用生物传感器方法测定正常小鼠肝脏中miR-21活性

目的:建立生物传感器检测小鼠肝脏中microRNA (miRNA)活性的方法,测定miR-21在正常小鼠肝脏中的活性。方法:首先,分子克隆方法构建检测miRNA活性的通用型质粒传感器Dsensor。将各种miRNA的互补序列插入Dsensor中构建成相应miRNA活性检测传感器。用水动力法将检测miR-21的Dsensor注射至正常小鼠体内,以miR-122 Dsensor为阳性对照,miR-206 Dsensor为阴性对照,以不插入任何miRNA互补序列的Dsensor为空白对照。不同时间点尾静脉采血测定Gluc表达,处死小鼠测定肝脏中Fluc表达,比较计算得出miRNA活性。最后,用QRT-PCR法测定小鼠肝脏组织中miR-21、miR-122和miR-206表达水平。结果:用RIF(Relative inhibiting fold)值表示miRNA活性,miR-21、miR-122和miR-206活性分别为80.03±21.25,29.90±5.90和0.92±0.29,表明小鼠正常肝脏中miR-21的负调控活性明显高于miR-122。然而,QRT-PCR法测定结果显示,miR-21的表达水平明显低于miR-122,而miR-206几乎没有表达。从miR-21与miR-122的活性与表达水平比较发现,miR-21的表达水平并没有真实反映其活性。结论:成功建立了一种小鼠肝脏中miRNA活性检测方法;首次发现小鼠正常肝脏中miR-21活性水平比miR-122更高,而表达水平却明显低于miR-122。提示miR-21对于维持肝脏的正常生理功能的重要作用,值得进一步研究其调控的靶基因和机理。

Detection of miR-21 Activity in Normal Mouse Liver Using Biosensors

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.