P71In vitro and in vivo direct monitoring of miRNA-22 expression in isoproterenol-induced cardiac hypertrophy by bioluminescence imaging

    loading  Checking for direct PDF access through Ovid

Abstract

Background: Growing evidence suggests that 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 miRNAs level, such as northern blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and microarrays can not provide real-time information of miRNAs biogenesis in vivo.

Methods: We constructed a miRNA reporter imaging system to monitor miR-22 expression in isoproterenol-induced cardiac hypertrophy repetitively and non-invasively. There were three copies of the antisence of miR-22 (3×PT_miR-22) cloned into 3′ UTR region 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/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 up-regulation of miR-22 and its prohypertrophic effects. Furthermore, knockdown of miR-22 by antagomir-22 could markedly reverse the repressed gaussia luciferase (Gluc) activities in vitro and in vivo. However, the firefly luciferase (Fluc) activity of CMV/Fluc was not affected with isoproterenol treatment.

Conclusion: This study elucidate the feasibility of using our constructed miRNA reporter imaging system to monitor the location and magnitude of expression level of miR-22 in cardiac hypertrophy in vitro and in vivo.

Related Topics

    loading  Loading Related Articles