miR-125b-5p and miR-125b-1-3p mediate TGF-beta response in ischemic heart failure

Background Myocardial fibrosis is a crucial factor in the progression of heart failure and transforming growth factor beta (TGF-β) signalling plays a key role in the development of cardiac fibrosis. MicroRNAs (miRNAs) are emerging as regulators of cardiac fibrosis through their effects on TGF-β signalling, fibroblast activation, and extracellular matrix protein expression. Targeting miRNAs holds promise for the prevention and treatment of cardiac fibrosis. Purpose We aimed to investigate the roles of miR-125b-5p and miR-125b-1-3p in TGF-β signalling and assess their therapeutic potential in ischemic heart failure (IHF). Methods RNA sequencing was performed to identify differentially expressed miRNAs and mRNAs in left ventricular tissues from end-stage IHF patients (n=8) compared to controls (n=8). Gene Set Enrichment Analysis (GSEA) was used to identify significantly enriched biological pathways and processes based on validated targets. MiRNA-mRNA interactions were predicted to identify their target genes and affected pathways. The identified miRNAs and their predicted target genes were validated in the left ventricles of IHF patients, and the antifibrotic potential of miRNAs was investigated in primary human cardiac fibroblasts. Results Principal component analysis revealed two distinct clusters in both the mRNA- and miRNA-ome between healthy and patient cardiac tissues, indicating significant transcriptomic alterations in failing hearts. RNA-seq data showed upregulation of miR-125b-1-3p (padj <0.007) in IHF patient hearts. In RT-qPCR both miR-125b-5p and miR-125b-1-3p (p<0.008; p<0.001) were upregulated. Predicted miRNA-mRNA interactions suggested that miR-125b-1-3p targets the plasminogen activator, tissue type (PLAT) and Apelin (APLN). Upregulation of both transcripts was observed when inhibiting miR-125b-1-3p (p=0.03; p=0.04) in human primary cardiac fibroblasts, suggesting that PLAT and APLN are potential targets. GSEA indicated the TGF-β signalling pathway as a target of these miRNAs. TGF-β2 stimulation in human cardiac fibroblasts led to upregulation of miR-125b-5p (p=0.049) and miR-125b-1-3p (p=0.015), along with downregulation of PLAT (p=0.0012) and APLN (p=0.0014). Importantly, TGF-β2 simulated an increase in α-SMA and COL1A1 at both the mRNA (p=0.0064; p=0.01) and protein (p=0.03; p<0.001) levels, confirming the fibroblast-to-myofibroblast transition (FMT). Furthermore, inhibition of miR-125b-1-3p resulted in downregulation of latent transforming growth factor beta binding protein 2 (p=0.02) and decreased thrombospondin 1 (p=0.02) accompanied by a trend of reduced phosphorylation of SMAD2/3, suggesting a potential role of miR-125b-1-3p in biosynthesis and activation of TGF-β. Indeed, inhibition of miR-125b-1-3p downregulated COL1A1 (p=0.01) and ACTA2 (p=0.001) in human cardiac fibroblasts, indicating reduced FMT. Conclusion Our results suggest that miR-125b-5p and miR-125b-1-3p may mediate TGF-β signaling in IHF.Figure