ABSTRACT
MicroRNAs are known to play a critical role in skeletal myogenesis and maintenance, and cofilin-2 (CFL2) is necessary for actin cytoskeleton dynamics and myogenic differentiation. Nonetheless, target molecules and the modes of action of miRNAs, especially those responsible for the inhibitory mechanism on the myogenesis by saturated fatty acids (SFA) or obesity, still remain unclear. Here, we reported the role played by miR-429-3p on CFL2 expression, actin filament dynamics, myoblast proliferation, and myogenic differentiation in C2C12 cells. Palmitic acid (PA), the most abundant SFA in diet, inhibited the myogenic differentiation of myoblasts, accompanied by CFL2 reduction and miR-429-3p induction. Interestingly, miR-429-3p suppressed the expression of CFL2 by targeting the 3'UTR of CFL2 mRNA directly. Transfection of miR-429-3p mimic in myoblasts increased F-actin formation and augmented nuclear YAP level, thereby promoting cell cycle progression and myoblast proliferation. Moreover, miR-429-3p mimic drastically suppressed the expressions of myogenic factors, such as MyoD, MyoG, and MyHC, and impaired myogenic differentiation of C2C12 cells. Therefore, this study unveiled the crucial role of miR-429-3p in myogenic differentiation through the suppression of CFL2 and provided implications of SFA-induced miRNA in the regulation of actin dynamics and skeletal myogenesis.
Subject(s)
Cell Differentiation/drug effects , Cofilin 2/metabolism , Down-Regulation/drug effects , MicroRNAs/metabolism , Muscle Development/drug effects , Palmitic Acid/pharmacology , 3' Untranslated Regions , Actins/metabolism , Animals , Antagomirs/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line , Cofilin 2/antagonists & inhibitors , Cofilin 2/genetics , Humans , Mice , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Myoblasts/cytology , Myoblasts/drug effects , Myoblasts/metabolism , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , YAP-Signaling Proteins/genetics , YAP-Signaling Proteins/metabolismABSTRACT
Understanding the multifaceted nature of microRNA (miRNA) function in mammalian cells is still a challenge. Commonly accepted principles of cooperativity and multiplicity of miRNA function imply that individual mRNAs can be targeted by several miRNAs whereas a single miRNA may concomitantly regulate a subset of different genes. However, there is a paucity of information whether multiple miRNAs regulate critical cellular events and thereby acting redundantly. To gain insight into this notion, we conducted an unbiased high-content miRNA screen by individually introducing 1139 miRNA mimics into Chinese hamster ovary (CHO) cells. We discovered that 66% of all miRNAs significantly impacted on proliferation, protein expression, apoptosis and necrosis. In summary, we provide evidence for a substantial degree of redundancy among miRNAs to maintain cellular homeostasis.
Subject(s)
Metabolic Networks and Pathways/genetics , MicroRNAs/genetics , RNA, Messenger/genetics , Animals , Apoptosis/genetics , CHO Cells , Cell Proliferation , Cofilin 2/antagonists & inhibitors , Cofilin 2/genetics , Cofilin 2/metabolism , Cricetulus , Gene Expression , Gene Expression Profiling , Homeostasis/genetics , MicroRNAs/metabolism , Molecular Mimicry , Necrosis/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , TransfectionABSTRACT
Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin ß3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin ß3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.
Subject(s)
Actins/metabolism , Crk-Associated Substrate Protein/metabolism , Muscle Development/physiology , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/metabolism , Actin Cytoskeleton/metabolism , Animals , Base Sequence , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Line , Cell Nucleus/metabolism , Cofilin 2/antagonists & inhibitors , Cofilin 2/genetics , Cofilin 2/metabolism , Crk-Associated Substrate Protein/antagonists & inhibitors , Crk-Associated Substrate Protein/genetics , DNA Primers/genetics , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Integrin beta3/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Models, Biological , Muscle Development/genetics , Mutagenesis , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA InterferenceABSTRACT
Cofilin is an actin regulatory protein that plays a critical role in actin filament dynamics in a variety of cells. We have previously demonstrated that excess cofilin in skeletal muscle cells leads to disruption of actin filaments, followed by actin-cofilin rod formation in the cytoplasm. In this study, to further clarify the role of cofilin in actin assembly during myofibrillogenesis, cofilin expression was suppressed in cultured chicken skeletal muscle cells. First, we confirmed that turnover of cofilin in myotubes was much higher than that of actin, and that the cofilin level could be decreased drastically within 2 days when cofilin de novo synthesis was suppressed. Next, cofilin expression in individual myotubes was suppressed by introducing antisense morpholino oligonucleotides into the cells by microinjection. Cofilin depletion at the early phase of myofibrillogenesis caused abnormal actin aggregates in myotubes and impaired actin organization into cross-striated myofibril structures. However, when cofilin expression was suppressed in developed myotubes, actin localization in striated myofibrils was scarcely affected. These results indicate that cofilin plays a critical role in the regulation of actin assembly at the early process of myofibrillogenesis.