PMID- 29689569 OWN - NLM STAT- MEDLINE DCOM- 20180705 LR - 20190212 IS - 1421-9778 (Electronic) IS - 1015-8987 (Linking) VI - 46 IP - 4 DP - 2018 TI - MicroRNA-223 Regulates Cardiac Fibrosis After Myocardial Infarction by Targeting RASA1. PG - 1439-1454 LID - 10.1159/000489185 [doi] AB - 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-beta1 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 alpha-actin, vimentin and alpha-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-beta1 treatment increased miR-223 expression in CFs. The miR-223 mimics enhanced cell proliferation and migration and collagen I, collagen III, and alpha-SMA protein expression in CFs, while the miR-223 inhibitors had contrasting effects and partially prevented the promoting effects of TGF-beta1. 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 alpha-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 alpha-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. CI - (c) 2018 The Author(s). Published by S. Karger AG, Basel. FAU - Liu, Xiaoxiao AU - Liu X FAU - Xu, Yifeng AU - Xu Y FAU - Deng, Yunfei AU - Deng Y FAU - Li, Hongli AU - Li H LA - eng PT - Journal Article DEP - 20180419 PL - Germany TA - Cell Physiol Biochem JT - Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology JID - 9113221 RN - 0 (3' Untranslated Regions) RN - 0 (Actins) RN - 0 (Antagomirs) RN - 0 (Collagen Type I) RN - 0 (Collagen Type II) RN - 0 (MIRN223 microRNA, rat) RN - 0 (MicroRNAs) RN - 0 (RNA, Small Interfering) RN - 0 (Transforming Growth Factor beta1) RN - 0 (Vimentin) RN - 0 (p120 GTPase Activating Protein) RN - 0 (smooth muscle actin, rat) RN - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt) RN - EC 2.7.12.2 (MAP Kinase Kinase 1) RN - EC 2.7.12.2 (MAP Kinase Kinase 2) SB - IM MH - 3' Untranslated Regions MH - Actins/metabolism MH - Animals MH - Antagomirs/metabolism MH - Base Sequence MH - Cell Differentiation/drug effects MH - Cell Movement/drug effects MH - Cell Proliferation MH - Cells, Cultured MH - Collagen Type I/metabolism MH - Collagen Type II/metabolism MH - Fibroblasts/cytology/metabolism MH - Fibrosis MH - MAP Kinase Kinase 1/metabolism MH - MAP Kinase Kinase 2/metabolism MH - Male MH - MicroRNAs/antagonists & inhibitors/genetics/*metabolism MH - Myocardial Infarction/metabolism/pathology/veterinary MH - Myocytes, Cardiac/cytology/metabolism MH - Phosphorylation MH - Proto-Oncogene Proteins c-akt/metabolism MH - RNA Interference MH - RNA, Small Interfering/metabolism MH - Rats MH - Rats, Sprague-Dawley MH - Sequence Alignment MH - Transforming Growth Factor beta1/pharmacology MH - Vimentin/metabolism MH - p120 GTPase Activating Protein/antagonists & inhibitors/genetics/*metabolism OTO - NOTNLM OT - Cardiac fibrosis OT - Microrna-223 OT - Myocardial infarction OT - RASA1 EDAT- 2018/04/25 06:00 MHDA- 2018/07/06 06:00 CRDT- 2018/04/25 06:00 PHST- 2017/11/07 00:00 [received] PHST- 2018/02/28 00:00 [accepted] PHST- 2018/04/25 06:00 [pubmed] PHST- 2018/07/06 06:00 [medline] PHST- 2018/04/25 06:00 [entrez] AID - 000489185 [pii] AID - 10.1159/000489185 [doi] PST - ppublish SO - Cell Physiol Biochem. 2018;46(4):1439-1454. doi: 10.1159/000489185. Epub 2018 Apr 19.