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Background: Long non-coding RNAs (lncRNAs) are deemed to be relevant to the tumorigenesis and development of a variety of tumors, containing gastric cancer (GC). The purpose of our investigations is to explore the character of HCP5 in GC. Methods: HCP5 expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) in 62 matched GC tissues and corresponding para-carcinoma tissues. In vitro and in vivo functional assays were subjected to verify the biological effects of HCP5 after alteration of HCP5. Chromatin immunoprecipitation assay (CHIP) assays were conducted to confirm that myocyte enhancer factor 2A (MEF2A) could bind to HCP5 promoter regions and thereby induce HCP5 expression. Analysis of the latent binding of miR-106b-5p to HCP5 and p21 was made by bioinformatics prediction and luciferase reporter assays. Results: Significant downregulation of HCP5 was detected in GC tissues. Negative correlation was determined between HCP5 expression level and tumor size and overall survival in GC patients. HCP5 depletion had a facilitating impact on proliferation, migration and invasion of GC cells. Consistently, overexpression of HCP5 came into an opposite effect. Moreover, we demonstrated that MEF2A could combine with the promoter region of HCP5 and thereby induce HCP5 transcription. Luciferase reporter assays revealed that HCP5 could compete with miR-106b-5p as a competing endogenous RNA (ceRNA) and upregulated p21 expression in GC. Conclusions: MEF2A-mediated HCP5 could exert an anti-tumor effect among the development of GC via miR-106b-5p/p21 axis, which provides a novel target for GC therapy.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by amyloid plaques and neurofibrillary tangles which significantly affects people’s life quality. Recently, AD has been found to be closely related to autophagy. The aim of this study was to identify autophagy-related genes associated with the pathogenesis of AD from multiple types of microarray and sequencing datasets using bioinformatics methods and to investigate their role in the pathogenesis of AD in order to identify novel strategies to prevent and treat AD. Our results showed that the autophagy-related genes were significantly downregulated in AD and correlated with the pathological progression. Furthermore, enrichment analysis showed that these autophagy-related genes were regulated by the transcription factor myocyte enhancer factor 2A (MEF2A), which had been confirmed using si-MEF2A. Moreover, the single-cell sequencing data suggested that MEF2A was highly expressed in microglia. Methylation microarray analysis showed that the methylation level of the enhancer region of MEF2A in AD was significantly increased. In conclusion, our results suggest that AD related to the increased methylation level of MEF2A enhancer reduces the expression of MEF2A and downregulates the expression of autophagy-related genes which are closely associated with AD pathogenesis, thereby inhibiting autophagy.

Objective Stroke is a severe complication of atrial fibrillation (AF). We aimed to discover key genes and microRNAs related to stroke risk in patients with AF using bioinformatics analysis. Methods GSE66724 microarray data, including peripheral blood samples from eight patients with AF and stroke and eight patients with AF without stroke, were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between AF patients with and without stroke were identified using the GEO2R online tool. Functional enrichment analysis was performed using the DAVID database. A protein–protein interaction (PPI) network was obtained using the STRING database. MicroRNAs (miRs) targeting these DEGs were obtained from the miRNet database. A miR–DEG network was constructed using Cytoscape software. Results We identified 165 DEGs (141 upregulated and 24 downregulated). Enrichment analysis showed enrichment of certain inflammatory processes. The miR–DEG network revealed key genes, including MEF2A, CAND1, PELI1, and PDCD4, and microRNAs, including miR-1, miR-1-3p, miR-21, miR-21-5p, miR-192, miR-192-5p, miR-155, and miR-155-5p. Conclusion Dysregulation of certain genes and microRNAs involved in inflammation may be associated with a higher risk of stroke in patients with AF. Evaluating these biomarkers could improve prediction, prevention, and treatment of stroke in patients with AF.

