TIAN Wen-hong, HU Jian-yang, DONG Xiao-yan, LI Ming-hao, WU Xiao-bing
China Biotechnology. 2013, 33(1): 60-66.
Biosensors were developed to detect miRNA activity in the liver of mouse. And miR-21 activity was assayed in normal mouse liver. A plasmid DNA based miRNA sensor (Dsensor) was designed for detection of miRNA activity. Control Dsensor carrying no miRNA target and three miRNA Dsensors (miR-122, miR-206, and miR-21) with a perfect complementary target were constructed using molecular cloning method in this study. Then Control Dsensor was injected into mouse by hydrodynamic method through tail-vein and Fluc activity was assayed for different tissues to validate specific transduction into liver for hydrodynamic injection. Subsequently, with miR-122 Dsensor as positive control and miR-206 Dsensor as negative control, Control, miR-122, miR-206, and miR-21 Dsensors were separately hydrodynamic injected into normal mice. Blood was taken from the tail-vein and Gluc activity was assayed at different time points. 10 d later, mice were sacrificed and Fluc activity was assayed in the liver. Gluc activity for miRNA Dsensor was compared with that for Control Dsensor. The ratio of Gluc activity (G) to Fluc activity (F) (G/F) for each Dsensor was computed and compared at 10 d. The ratio of G/F value for Control Dsensor to that for miRNA Dsensor was further computed which was nominated as relative inhibiting fold (RIF) to indicate miRNA activity. Lastly miR-21, miR-122, and miR-206 expression levels were detected by QRT-PCR. Results demonstrated that Fluc was primarily expressed in liver and that Gluc activity for miR-122 and miR-21 Dsensor were less than that for Control Dsensor while Gluc activity for miR-206 was as much as that for Control Dsensor. G/F values were similar to Gluc activity. RIF values for miR-21, miR-122, and miR-206 were separately 80.03?21.25, 29.90?5.90, and 0.92?0.29, suggesting that miR-21 activity was higher than miR-122 activity while miR-206 activity was not detected in normal mouse liver. However, expression level of miR-21 was lower than that of miR-122 by QRT-PCR, indicating that miR-21 expression level did not authentically reflect its expression level. In conclusion, the method for detection of miRNA activity in mouse liver was successfully established and it was firstly found that miR-21 activity was higher than miR-122 while less expression of miR-21 compared with miR-122 in normal mouse liver, which indicated important functions of miR-21 for normal liver and provided a basis for further miR-21 research in mouse liver.
