Effect of miR-18a-5p, miR-19a-3p, and miR-20a-5p on In Vitro Cardiomyocyte Differentiation of Human Endometrium Tissue-Derived Stem Cells Through Regulation of Smad4 Expression.

Background: Smad4 regulates the expression of the genes required for heart homeostasis. Regarding the central role of microRNAs in cardiac biology, we investigated the expression of the three Smad4-targeting miRNAs, namely miR-18a-5p, miR-19a-3p, and miR-20a-5p, as well as Smad4 during differentiation of human endometrium-derived mesenchymal stem cells (hEMSCs) into cardiomyocytes (CMs). Methods: To evaluate mesenchymal phenotype and multi-lineage differentiation ability of hEMSCs, immunophenotyping by flow cytometry and differentiation into osteoblasts and adipocytes were performed, respectively. For transdifferentiation into CMs, hEMSCs were exposed to a cardiomyogenic medium composed of 5-aza and bFGF for 30 days. The comparison between transcriptional expression levels of Nkx2-5, GATA4, Smad4, TNNT2, TBX5, miR-18a-5p, miR-19a-3p, and miR-20a-5p by qRT-PCR, as well as protein levels of Nkx2-5, Smad4, and cTnT by immunofluorescence staining, was conducted in every 6 days. Results: In vitro, the mesenchymal stem cell phenotype of hEMSCs and their potency for differentiation into other MSCs were confirmed. Differentiated hEMSCs had morphological characteristics of CMs. The percentage of positive cells for Nkx2-5, Smad4, and cTnT proteins was increased following induction and culminated on the 24th day. Also, mRNA levels of Nkx2-5, GATA4, Smad4, TNNT2, and TBX5 exhibited the same trend. The expression of investigated miRNAs was significantly decreased sequentially. A significant negative correlation between expressions of Smad4 and investigated miRNAs was observed. Conclusion: Our results indicate that miR-18a-5p, miR-19a-3p, and miR-20a-5p are involved in the cardiac differentiation propensity of hEMSCs potentially by regulation of Smad levels. Although, more mechanistic experiments are required to confirm this idea.

MicroRNAs and exosomes: Cardiac stem cells in heart diseases.

Treating cardiovascular diseases with cardiac stem cells (CSCs) is a valid treatment among various stem cell-based therapies. With supplying the physiological need for cardiovascular cells as their main function, under pathological circumstances, CSCs can also reproduce the myocardial cells. Although studies have identified many of CSCs' functions, our knowledge of molecular pathways that regulate these functions is not complete enough. Either physiological or pathological studies have shown, stem cells proliferation and differentiation could be regulated by microRNAs (miRNAs). How miRNAs regulate CSC behavior is an interesting area of research that can help us study and control the function of these cells in vitro; an achievement that may be beneficial for patients with cardiovascular diseases. The secretome of stem and progenitor cells has been studied and it has been determined that exosomes are the main source of their secretion which are very small vesicles at the nanoscale and originate from endosomes, which are secreted into the extracellular space and act as key signaling organelles in intercellular communication. Mesenchymal stem cells, cardiac-derived progenitor cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), and iPSC-derived cardiomyocytes release exosomes that have been shown to have cardioprotective, immunomodulatory, and reparative effects. Herein, we summarize the regulation roles of miRNAs and exosomes in cardiac stem cells.

Pivotal Role of TGF-ß/Smad Signaling in Cardiac Fibrosis: Non-coding RNAs as Effectual Players.

Unintended cardiac fibroblast proliferation in many pathophysiological heart conditions, known as cardiac fibrosis, results in pooling of extracellular matrix (ECM) proteins in the heart muscle. Transforming growth factor ß (TGF-ß) as a pivotal cytokine/growth factor stimulates fibroblasts and hastens ECM production in injured tissues. The TGF-ß receptor is a heterodimeric receptor complex on the plasma membrane, made up from TGF-ß type I, as well as type II receptors, giving rise to Smad2 and Smad3 transcription factors phosphorylation upon canonical signaling. Phosphorylated Smad2, Smad3, and cytoplasmic Smad4 intercommunicate to transfer the signal to the nucleus, culminating in provoked gene transcription. Additionally, TGF-ß receptor complex activation starts up non-canonical signaling that lead to the mitogen-stimulated protein kinase cascade activation, inducing p38, JNK1/2 (c-Jun NH2-terminal kinase 1/2), and ERK1/2 (extracellular signal-regulated kinase 1/2) signaling. TGF-ß not only activates fibroblasts and stimulates them to differentiate into myofibroblasts, which produce ECM proteins, but also promotes fibroblast proliferation. Non-coding RNAs (ncRNAs) are important regulators of numerous pathways along with cellular procedures. MicroRNAs and circular long ncRNAs, combined with long ncRNAs, are capable of affecting TGF-ß/Smad signaling, leading to cardiac fibrosis. More comprehensive knowledge based on these processes may bring about new diagnostic and therapeutic approaches for different cardiac disorders.