Non-coding RNAs in Development and Disease: Background, Mechanisms, and Therapeutic Approaches.

Advances in RNA-sequencing techniques have led to the discovery of thousands of non-coding transcripts with unknown function. There are several types of non-coding linear RNAs such as microRNAs (miRNA) and long non-coding RNAs (lncRNA), as well as circular RNAs (circRNA) consisting of a closed continuous loop. This review guides the reader through important aspects of non-coding RNA biology. This includes their biogenesis, mode of actions, physiological function, as well as their role in the disease context (such as in cancer or the cardiovascular system). We specifically focus on non-coding RNAs as potential therapeutic targets and diagnostic biomarkers.

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

Inhibition of the Cardiac Fibroblast-Enriched lncRNA Meg3 Prevents Cardiac Fibrosis and Diastolic Dysfunction.

RATIONALE: Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. Recent studies described the role of long noncoding RNAs (lncRNAs) in cardiac pathologies. Nevertheless, detailed reports on lncRNAs regulating CF biology and describing their implication in cardiac remodeling are still missing. OBJECTIVE: Here, we aimed at characterizing lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction. METHODS AND RESULTS: Global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp-2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-ß I. Chromatin immunoprecipitation showed that further induction of Mmp-2 expression by transforming growth factor-ß I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance. CONCLUSIONS: Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling.

Non-coding RNAs as modulators of the cardiac fibroblast phenotype.

Cardiac fibroblasts represent one of the most frequent cell type in the heart of rodents and humans and alterations of their phenotype have a great impact on cardiac function. Due to aging, ischemic injuries, valvular dysfunctions, hypertension and aortic stenosis, multiple signals trigger the accumulation of extracellular matrix in the cardiac interstitium and perivascular space, leading to structural and functional detrimental changes in the heart. Cardiac fibroblasts are the principal orchestrators of matrix formation and degradation and indirectly regulate cardiac hypertrophy and inflammation. Understanding the molecular bases of their action could provide tools for the treatment of cardiac remodeling. This review summarizes recent evidences on non-coding RNAs, including microRNAs and long non-coding RNAs that modulate the phenotype of cardiac fibroblasts and may serve in the future as targets for novel therapeutic strategies against cardiac fibrosis.

Noncoding RNAs as regulators of cardiomyocyte proliferation and death.

Cardiovascular diseases are currently the main cause of morbidity and mortality worldwide. Ischemic heart disease, in particular, is responsible for the majority of cardiac-related deaths. Given the negligible regenerative potential of the human myocardium, there is a strong need for therapeutic strategies aiming at enhancing cardiomyocyte survival and proliferation following injury or at inhibiting their death. MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression at a post-transcriptional level with important functions in cardiovascular physiology and disease. It has been demonstrated that miRNAs can influence the ability of cardiomyocytes to enter the cell cycle and/or escape from death pathways. Additionally, long non coding-RNAs could be involved in such pathways. This review summarizes recent evidences on noncoding RNAs regulating proliferation and death of cardiomyocytes representing a future therapeutic for the treatment of heart diseases. This article is part of a Special Issue entitled SI: Non-coding RNAs.

miR-21 promotes fibrosis in an acute cardiac allograft transplantation model.

AIMS: Cardiac transplantation is the only curative therapy for end-stage heart failure. Fibrosis is one of the major causes for impaired function of cardiac allografts. MicroRNAs, a class of small non-coding RNAs, play a critical role in the development of cardiovascular disease, but the role of microRNAs in cardiac allograft failure is not well understood. METHODS AND RESULTS: To uncover a role of microRNAs during cardiac graft fibrosis, we generated global microRNA profiles in allogeneic (BALB/c in C57BL/6N) and isogeneic (C57BL/6N in C57BL/6N) murine hearts after transplantation. miR-21 together with cardiac fibrosis was increased in cardiac allografts compared with isografts. Likewise, patients with cardiac rejection after heart transplantation showed increased cardiac miR-21 levels. miR-21 was induced upon treatment with IL-6 in a monocyte cell line. Overexpression of miR-21 in this monocyte cell line activated a fibrotic gene programme and promoted monocyte-to-fibrocyte transition together with activation of chemokine (C-C) motif ligand 2 (monocyte chemoattractant protein 1) via the phosphatase and tensin homologue/activator protein 1 regulatory axis. In vivo, both genetic and pharmacological inhibition of miR-21 successfully reduced fibrosis and fibrocyte accumulation in cardiac allografts. CONCLUSION: Thus, inhibition of miR-21 is a novel strategy to target fibrosis development in cardiac allografts.

Non-coding RNAs in cardiovascular ageing.

The increasing burden of ageing populations and their healthcare expenditure is a major challenge worldwide. Ageing is a complex disorder and can be defined as progressive decline in function with time leading to increased incidence of various cardiovascular, neurological and immunological diseases. The human genome comprises of many protein coding and even more non-coding RNA genes. MicroRNAs, a class of non-coding RNA, regulate the expression of multiple messenger RNAs post-transcriptionally and are reported to be involved in crucial aspects of cell biology encompassing ageing. Recently, several studies have reported the regulation of microRNAs with ageing and microRNAs like miR-34 have emerged as critical regulator of ageing extending from Caenorhabditis elegans to mammals. Here, we summarize the reported role of microRNAs as well as long noncoding RNAs (lncRNAs) in the process of ageing with a special emphasis on cardiovascular ageing.