Tian Wenhong, Dong Xiaoyan, Wang Gang, Wu Xiaobing
State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Disease Prevention and Control, Chinese Center for Disease Prevention and Control, Beijing 100052, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Jun;26(6):809-16.
We developed a method for monitoring of miRNA activity in live cells by a secreted luciferase gene based plasmid sensor named as Gsensor. Firstly, we constructed pAAV2neo-Gluc-MCS-polyA as "empty Gsensor", which contained multiple cloning sites (MCS) for miRNA target inserted. To detect miR142-3p activity, miR142-3p Gsensor and miR142-3p Gsensor-3 were constructed by inserting one or three complementary miR142-3p targets into pAAV2neo-Gluc-MCS-ployA. Subsequently, miR142-3p Gsensor and miR142-3p Gsensor-3 were respectively transfected into U937 cells and Gluc activity was assayed in the supernatant 48 h post transfection. Results showed that both of them effectively indicated miR142-3p activity of inhibiting Gluc expression compared with empty Gsensor. Simultaneously, miR142-3p Gsensor also demonstrated the inhibition of miR142-3p activity by Anti-miR142 when they were cotransfected into U937 cells. This implied one copy of miRNA target in Gsensor was sensitive enough for investigation of miRNA activity. We further analyzed factors affecting Gsensor function including time and dose, and found that miR142-3p activity sensed by miR142-3p Gsensor rose within 48 h post transfection and approached stable thereafter. Transfected dose varying among 0.001-0.05 pg/cell had little effect on its function. Using miR142-3p Gsensor, we further detected miR142-3p activity in HEK293, U937, K562, SP2/0 and P815 cells. Results suggested that miR142-3p activity was high in U937, K562, SP2/0 and P815 cells and almost negative in HEK293. miR142-3p activity was positively correlated with its relative copies in HEK293, U937 and K562 detected by QRT-PCR. In conclusion, Gsensor proved to be an effective tool for monitoring of miRNA activity in live cells, and provide a new method for monitoring miRNA activity in vitro.
我们开发了一种通过基于分泌型荧光素酶基因的质粒传感器Gsensor监测活细胞中miRNA活性的方法。首先，我们构建了pAAV2neo-Gluc-MCS-polyA作为“空的Gsensor”，它包含用于插入miRNA靶标的多个克隆位点（MCS）。为了检测miR142-3p的活性，通过将一个或三个互补的miR142-3p靶标插入pAAV2neo-Gluc-MCS-ployA构建了miR142-3p Gsensor和miR142-3p Gsensor-3。随后，将miR142-3p Gsensor和miR142-3p Gsensor-3分别转染到U937细胞中，并在转染后48小时测定上清液中的Gluc活性。结果表明，与空的Gsensor相比，它们两者都有效地表明了miR142-3p抑制Gluc表达的活性。同时，当将miR142-3p Gsensor与Anti-miR142共转染到U937细胞中时，miR142-3p Gsensor也证明了Anti-miR142对miR142-3p活性的抑制作用。这意味着Gsensor中的一个miRNA靶标拷贝对于研究miRNA活性足够敏感。我们进一步分析了影响Gsensor功能的因素，包括时间和剂量，发现miR142-3p Gsensor检测到的miR142-3p活性在转染后48小时内升高，此后趋于稳定。转染剂量在0.001-0.05 pg/细胞之间变化对其功能影响很小。使用miR142-3p Gsensor，我们进一步检测了HEK293、U937、K562、SP2/0和P815细胞中miR142-3p的活性。结果表明，miR142-3p活性在U937、K562、SP2/0和P815细胞中较高，而在HEK293细胞中几乎为阴性。通过QRT-PCR检测，miR142-3p活性与其在HEK293、U937和K562中的相对拷贝数呈正相关。总之，Gsensor被证明是监测活细胞中miRNA活性的有效工具，并为体外监测miRNA活性提供了一种新方法。
