid and peptide nucleic acid-modified antisense oligonucleotides inhibition of miR-125b were significantly higher than methylation; The ability of peptide nucleic acid-modified antisense oligonucleotides on inhibition and persistence was significantly higher than the methylation and locked nucleic acid-modified. Conclusions Peptide nucleic acid-modified antisense oligonucleotides on the inhibition of miRNAexpression have significant capacity of efficiency ,safety and sustainability.