State Key Laboratory of Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China.
Sci China Life Sci. 2011 May;54(5):418-25. doi: 10.1007/s11427-011-4171-0. Epub 2011 May 15.
Technology for monitoring in vivo microRNA (miRNA) activity is extremely important for elucidating miRNA biology. However, in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably reflect real-time functional changes in miRNAs. Sensors for miRNA were developed by adding miRNA target sequences to the 3'-untranslated region of Gaussia princeps luciferase (Gluc) mRNA. These sensors were then evaluated in vitro and in vivo by measuring Gluc activity in cell supernatants and in peripheral blood. Sensors driven by the CMV promoter were effective for monitoring miR-122 in living cells, but not for the long-term monitoring of miR-122 or miR-142 in mouse liver because of CMV-promoter silencing. Replacing the CMV promoter with a CAG promoter rendered these sensors effective for the long-term monitoring of relevant liver miRNA activities. We subsequently used the CAG-promoter-based sensor for the long-term monitoring of endogenous liver miR-122, miR142 and miR-34a activities, as well as for exogenous miR-34a activity. Our study demonstrates that real-time in vivo activities of miRNAs can be continuously and conveniently detected in mouse liver using the sensors that we have developed.
监测体内 microRNA (miRNA) 活性的技术对于阐明 miRNA 生物学至关重要。然而，由于缺乏可靠反映 miRNA 实时功能变化的简便方法，miRNA 的体内研究受到了阻碍。通过将 miRNA 靶序列添加到 Gaussia princeps 荧光素酶 (Gluc) mRNA 的 3'-非翻译区，开发了用于 miRNA 的传感器。然后通过测量细胞上清液和外周血中的 Gluc 活性，在体外和体内评估这些传感器。由 CMV 启动子驱动的传感器可有效用于监测活细胞中的 miR-122，但不能用于监测小鼠肝脏中的 miR-122 或 miR-142 的长期变化，因为 CMV 启动子沉默。用 CAG 启动子替代 CMV 启动子使这些传感器能够有效地长期监测相关的肝 miRNA 活性。随后，我们使用基于 CAG 启动子的传感器长期监测内源性肝 miR-122、miR142 和 miR-34a 活性以及外源性 miR-34a 活性。我们的研究表明，使用我们开发的传感器可以连续方便地检测小鼠肝脏中 miRNA 的实时体内活性。
