在Hela细胞中验证miRNA-21对TLR4的调控  

Micro RNA-21 for regulation of TLR4 in Hela cells

作　　者：赵婧[1] 岳鹏 黄家芳[1] 汪玉涛[1] 马婷[1] 陈宝荣[1] 

机构地区：[1]北京航天总医院检验科,北京市100076 [2]防化学院门诊部,北京市102205

出　　处：《中国组织工程研究》2015年第2期220-224,共5页Chinese Journal of Tissue Engineering Research

摘　　要：背景:mi RNA通过调节特异靶基因的表达,在疾病的发展及预后中起着重要作用。目的:探索mi RNA-21在宫颈癌Hela细胞中对TLR4基因的调控关系。方法:通过miR NA靶基因预测网站寻找可能与miR NA-21相互作用的靶基因,利用已构建的携带pri-mi RNA-21基因重组腺病毒载体,包装并大量扩增病毒感染Hela细胞,检测荧光蛋白的表达水平,提取感染48 h蛋白,通过Western印迹检测TLR4蛋白表达。从蛋白水平验证在Hela细胞中mi RNA-21与靶基因TLR4的靶向调控关系。结果与结论:100 MOI的重组腺病毒p Ad/pri-miR NA-21、p Ad/neg可以成功感染Hela细胞。生物信息学方法显示mi RNA-21和TLR4存在可能的结合位点。发现感染pA d/pri-miR NA-21组与对照组pA d/neg及空白组相比,TLR4蛋白的表达量明显降低。证实了mi RNA-21可以负调节靶基因TLR4的表达。BACKGROUND：MicroRNAs （miRNA） through regulating specific target gene mRNA expression play important roles in different processes of diseases. OBJECTIVE：To study the interaction of miRNA-21 with its target gene TLR4 in Hela cels. METHODS：The candidate target gene of miRNA-21 was determined according to miRNA analysis databases. The constructed recombinant adenovirus vector carrying pri-miRNA-21 gene was used, which could package and amplify viruses to transfect Hela cels. Then, the expression of fluorescent proteins was detected. Forty-eight hours after transfection of miRNA-21 or control, extracted proteins were used for detection of TLR4 protein using western blot assay. RESULTS AND CONCLUSION：Recombinant adenoviruses pAd/pri-miRNA-21 and pAd/neg at 100 MOI could successfuly infect Hela cels. Bioinformatic analysis suggested several possible binding sites between miRNA-21 and TLR4. The experimental results showed that miRNA-21 down-regulated TLR4 at protein levels, indicating that miRNA-21 can interfere with the expression of TLR4 target gene.

关 键 词：组织构建 组织工程 重组腺病毒载体 pri-miRNA-21 TLR4基因 HELA细胞 靶基因 

分 类 号：R318[医药卫生—生物医学工程]