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| Detection of miR-21 Activity in Normal Mouse Liver Using Biosensors |
| TIAN Wen-hong1, HU Jian-yang2, DONG Xiao-yan3, LI Ming-hao4, WU Xiao-bing5 |
1. School of Life Science, Jilin University, Changchun 130012, China;
2. The Children’s Hospital of Chongqing Medical University, Chongqing 400000, China;
3. Beijing FivePlus Molecular Medicine Institute, Beijing 100176, China;
4. People Hospital, the Ningxia Hui Autonomous Region, Yinchuan 750001, China;
5. Beijing Yizhuang International Biomedical Investment & Management Co. Ltd., Beijing 101111, China |
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Abstract 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.
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Received: 25 October 2012
Published: 25 January 2013
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[1] Bartel D P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 2004, 116(27): 281-297.
[2] Shivdasani R A. MicroRNAs: regulators of gene expression and cell differentiation. Blood, 2006, 108(12): 3646-3653.
[3] Brennecke J, Hipfner D R, Stark A, et al. bantam encodes a developmentally regulated microRNA that control cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell, 2003, 113(1): 25-36.
[4] Xu P, Guo M, Hay B A. MicroRNAs and the regulation of cell death. Trends Genet, 2004, 20(12): 617-624.
[5] Tian W, Dong X, Liu X, et al. High-throughput functional mircroRNAs profiling by recombinant AAV-based microRNA sensor arrays. PLoS ONE, 2012, 7(1): e29551.
[6] 田文洪,王刚,罗顺涛,等. 体外实时动态监测水动力注射分泌型荧光素酶基因的表达. 生物工程学报,2009,25(10):1552-1557. Tian W H, Wang G, Luo S T, et al. Real time ex vivo detection and dynamic monitoring of in vivo expression of secreted luciferase gene injected by hydrodynamic method. Chin J Biotech, 2009, 25(10):1552-1557.
[7] Andrianaivo F, Lecocq M, Wattiaux-De Coninck S, et al. Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection. J Gene Med, 2004, 6(8): 877-883.
[8] Suda T, Gao X, Stolz D B, et al. Structural impact of hydrodynamic injection on mouse liver. Gene Ther, 2007, 14(2): 129-137.
[9] Chang J, Nicolas E, Marks D, et al. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biol, 2004, 1(2): 106-113.
[10] Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of tissue-specific microRNAs from mouse. Curr Biol, 2002, 12(9): 735-739.
[11] Triger D R. Physiological functions of the liver. Br J Hosp Med, 1979, 22(5): 424, 429-430,432.
[12] Selcuklu S D, Donoghue M T, Spillane C. miR-21 as key regulator of oncogenic processes. Biochem Soc Trans, 2009, 37(Pt 4): 918-925.
[13] Ng R, Song G, Roll G R, et al. A microRNA-21 surge facilitates rapid cyclin D1 translation and cell cycle progression in mouse liver regeneration. J Clin Invest, 2012, 122(3): 1097-1108.
[14] 王刚,董小岩,胡键阳,等. 利用分泌型荧光素酶体外长期检测小鼠肝脏microRNA活性. 中国科学:生命科学,2011,41(4):273-280. Wang G, Dong X Y, Hu J Y, et al. Long-term ex vivo monitoring of in vivo microRNA activity in liver using a secreted luciferase sensor. Sci China Life Sci, 2011, 54(5): 418-425.
[15] Brown B D, Gentner B, Cantore A, et al. Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue lineage and differentiation state. Nat Biotechnol, 2007, 25(12): 1457-1467.
[16] Mullokandov G, Baccarini A, Ruzo A, et al. High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries. Nat Methods, 2012, 9(8): 840-846. |
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