MicroRNAs are posttranscriptional gene regulators that are differentially expressed during various diseases and have been implicated in the underlying pathogenesis. We report here that miR-199a is acutely downregulated in cardiac myocytes on a decline in oxygen tension. This reduction is required for the rapid upregulation of its target, hypoxia-inducible factor (Hif)-1α. Replenishing miR-199a during hypoxia inhibits Hif-1α expression and its stabilization of p53 and, thus, reduces apoptosis. On the other hand, knockdown of miR-199a during normoxia results in the upregulation of Hif-1α and Sirtuin (Sirt)1 and reproduces hypoxia preconditioning. Sirt1 is also a direct target of miR-199a and is responsible for downregulating prolyl hydroxylase 2, required for stabilization of Hif-1α. Thus, we conclude that miR-199a is a master regulator of a hypoxia-triggered pathway and can be exploited for preconditioning cells against hypoxic damage. In addition, the data demonstrate a functional link between 2 key molecules that regulate hypoxia preconditioning and longevity.
The posttranscriptional regulator, microRNA-21 (miR-21), is up-regulated in many forms of cancer, as well as during cardiac hypertrophic growth. To understand its role, we overexpressed it in cardiocytes where it revealed a unique type of cell-to-cell "linker" in the form of long slender outgrowths and branches. We subsequently confirmed that miR-21 directly targets and down-regulates the expression of Sprouty2 (SPRY2), an inhibitor of branching morphogenesis and neurite outgrowths. We found that beta-adrenergic receptor (betaAR) stimulation induces up-regulation of miR-21 and down-regulation of SPRY2 and is, likewise, associated with connecting cell branches. Knockdown of SPRY2 reproduced the branching morphology in cardiocytes, and vice versa, knockdown of miR-21 using a specific 'miRNA eraser' or overexpression of SPRY2 inhibited betaAR-induced cellular outgrowths. These structures enclose sarcomeres and connect adjacent cardiocytes through functional gap junctions. To determine how this aspect of miR-21 function translates in cancer cells, we knocked it down in colon cancer SW480 cells. This resulted in disappearance of their microvillus-like protrusions accompanied by SPRY2-dependent inhibition of cell migration. Thus, we propose that an increase in miR-21 enhances the formation of various types of cellular protrusions through directly targeting and down-regulating SPRY2.
MicroRNA-21 (miR-21) is highly up-regulated during hypertrophic and cancerous cell growth. In contrast, we found that it declines in cardiac myocytes upon exposure to hypoxia. Thus, the objective was to explore its role during hypoxia. We show that miR-21 not only regulates phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but also targets Fas ligand (FasL). During prolonged hypoxia, down-regulation of miR-21 proved necessary and sufficient for enhancing expression of both proteins. We demonstrate here for the first time that miR-21 is positively regulated via an AKT-dependent pathway, which is depressed during prolonged hypoxia. Accordingly, hypoxia-induced down-regulation of miR-21 and up-regulation of FasL and PTEN were reversed by activated AKT and reproduced by a dominant negative mutant, wortmannin, or PTEN. Moreover, the antiapoptotic function of AKT partly required miR-21, which was sufficient for inhibition of caspase-8 activity and mitochondrial damage. In consensus, overexpression of miR-21 in a transgenic mouse heart resulted in suppression of ischemia-induced up-regulation of PTEN and FasL expression, an increase in phospho-AKT, a smaller infarct size, and ameliorated heart failure. Thus, we have identified a unique aspect of the function of AKT by which it inhibits apoptosis through miR-21-dependent suppression of FasL. MicroRNA (miRNA)3 are molecules approximately twenty ribonucleotides long that specifically target mRNA through partial complementarity and, thereby, inhibit translation and/or induce their degradation. miR-21 is one of the most commonly and dramatically up-regulated miRNA in many cancers (1, 2) and has been implicated in the inhibition of programmed cell death (2). Some of its validated targets include tropomyosin 1 (3), PTEN (2, 4, 5), programmed cell death 4 (Pdcd4) (6, 7), TAp63 isoform of p53 family, and LRRFIP1, an inhibitor of NFB signaling (8). Similarly, miR-21 is one of the most highly and consistently up-regulated miRNA during cardiac hypertrophy (9 -12). Thum et al. (13) show that miR-21 is predominantly up-regulated in the myofibroblasts where it targets sprouty1 and enhances their survival and, thereby, fibrosis in the heart. Similarly, Roy et al. (14) show that miR-21 is elevated in the myofibroblast-infiltrated area 7 days after ischemia/ reperfusion and suppresses metalloprotease-2 via targeting PTEN. More recently, studies have shown that miR-21 exerts an antiapoptotic function in cardiac myocytes via inhibiting PDCD4 (15) and reduces infarct size via local viral delivery to the heart (16). However, the signaling pathway that regulates miR-21 has not been identified.Two of the molecules that play a major role in ischemic injury of the heart include PTEN and FasL. PTEN is a major negative regulator of AKT (17) whose activity is modulated by its abundance, oxidation, or phosphorylation (18). It is also targeted by miR-21, which provides a specific post-transcriptional mechanism for regulating its expression (2, 4, 5). PTEN has been regarded as the A...
We have recently reported that downregulation of miR-199a-5p is necessary and sufficient for inducing upregulation of its targets, including hypoxia-inducible factor-1alpha (Hif-1α) and Sirt1, during hypoxia preconditioning (HPC). Conversely, others and we have reported that miR-199a-5p is upregulated during cardiac hypertrophy. Thus, the objective of this study was to delineate the signaling pathways that regulate the expression of miR-199a-5p and its targets, and their role in myocyte survival during hypoxia. Since HPC is mediated through activation of the AKT pathway, we questioned if AKT is sufficient for inducing downregulation of miR-199a-5p. Our present study shows that overexpression of a constitutively active AKT (caAKT) induced 70% reduction in miR-199a-5p and was associated with a robust increase in HiF-1α (10 ± 2 fold) and Sirt1 (4 ± 0.8 fold) that was reversed by overexpression of miR-199a-5p. Similarly, insulin receptor-stimulated activation of the AKT pathway induced downregulation of miR-199a-5p and upregulation of its targets. In contrast, β-adrenergic receptor (βAR) activation in vitro and in vivo, induced 1.8-3.5-fold increase in miR-199a-5p. Accordingly, we predicted that βAR would antagonize AKT-induced, miR-199a-5p-dependent, upregulation of Hif-1α and Sirt1. Indeed, pre-treating the myocytes with isoproterenol before applying HPC, caAKT, or insulin resulted in 87 ± 3%, 75 ± 15 %, and 100% reductions in Hif-1α expression, respectively, and sensitized the cells to hypoxic injury. Thus, activation of beta-adrenergic signaling counteracts the survival effects of the AKT pathway via upregulating miR-199a-5p.
With the advent of microRNA (miRNA) we are compelled to revise our understanding of the mechanisms underlying gene regulation during health and disease. A miRNA is approximately 21 ribonucleotides-long, genetically encoded, with a potential to recognize multiple mRNA targets guided by sequence complementarity and RNA-binding proteins. This class of molecules is functionally versatile, with the capacity to specifically inhibit translation initiation or elongation, as well as, induce mRNA degradation, through predominantly targeting the 3'-untranslated regions of mRNA. Early on it was realized that the levels of individual miRNA varied under different developmental, biological, or pathological conditions, thus, implicating these molecules in normal and pathological cellular attributes. In this article we will share our views on how the functions of miRNA conform to our existing knowledge on post-transcriptional regulation of gene expression and disease mechanisms, and their potential as biomarkers and therapeutic targets in diseases.
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