在Hela细胞中验证miRNA-21对TLR4的调控

背景：miRNA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miRNA-21在宫颈癌Hela细胞中对TLR4基因的调控关系。 方法：通过miRNA靶基因预测网站寻找可能与miRNA-21相互作用的靶基因，利用已构建的携带pri-miRNA-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miRNA-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pAd/pri-miRNA-21、pAd/neg可以成功感染Hela细胞。生物信息学方法显示miRNA-21和TLR4存在可能的结合位点。发现感染pAd/pri-miRNA-21组与对照组pAd/neg及空白组相比，TLR4蛋白的表达量明显降低。证实了miRNA-21可以负调节靶基因TLR4的表达。  中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程全文链接：

MicroRNA-21 for regulation of TLR4 in Hela cells

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 cells. 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 cells. 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 successfully infect Hela cells. 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.