靶向COX-2基因的microRNA真核表达载体的构建及其活性鉴定Construction and activity identification of COX-2-targeting microRNA expressing eukaryotic vector
赵庆华;田纪伟;王雷;董双海;王震;刘诚袆;
摘要(Abstract):
[目的]构建靶向COX-2基因的microRNA真核表达载体,评估其转染人喉癌细胞株Hep-2细胞后对COX-2基因的干扰效果。[方法]根据COX-2 mRNA序列设计合成4对microRNA片段,定向克隆到pcDNA6.2GW/EmGFPmiR真核表达载体上,采用测序分析鉴定插入序列的完整性;并将其转染至Hep-2细胞株中。采用实时荧光定量PCR检测其对COX-2基因表达的干扰效果以得到最佳干预效果的RNAi片段。[结果]构建的4组重组体插入片段的碱基序列完全正确。4组microRNA片段对Hep-2细胞中COX-2基因mRNA表达水平均有下降。其中COX-2 mi-1组的表达水平最低。[结论]从构建的4组COX-2 microRNA重组体中成功的筛选出最佳干扰靶点质粒,即COX-2 mi-1组,为应用microRNA靶向COX-2的基因治疗的研究奠定了基础。
关键词(KeyWords): 环氧化酶;软骨;microRNA;RNA;小分子干扰
基金项目(Foundation): 上海市卫生局青年项目(编号:2009-37);; 上海交通大学医学院自然科学基金项目(编号:09XJ21048)
作者(Author): 赵庆华;田纪伟;王雷;董双海;王震;刘诚袆;
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