[A novel method for monitoring miRNA activity by expression changes of secreted luciferase gene in live cells].
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.
Tian
Wenhong
W
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.
Dong
Xiaoyan
X
Wang
Gang
G
Wu
Xiaobing
X
D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D035683:MicroRNAs / Q000378:metabolism*; D014162:Transfection; D020298:U937 Cells
D035683:MicroRNAs; D008156:Luciferases