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EphrinA1基因小干扰RNA的构建及其在Huh-7细胞中的沉默效应

Construction of EphrinA1 SiRNA and analysis of its silenced effect in hepatoma cell line Huh-7
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摘要 目的:构建针对EphrinA1基因的发夹状RNA(ShRNA)表达载体pSilencer2.1-EphrinA1-SiRNA,观察其对人肝癌细胞Huh-7中EphrinA1基因表达的特异性抑制效应。方法:设计合成针对EphrinA1 mRNA的RNAi核苷酸片段,并定向克隆到pSilencer2.1-U6载体中,构建重组质粒pSilencer2.1-EphrinA1-SiRNA,同时构建不针对任何序列的重组质粒pSilencer2.1-EphrinA1-ScrRNA,通过脂质体介导转染入Huh-7细胞,应用逆转录聚合酶链反应检测小干扰RNA的沉默效应。结果:酶切鉴定证实pSilencer2.1-EphrinA1-SiRNA和pSilencer2.1-EphrinA1-ScrRNA重组质粒构建成功,转染Huh-7细胞后,pSilencer2.1-EphrinA1-SiRNA组EphrinA1 mRNA表达较pSilencer2.1-EphrinA1-ScrRNA组和空白对照组明显下调,差异有统计学意义(P<0.05)。结论:pSilencer2.1-EphrinA1-SiRNA载体的成功构建为下一步研究以EphrinA1基因为靶位点的肝细胞癌基因治疗奠定了重要的实验基础。 Objective：To observe the inhibition effect of ShRNA on EphrinA1 expression in hepatoma cell line Huh-7 through construction of the expressed carrier pSilencer2.1-EphrinA1-ScrRNA of EphrinAI.Methods：Two pairs of SiRNA were designed and synthesized according to the EphrinA1 mRNA sequence in GenBank,and then were inserted into pSilencer2.1-U6 plasmids to create recombinant plasmids pSilencer2.1-EphrinA1-SiRNA and pSilencer2.1-EphrinA1-ScrRNA（control）.After being transfected into Huh-7 cells,EphrinA1 expressed by ShRNAs was detected to be down regulated by RTPCR. Results： All plasmids were constructed successfully. Expression of EphrinA1 in Huh-？ cells infected with pSilencer2.1-EphrinA1-SiRNA plasmids was significantly down regulated compared with that is infected with pSilencer2.1-EphrinA1-ScrRNA plasmids （P〈 0.05）. Conclusion： Con-struction of pSilencer2.1-EphrinA1-SiRNA lays an important experimental foundation for gene therapy of hepatocellular carcinoma （HCC） by targeting the EphrinA1 gene.
作者 王怡 陈钢 陈必成 刘佳明 黄陈平
机构地区 温州医学院环境与公共卫生学院 温州医学院附属第一医院肝胆外科
出处 《温州医学院学报》 CAS 2009年第6期555-557,共3页 Journal of Wenzhou Medical College
基金 温州市科技局科研基金资助项目(Y20060074)
关键词 EPHRINA1 小干扰RNA 肝细胞癌 EphrinA1 SiRNA HCC
分类号 R735.7 [医药卫生—肿瘤]
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参考文献10

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二级参考文献14

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共引文献5

温州医学院学报
2009年 第6期

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