一种利用分泌型荧光素酶基因表达变化监测活细胞中miRNA活性的新方法

A novel method for monitoring miRNA activity by expression changes of secreted luciferase gene in live cells

摘要:

建立了一种以分泌型的荧光素酶Gluc为报告基因的miRNA传感器质粒 (命名为Gsensor) 监测活细胞中miRNA (microRNA) 活性的方法。首先构建了pAAV2neo-Gluc-MCS-polyA质粒作为Gsensor的空载体，同时其中的MCS位点可供插入miRNA的靶序列。以miR142-3p为检测对象，将1个和3个拷贝的与miR142-3p完全互补靶序列分别插入pAAV2neo-Gluc-MCS-polyA中，构建成miR142-3p Gsensor和miR142-3p Gsensor-3。将它们分别转染至U937细胞中,检测培养上清中Gluc的表达水平。结果显示二者均可有效反映U937细胞中miR 142-3p的抑制活性 (分别与Gsensor空载体相比)，提示Gsensor中采用一个拷贝的miRNA靶序列即可满足检测要求。并且miR142-3p Gsensor也能有效地反映出Anti-miR142对miR142-3p活性的抑制作用。随后，分析了时间、转染剂量对Gsensor检测结果的影响。结果表明，在U937细胞中miR142-3p Gsensor表现的miR142-3p活性在48 h后趋于稳定；Gsensor转染剂量在0.001~0.05 pg/cell范围内不影响其功能。最后，利用miR142-3p Gsensor 检测了HEK293、U937、K562、SP2/0和P815细胞内miR142-3p活性，结果发现miR142-3p活性在U937、K562、SP2/0和P815细胞中均较高，而在HEK293中几乎没有活性。用QRT-PCR方法检测miR142-3p的相对拷贝数。结果表明，在HEK293、U937和K562细胞中，miR142-3p活性与其相对拷贝数呈正相关。本研究表明Gsensor可作为一种有效的miRNA活性检测工具，为体外实时动态监测miRNA活性提供了一种新方法。

Abstract:

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.