The MiR-17-92Cluster Post-transcriptionally Regulates FOG-2Expression In Mouse Embryonic Cardiomyocytes

In humans, congenital heart disease usually refers to abnormalities inthe heart's structure or function, which are generated before birth and occurin approximately12of1000live births. The development of the heart is afascinating process. It involves the differentiation of cardiac precursors intomany kinds of cell types including myocardium, cardiac mesenchyme, andendocardium. Each of these cell types contributes in its own specific way toform the cardiac chambers, conduction system, and a highly specializedvalvuloseptal apparatus. It is clear that precise regulation of itsdevelopment is critical for normal functioning of the heart.Friend of Gata-2(FOG-2, also called zfpm2) is a multizinc finger nuclear corepressorprotein, which is necessary for cardiac development. FOG-2interactsdirectly with GATA factors to modulate GATA-dependent transcriptionalactivation and can act as an activator or repressor, depending on thepromoter and cell type. FOG-2–/–embryos die around day E13.5of cardiacdefects, including thin myocardium, large ventricular septal defect, severeatrioventricular endocardial cushion defect, overriding aorta, and severeunderdevelopment of the coronary vascular plexus.MicroRNAs (miRNAs)are a class of small singlestranded (~20nucleotides; nt), non-coding RNAgenes that, by binding complementary sequences in the3' untranslatedregions (3'UTR) of messenger RNAs (mRNAs), mediate translational repression or direct mRNA cleavage. miRNAs have gradually beenrecognized as regulators of embryonic development; however, relativelyfew miRNAs have been identified that regulate cardiac development.Previous research has shown that the FOG-2is critical for cardiacdevelopment. We initially used bioinformatics to analyze3'UTR of FOG-2to predict the potential of miRNAs to target it. As a result, the entire mouseFOG-23' UTR was found to contain62different miRNAs target sites. Ofthe62miRNAs,34miRNAs was found to contain17conservative targetsites. The miR-17-92cluster includes five miRNAs (miR-17-5p, miR-19a,miR-19b, miR-20a, and miR-92a) in3conservative target sites. Moreover,a series of recent papers have established an essential role for themiR-17-92cluster of miRNAs in the development of the heart. Toinvestigate the possibility that the miR-17-92cluster regulates FOG-2expression and inhibits proliferation in mouse embryonic cardiomyocytesWe used luciferase assays to demonstrate that the microRNAs ofmiR-17-92interact with the predicted target sites in the3'UTR of FOG-2.Furthermore, RT-PCR and Western blot were used to demonstrate thepost-transcriptional regulation of FOG-2by miR-17-92in embryoniccardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdUcell assays together with the FOG-2rescue strategy were employed toevaluate the effect of proliferation on embryonic cardiomyocytes andTUNEL cell assays were employed to evaluate the effect of apoptosis onembryonic cardiomyocytes. We first found that the miR-17-5p andmiR-20a of miR-17-92directly target the3'UTR of FOG-2andpost-transcriptionally repress the expression of FOG-2. Moreover, ourfindings demonstrated that over-expression of miR-17-92may inhibit cellproliferation via post-transcriptional repression of FOG-2in embryoniccardiomyocytes. We also found that over-expression of miR-17-92may promote cell apoptosis but not via post-transcriptional repression of FOG-2in embryonic cardiomyocytes. These results indicate that the miR-17-92cluster regulates the expression of FOG-2protein and inhibitscardiomyocytes prolifetation. It's suggest that the miR-17-92cluster mightplay an important role in heart development.