人Hiwi miRNA干扰质粒的构建及其鉴定
Journal of Clinical Medicine in Practice(2013)
Abstract
目的构建人Hiwi基因miRNA干扰质粒,探讨其对肝癌7721细胞株中Hiwi基因和蛋白表达的阻抑作用。方法根据Homo sapiens Hiwi基因序列,设计RNA干扰靶点,构建4对Hiwi的pcDNATM6.2-GW/EmGFPmiR miRNA及1对无效对照miRNA干扰质粒并将其转染至肝癌7721细胞中,通过RT-PCR和Western blot检测7721细胞中HiwimRNA和蛋白的表达水平。结果成功构建了4对Hiwi miRNA干扰质粒及无效干扰质粒,测序表明Hiwi干扰序列及读框完全正确。RT-PCR和Western blot结果显示,与未转染组及阴性对照组比较,转染该载体能有效下调7721细胞株中mRNA和蛋白的表达。结论成功构建了Hiwi的干扰表达载体,为进一步探讨Hiwi基因在肝癌中的相关机制提供了实验基础。
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Key words
plasmid construction,Hiwi gene,miRNA,hepatocellular carcinoma
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