The miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation of mouse embryonic cardiomyocytes

Abstract

MicroRNAs (miRNAs) have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92) cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2) is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation in mouse embryonic cardiomyocytes we initially used bioinformatics to analyze 3' untranslated regions (3'UTR) of FOG-2 to predict the potential of miR-17-92 to target it. We used luciferase assays to demonstrate that miR-17-5p and miR-20a of miR-17-92 interact with the predicted target sites in the 3'UTR of FOG-2. Furthermore, RT-PCR and Western blot were used to demonstrate the post-transcriptional regulation of FOG-2 by miR-17-92 in embryonic cardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdU cell assays together with the FOG-2 rescue strategy were employed to evaluate the effect of proliferation on embryonic cardiomyocytes. We first found that the miR-17-5p and miR-20a of miR-17-92 directly target the 3'UTR of FOG-2 and post-transcriptionally repress the expression of FOG-2. Moreover, our findings demonstrated that over-expression of miR-17-92 may inhibit cell proliferation via post-transcriptional repression of FOG-2 in embryonic cardiomyocytes. These results indicate that the miR-17-92 cluster regulates the expression of FOG-2 protein and suggest that the miR-17-92 cluster might play an important role in heart development.

Figure 1.. Bioinformatic analysis of the 3′ untranslated regions (3′UTR) of Friend of Gata-2 (FOG-2). A, The figure shows that the 3′UTR of the FOG-2 is highly conserved across mammals, as shown by the UC Santa Cruz genome database. The chromosome tracks show the location of the 3′UTR of FOG-2. In the dense display tracks, conservation is shown in grayscale using darker values to indicate higher levels of overall conservation as scored by phastCons. B, Schematic diagrams of the 3′UTR of FOG-2 and the predicted binding sites for the miR-17-92 cluster via the TargetScan release 5.1 database. The upper panel shows three putative target sites predicted on FOG-2 3′UTR and the lower panel shows multiple sequence alignment of miR-17-92 with the three binding sites on 3′UTR of FOG-2. The underlined bases represent the location of predicted miR-17-92 seed sites and spaces are added where needed to facilitate the alignment. The number in parentheses (below the yellow arrows) represents the location of miR-17-92 potential binding sites. Nucleotides in dark-shaded boxes indicate sequences that are present in all five species.

Figure 2.. miR-17-5p and miR-20a directly targeted the 3′ untranslated regions (3′UTR) of Friend of Gata-2 (FOG-2). A, A schematic diagram of the constructs used to evaluate the function of the conserved region of the 3′UTR of FOG-2. B, HEK 293T cell lines were transfected with pRL-SV40 and a luciferase reporter containing the 3′UTR of FOG-2. Forty-eight hours after transfection, cell lysates were assayed for luciferase activity and normalized to Renilla luciferase activity. Data are reported as means ± SEM for three independent experiments. *P < 0.05 (ANOVA).

Figure 3.. miR-17-5p and miR-20a were expressed in the embryonic cardiomyocytes (EC). miR-17-5p and miR-20a were overexpressed with the transfected pcDNA3.1-miR-17-92 vector in embryonic cardiomyocytes. *P < 0.05 (t-test).

Figure 4.. miR-17-92 post-transcriptionally down-regulates FOG-2 protein in cardiomyocytes 48 h after transfection. A, Detection of Friend of Gata-2 (FOG-2) mRNA expression by RT-PCR. Overexpression of miR-17-92 did not reduce the level of endogenous FOG-2 mRNA. Overexpression of FOG-2 increased the level of exogenous FOG-2 mRNA. Data were normalized to the GAPDH level. B, Detection of FOG-2 protein expression by Western blotting. Overexpression of miR-17-92 reduced the levels of endogenous FOG-2 protein. Overexpression of FOG-2 increased the level of exogenous FOG-2 protein. Data were normalized to the level of GAPDH. Data are reported as means ± SEM for three independent experiments. *P < 0.05 (ANOVA).

Figure 5.. Expression of miR-17-92 inhibited cardiomyocyte proliferation. A, Cardiomyocyte morphology was assessed by fluorescence microscopy (400X). Cardiomyocyte proliferation was determined by the 5-ethynyl-2′-deoxyuridine (EdU) assay. Forty-eight hours after transfection, cardiomyocytes were stained with EdU and DAPI. The arrowheads indicate cells undergoing proliferation. B, Cell proliferation rate was decreased by overexpression of miR-17-92, but might be rescued by overexpression of Friend of Gata-2 (FOG-2). Data are reported as means ± SEM for three independent experiments. *P < 0.05 (ANOVA).