HeLa细胞中miRNA-214靶基因Mek3的确定

目的探索miRNA-214在HeLa细胞中的与其靶基因Mek3相互作用。方法通过miRNA靶基因预测网站寻找可能与miRNA-214相互作用的靶基因，合成miRNA-214和对照序列，将miRNA-214、对照序列、Mek3的3’非翻译区（3’UTR）以及突变的Mek3 3’UTR分别克隆到表达载体上，转染HeLa细胞，转染48h后提取蛋白，检测绿色荧光蛋白的表达水平；HeLa细胞转染miRNA-214后，Trizol抽提RNA，通过荧光定量PCR检测Mek3mRNA的表达水平；Western印迹检Mek3的蛋白表达水平。经过以上实验从mRNA和蛋白水平上验证了在HeLa细胞中miRNA-214对靶基因Mek3的作用效应。结果生物信息学方法显示miRNA-214和Mek3存在可能的结合位点。经过实验验证了miRNA-214可以下调Mek3的mRNA和蛋白水平。结论miRNA-214可以负调节靶基因Mek3的表达。

Interaction of miRNA-214 with its Target Gene Mek3 in HeLa Cells

Objective To study the interaction of miRNA-214 with its target gene Mek3. Methods The candidate target gene of miRNA-214 was determined according to miRNA analysis databases. The corresponding miRNA-214 and control sequence were synthesized, miRNA- 214, control sequence, 3＇UTR of Mek3 and mutant Mek3 3＇UTR were cloned into expression vectors respectively, and then co-transfeeted into HeLa cells. Forty-eight hours after transfection, cells were lysed for detection of relative expression of Mek3 3＇UTR by determining eGFP expressing levels. After transfeetion of miRNA-214 or control, RNA was extracted from Hela cells by Trizol agent, and real-time PCR was used for detection of Mek3 mRNA. Western blotting was used for detection of Mek3 protein. Results Bioinformatic analysis suggested several possible binding sites between miRNA-214 and Mek3. The experimental results showed that miRNA-214 down-regulated Mek3 at both mRNA and protein levels. Conclusions MicroRNA-214 can interfere with the expression of Mek3 target gene.