PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
PURPOSE: 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. METHODS: 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 mousecardiac 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. RESULTS: 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. CONCLUSION: 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.
Authors: Raghu S Nagalingam; Nagalingam R Sundaresan; Mahesh P Gupta; David L Geenen; R John Solaro; Madhu Gupta Journal: J Biol Chem Date: 2013-02-27 Impact factor: 5.157
Authors: Mariko Tatsuguchi; Hee Young Seok; Thomas E Callis; J Michael Thomson; Jian-Fu Chen; Martin Newman; Mauricio Rojas; Scott M Hammond; Da-Zhi Wang Journal: J Mol Cell Cardiol Date: 2007-04-14 Impact factor: 5.000
Authors: Ahmet Ucar; Shashi K Gupta; Jan Fiedler; Erdem Erikci; Michal Kardasinski; Sandor Batkai; Seema Dangwal; Regalla Kumarswamy; Claudia Bang; Angelika Holzmann; Janet Remke; Massimiliano Caprio; Claudia Jentzsch; Stefan Engelhardt; Sabine Geisendorf; Carolina Glas; Thomas G Hofmann; Michelle Nessling; Karsten Richter; Mario Schiffer; Lucie Carrier; L Christian Napp; Johann Bauersachs; Kamal Chowdhury; Thomas Thum Journal: Nat Commun Date: 2012 Impact factor: 14.919