小鼠胚胎着床相关基因中mir-21靶基因的初步验证

Primary test of the targets of mouse mir-21,a mouse embryo implantation-related gene

利用microRNA靶位点预测网站结合小鼠胚胎着床芯片数据预测了mir-21的5个与着床相关的靶基因:Reck、F3、Upk1 b、Ggh、Tmed6,通过分别构建含有靶基因3′UTR的重组报告载体、pre-mir-21或/和mir-21inhibitor体外转染细胞、双荧光素酶活性分析初步确定mir-21的靶位点。pre-mir-21可显著降低转染上述5个重组载体的细胞内荧光素酶活性;mir-21 inhibitor可显著增加转染Reck、Upk1 b和Tmed63′UTR的重组载体的细胞内荧光素酶活性;pre-mir-21和mir-21 inhibitor共转染可分别显著增加转染含有Reck3′UTR、F33′UTR、Upk1 b3′UTR和Ggh3′UTR的重组载体的细胞内荧光素酶活性。Reck和Upk1b可能是mir-21的靶基因。

To dissect the molecular basis underlying mir-21 associated embryo implantation, putative mir-21 targets （Reck, F3, Ggh and Tmed6）that are likely involved in mouse embryo implantation were examined by the establishment of in vitro transfection cells and dual-luciferase activity assay. These transfection cells contain a recombinant reporter with 3′UTR of the targets downstream of luciferase gene, and pre-mir-21 and/or mir-21 inhibitor. The results showed that pre-mir-21 can significantly decrease the luciferase activity in all the cells transfected with the reporter, whereas mir-21 inhibitor can only significantly increase the fluorescence activity in Reck, Upk1b and Tmed6. Pre-mir-21 and mir-21 inhibitor cotransfection can significantly decrease the fluorescence activity in Reck, F3, Upklb and Ggh. In conclusion, Reck and Upklb might be the targets of mir-21.