In vitro and in vivo direct monitoring of miRNA-22 expression in isoproterenol-induced cardiac hypertrophy by bioluminescence imaging
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Growing evidence suggests that microRNAs (miRNAs) play key roles in cardiac hypertrophy. To measure the expression of endogenous miRNAs is very conducive to understanding the importance of miRNAs in cardiac hypertrophy. However, current methods to monitor endogenous miRNA levels, such as Northern blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and microarrays cannot provide real-time information on miRNA biogenesis in vivo.
We constructed a miRNA reporter imaging system to monitor miR-22 expression in isoproterenol-induced cardiac hypertrophy repetitively and noninvasively. There were three copies of the antisense of miR-22 (3×PT_miR-22) cloned into the 3′ untranslated region (UTR) of the Gaussia luciferase (Gluc) reporter genes under the control of the cytomegalovirus (CMV) promoter in this miRNA reporter system (CMV/Gluc/3×PT_miR-22). CMV/firefly luciferase (Fluc) was used as a positive control for imaging of miR-22 expression. Meanwhile, quantifications of miR-22 in cardiomyocyte hypertrophy and in mouse cardiac hypertrophy induced by isoproterenol stimulation were measured by qRT-PCR. Furthermore, we used this miRNA reporter imaging system to appraise the antihypertrophic effect of antagomir-22 in vitro and in vivo.
The bioluminescence signals of the CMV/Gluc/3×PT_miR-22 were gradually decreased with prolongation of isoproterenol intervention in vitro and in vivo. Overexpression of miR-22 was observed in cardiac hypertrophy, and markedly administration of antagomir-22 could reverse the upregulation of miR-22 and its prohypertrophic effects. Furthermore, knockdown of miR-22 by antagomir-22 could markedly reverse the repressed Gluc activities in vitro and in vivo. However, the Fluc activity of CMV/Fluc was not affected with isoproterenol treatment.
This study elucidates the feasibility of using our constructed miRNA reporter imaging system to monitor the location and magnitude of expression levels of miR-22 in cardiac hypertrophy in vitro and in vivo.
KeywordsCardiac hypertrophy miRNA-22 Bioluminescence imaging Firefly luciferase Gaussia luciferase
Quantitative real-time polymerase chain reaction
Green fluorescent protein
Red fluorescent protein
Atrial natriuretic peptide
Brain natriuretic peptide
Beta-myosin heavy chain
The authors thank Dr. Tao Huang and Dr. Kai Wang for valuable suggestions for the design and manuscript revision. We also thank Xilin Sun for technical assistance in bioluminescence imaging study. This work was supported by key science and technology research projects of Harbin Municipal (2007AA3CS085), China Postdoctoral Science Foundation (20070420105), National Natural Science Foundation of China (30970807), the International Cooperation and Exchanges Program of the National Ministry of Science and Technology (2009DFB30040), China Postdoctoral Special Science Foundation, and the Ph.D. Programs Foundation of the Ministry of Education of China (200801305), National Natural Science Foundation of China (31210103913).
Conflicts of interest
- 10.Knezevic I, Patel A, Sundaresan NR, Gupta MP, Solaro RJ, Nagalingam RS, et al. A novel cardiomyocyte-enriched microRNA, miR-378, targets insulin-like growth factor 1 receptor: implications in postnatal cardiac remodeling and cell survival. J Biol Chem 2012;287:12913–26. doi: 10.1074/jbc.M111.331751.PubMedCentralPubMedCrossRefGoogle Scholar
- 25.Berger F, Paulmurugan R, Bhaumik S, Gambhir SS. Uptake kinetics and biodistribution of 14C-D-luciferin–a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction: impact on bioluminescence based reporter gene imaging. Eur J Nucl Med Mol Imaging 2008;35:2275–85. doi: 10.1007/s00259-008-0870-6.PubMedCrossRefGoogle Scholar