L affiliations.Glycol chitosan MedChemExpress Copyright: 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access write-up distributed under the terms and circumstances on the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ four.0/).Cells 2021, ten, 2725. https://doi.org/10.3390/cellshttps://www.mdpi.com/journal/cellsCells 2021, 10,2 ofRecently, several research have focused on the regulatory roles of miRNAs in muscle homeostasis, muscle wasting, along with other myopathies [14,15]. Accumulating evidence indicates that many miRNAs are involved in muscle wasting through their inhibitory effects on myogenesis [9,16]. Nevertheless, the molecular mechanism whereby SFA-induced miRNAs suppress myogenic differentiation remains largely unknown. Actin remodeling, coordinated by actin-binding proteins, modulates the cytoskeletal dynamics important for myoblast proliferation and differentiation [17,18]. Cofilin two (CFL2) is often a skeletal muscle-specific actin-binding protein and belongs to the actin-depolymerizing factor (ADF)/cofilin family [19,20]. CFL2 plays an essential role in actin remodeling by severing or depolymerizing filamentous actin (F-actin), which can be involved in muscle improvement and upkeep [19,20]. Inside a mouse model, the functional ablation of CFL2 was associated with skeletal muscle wasting accompanied by Dorsomorphin supplier F-actin accumulation [21]. Moreover, CFL2 knockout disrupted sarcomere structure and integrity with enhanced actin polymerization [22]. Furthermore, CFL1-mediated actin remodeling has been shown to regulate cell proliferation associated with myogenic differentiation [23,24]. In a previous study, we identified that CFL2 knockdown by siRNA promoted myoblast proliferation and consequently inhibited myogenic differentiation in C2C12 cells [25]. Although CFL2 is identified to be essential for skeletal myogenesis and maintenance, its regulation by miRNAs in the course of myogenic differentiation has not been explored. Here, we investigated the role of SFA-induced miRNA on myogenic differentiation. We discovered that miR-325-3p, markedly induced by palmitic acid (PA) in myoblasts, regulates CFL2 expression straight. We also showed that miR-325-3p plays a essential role in cell proliferation, myogenic factors expressions, and differentiation in myoblasts. Our findings relating to the regulatory functions of miR-325-3p on myogenesis improve understanding with the mechanism of muscle wasting inside the background of obesity and will deliver a novel diagnostic and therapeutic target for muscle wasting and sarcopenic obesity. 2. Components and Solutions 2.1. Cell Culture, Differentiation and PA Treatment C2C12 myoblasts, an immortalized murine muscle progenitor cell line (ATCC), were maintained in a growth medium (GM; Dulbecco’s modified Eagle’s medium (DMEM) containing 10 fetal bovine serum and 1 penicillin/streptomycin) (Gibco, Carlsbad, CA, USA) at 37 C within a 5 CO2 humidified incubator. For the biochemical study, cells have been seeded on 6-well plates (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 1.three 105 cells/well in two mL of GM. Just after 24 h, cells were transiently transfected with indicated oligonucleotides making use of Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) in line with the manufacturer’s guidelines. When cells reached 800 confluence, myoblasts have been differentiated to myotubes by switching to a differentiation medium (DM; DMEM containing 2 dialyzed horse serum and 1 penicillin/streptomycin). When necessary, cells had been treated w.