LncRNA-<i>Safe</i> contributes to cardiac fibrosis through <i>Safe</i>-<i>Sfrp2</i>-HuR complex in mouse myocardial infarction

Abstract

<b>Rationale</b>: As a hallmark of various heart diseases, cardiac fibrosis ultimately leads to end-stage heart failure. Anti-fibrosis is a potential therapeutic strategy for heart failure. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of heart diseases that promise to serve as therapeutic targets. However, few lncRNAs have been directly implicated in cardiac fibrosis. <b>Methods</b>: The lncRNA expression profiles were assessed by microarray in cardiac fibrotic and remote ventricular tissues in mice with myocardial infarction. The mechanisms and functional significance of lncRNA-<i>AK137033</i> in cardiac fibrosis were further investigated with both <i>in vitro</i> and <i>in vivo</i> models. <b>Results</b>: We identified 389 differentially expressed lncRNAs in cardiac fibrotic and remote ventricular tissues in mice with myocardial infarction. Among them, a lncRNA (<i>AK137033</i>) we named <i>Safe</i> was enriched in the nuclei of fibroblasts, and elevated in both myocardial infarction and TGF-β-induced cardiac fibrosis. Knockdown of <i>Safe</i> prevented TGF-β-induced fibroblast-myofibroblast transition, aberrant cell proliferation and secretion of extracellular matrix proteins <i>in vitro</i>, and mended the impaired cardiac function in mice suffering myocardial infarction. <i>In vitro</i> studies indicated that knockdown of <i>Safe</i> significantly inhibited the expression of its neighboring gene <i>Sfrp2</i>, and vice versa. The <i>Sfrp2</i> overexpression obviously disturbed the regulatory effects of <i>Safe</i> shRNAs in both the <i>in vitro</i> cultured cardiac fibroblasts and myocardial infarction-induced fibrosis. Dual-Luciferase assay demonstrated that <i>Safe</i> and <i>Sfrp2</i> mRNA stabilized each other via their complementary binding at the 3'-end. RNA electrophoretic mobility shift assay and RNA immunoprecipitation assay indicated that RNA binding protein HuR could bind to <i>Safe</i>-<i>Sfrp2</i> RNA duplex, whereas the knockdown of <i>HuR</i> dramatically reduced the stabilization of <i>Safe</i> and <i>Sfrp2</i> mRNAs, down-regulated their expression in cardiac fibroblasts, and thus inhibited TGF-β-induced fibrosis. The <i>Safe</i> overexpression partially restrained the phenotype change of cardiac fibroblasts induced by <i>Sfrp2</i> shRNAs, but not that induced by <i>HuR</i> shRNAs. <b>Conclusions</b>: Our study identifies <i>Safe</i> as a critical regulator of cardiac fibrosis, and demonstrates <i>Safe</i>-<i>Sfrp2</i>-HuR complex-mediated <i>Sfrp2</i> mRNA stability is the underlying mechanism of <i>Safe</i>-regulated cardiac fibrosis. Fibroblast-enriched <i>Safe</i> could represent a novel target for anti-fibrotic therapy in heart diseases.