MicroRNA-223 Regulates Cardiac Fibrosis After Myocardial Infarction by Targeting RASA1

• Published in: Cellular physiology and biochemistry Vol. 46; no. 4; pp. 1439 - 1454
• Main Authors: Liu, Xiaoxiao, Xu, Yifeng, Deng, Yunfei, Li, Hongli
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland S. Karger AG 01.05.2018; Cell Physiol Biochem Press GmbH & Co KG
• Subjects: 3' Untranslated Regions; Actins - metabolism; Animals; Antagomirs - metabolism; Base Sequence; Cardiac fibrosis; Cardiac function; Cardiomyocytes; Cell adhesion & migration; Cell Differentiation - drug effects; Cell Movement - drug effects; Cell Proliferation; Cells, Cultured; Collagen Type I - metabolism; Collagen Type II - metabolism; Coronary vessels; Experiments; Fibroblasts; Fibroblasts - cytology; Fibroblasts - metabolism; Fibrosis; Gene expression; Growth factors; Heart attacks; Heart failure; Kinases; Male; MAP Kinase Kinase 1 - metabolism; MAP Kinase Kinase 2 - metabolism; Microrna-223; MicroRNAs; MicroRNAs - antagonists & inhibitors; MicroRNAs - genetics; MicroRNAs - metabolism; Myocardial infarction; Myocardial Infarction - metabolism; Myocardial Infarction - pathology; Myocardial Infarction - veterinary; Myocytes, Cardiac - cytology; Myocytes, Cardiac - metabolism; Original Paper; Ostomy; p120 GTPase Activating Protein - antagonists & inhibitors; p120 GTPase Activating Protein - genetics; p120 GTPase Activating Protein - metabolism; Phosphorylation; Prevention; Proteins; Proto-Oncogene Proteins c-akt - metabolism; RASA1; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering - metabolism; Rodents; Sequence Alignment; Signal transduction; Transforming Growth Factor beta1 - pharmacology; Vimentin - metabolism; Grand Island; United States > US; Myocardial infarction; Microrna-223; Cardiac fibrosis; RASA1
• ISSN: 1015-8987; 1421-9778
• DOI: 10.1159/000489185

Background/Aims: Percutaneous coronary intervention reduces acute myocardial infarction (MI)-induced mortality to a great extent, but effective treatments for MI-induced cardiac fibrosis and heart failure are still lacking. MicroRNAs (miRNAs) play a variety of roles in cells and have thus been investigated extensively. MicroRNA-223 (miR-223) expression has been reported to be altered in post-MI heart failure in humans; however, the roles of miR-223 in MI remain unknown. Our study aimed to elucidate the roles of miR-223 in cardiac fibrosis. Methods: Cultured cardiac fibroblasts (CFs) were activated by TGF-β1 stimulation. Gain and loss of miR-223 and RAS p21 protein activator 1 (RASA1) knockdown in CFs were achieved by transfecting the cells with miR-223 mimics and inhibitors, as well as small interfering RNA-RASA1 (siRASA1), respectively. Quantitative real-time reverse transcriptase-polymerase chain reactions (qRT-PCR) was used to determine miR-223-3p and RASA1 expression levels, and Cell Counting Kit-8 (CCK-8), transwell migration and scratch assays were performed to assess CFs viability and migration, respectively. Western blotting was used to detect collagen I, collagen III, alpha-smooth muscle actin (a-SMA), RASA1, p-Akt/t-Akt, p-MEK1/2/t-MEK1/2, and p-ERK1/2/t-ERK1/2 protein expressions, and immunofluorescence assays were used to detect the expression of α-actin, vimentin and α-SMA. Luciferase assays were carried out to determine whether miR-223 binds to RASA1. Rat models of MI were established by the ligation of the left anterior descending (LAD) coronary artery. MiR-223 inhibition in vivo was achieved via intramyocardial injections of the miR-223 sponge carried by adeno-associated virus 9 (AAV9). The cardiac function was detected by echocardiography, and cardiac fibrosis was shown by Masson’s trichrome staining. Results: miR-223 was increased in CFs compared to cardiomypcytes, and TGF-β1 treatment increased miR-223 expression in CFs. The miR-223 mimics enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs, while the miR-223 inhibitors had contrasting effects and partially prevented the promoting effects of TGF-β1. qRT-PCR and western blotting revealed that miR-223 negatively regulated RASA1 expression, and the luciferase assays showed that miR-223 suppressed the luciferase activity of the RASA1 3’ untranslated region (3'UTR), indicating that miR-223 binds directly to RASA1. Similar to transfection with the miR-223 mimics, RASA1 knockdown enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs. Moreover, RASA1 knockdown partially reversed the inhibitory effects of the miR-223 inhibitor on cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression, indicating that the effects of miR-223 in CFs are partially mediated by the regulation of RASA1 expression. Further exploration showed that miR-223 mimics and siRASA1 promoted MEK1/2, ERK1/2 and AKT phosphorylation, while the miR-223 inhibitors had contrasting effects. The in vivo experiments confirmed the results of the in vitro experiments and showed that miR-223 inhibition prevented cardiac functional deterioration and cardiac fibrosis. Conclusions: miR-223 enhanced cell proliferation, migration, and differentiation in CFs, thus mediated cardiac fibrosis after MI partially via the involvement of RASA1.