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.

miR181c promotes apoptosis and suppresses proliferation of metanephric mesenchyme cells by targeting Six2 in vitro

“…Forty‐eight hours after being transfected with plasmid or miRNA mimic, mK3 cells were briefly rinsed with PBS and lysed with 200 µl of radioimmunoprecipitation assay cell lysis buffer per well of a six‐well plate. Western blotting was performed as previously described . The lysate was resolved in 12% sodium dodecyl sulphate PAGE gel, and protein bands were transferred to the polyvinylidene difluoride membrane.…”

“…Western blotting was performed as previously described. 15 The lysate was resolved in 12% sodium dodecyl sulphate PAGE gel, and protein bands were transferred to the polyvinylidene difluoride membrane. The membrane was hatched with primary antibody (rabbit anti-Six2, 1:600 dilution, Proteintech, USA, and mouse anti-β-tubulin, 1:3000 dilution, TransGen, China) at 4°overnight.…”

Roles of miR-17-92 Cluster in Cardiovascular Development and Common Diseases

“…Another study based on embryonic cardiomyocytes reported that overexpression of miR-17-92 would reduce the cell proliferation through posttranscriptional repression of Friend of Gata-2 (FOG-2, a nuclear corepressor protein), which is a critical factor for cardiac development. Moreover, FOG-2 could partially rescue the abnormal proliferation induced by miR-17-92 [12]. In addition, miR-17-92 cluster member miR-19 promoted cardiomyocyte proliferation through direct downregulation of PTEN in vitro [21].…”

“…Among all miRNA clusters, miR-17-92, which was called oncomir-1 at first, is a highly conserved cluster that is essential in adipocyte differentiation [8], lung development [9], angiogenesis [10], tumorigenesis [11], and especially heart development [12, 13]. miR-17-92 cluster has been confirmed to be involved in normal cardiac development and its change took part in the genesis of many cardiovascular diseases, including heart failure [14], cardiomyopathy, arrhythmias, and cardiac hypertrophy [15], while its mechanisms of action in these contexts are not fully understood (Figure 1).…”

Discrimination of the Expression of Paralogous microRNA Precursors That Share the Same Major Mature Form

“…miRNA clusters perform important functions by regulating various cellular processes. For example, the most frequently studied miR-17-92 cluster is involved in the development of the heart, lungs, and immune system and in the formation of tumors [13]–[15]. Clustered miRNAs are usually derived from a common primary transcript and coexpressed [16].…”