The function of nuclear receptor corepressor 1 (NCoR1) in cardiomyocytes is unclear, and its physiological and pathological implications are unknown. Here, we found that cardiomyocyte‐specific NCoR1 knockout (CMNKO) mice manifested cardiac hypertrophy at baseline and had more severe cardiac hypertrophy and dysfunction after pressure overload. Knockdown of NCoR1 exacerbated whereas overexpression mitigated phenylephrine‐induced cardiomyocyte hypertrophy. Mechanistic studies revealed that myocyte enhancer factor 2a (MEF2a) and MEF2d mediated the effects of NCoR1 on cardiomyocyte hypertrophy. The receptor interaction domains (RIDs) of NCoR1 interacted with MEF2a to repress its transcriptional activity. Furthermore, NCoR1 formed a complex with MEF2a and class IIa histone deacetylases (HDACs) to suppress hypertrophy‐related genes. Finally, overexpression of RIDs of NCoR1 in the heart attenuated cardiac hypertrophy and dysfunction induced by pressure overload. In conclusion, NCoR1 cooperates with MEF2 and HDACs to repress cardiac hypertrophy. Targeting NCoR1 and the MEF2/HDACs complex may be an attractive therapeutic strategy to tackle pathological cardiac hypertrophy.

Early ViewOnline Version of Record before inclusion in an issue; Hibernating brown bears, Ursus arctos, undergo extended periods of inactivity and yet these large hibernators are resilient to muscle disuse atrophy. Physiological characteristics associated with atrophy resistance in bear muscle have been examined (e.g., muscle mechanics, neural activity) but roles for molecular signaling/regulatory mechanisms in the resistance to muscle wasting in bears still require investigation. Using quantitative reverse transcription PCR (RT-qPCR), the present study characterized the responses of 36 microRNAs linked with development, metabolism, and regeneration of skeletal muscle, in the vastus lateralis of brown bears comparing winter hibernating and summer active animals. Relative levels of mRNA of selected genes (mef2a, pax7, id2, prkaa1, and mstn) implicated upstream and downstream of the microRNAs were examined. Results indicated that hibernation elicited a myogenic microRNA, or "myomiR", response via MEF2A-mediated signaling. Upregulation of MEF2A-controlled miR-1 and miR-206 and respective downregulation of pax7 and id2 mRNA are suggestive of responses that promote skeletal muscle maintenance. Increased levels of metabolic microRNAs, such as miR-27, miR-29, and miR-33, may facilitate metabolic suppression during hibernation via mechanisms that decrease glucose uptake and fatty acid oxidation. This study identified myomiR-mediated mechanisms for the promotion of muscle regeneration, suppression of ubiquitin ligases, and resistance to muscle atrophy during hibernation mediated by observed increases in miR-206, miR-221, miR-31, miR-23a, and miR-29b. This was further supported by the downregulation of myomiRs associated with a muscle injury and inflammation (miR-199a and miR-223) during hibernation. The present study provides evidence of myomiR-mediated signaling pathways that are activated during hibernation to maintain skeletal muscle functionality in brown bears.

The aim of the present study was to identify the genetic defect responsible for familial coronary artery disease/myocardial infarction (CAD/MI), which exhibited an autosomal dominant pattern of inheritance, in an extended Chinese Han pedigree containing 34 members. Using exome and Sanger sequencing, a novel 6‑base pair (bp) 'CAGCCG' deletion in exon 11 of the myocyte enhancer factor 2A (MEF2A) gene was identified, which cosegregated with CAD/MI cases in this family. This 6‑bp deletion was not detected in 311 sporadic cases of premature CAD/MI or in 323 unrelated healthy controls. Determination of a genetic risk profile has a key role in understanding the pathogenesis of CAD and MI. Among the reported risk‑conferring genes and their variants, mutations in MEF2A have been reported to segregate with CAD/MI in Caucasian families. Causative missense mutations have also been detected in sporadic CAD/MI cases. However, this suggested genetic linkage is controversial, since it could not be confirmed by ensuing studies. The discovery of a novel MEF2A mutation in a Chinese family with premature CAD/MI suggests that MEF2A may have a significant role in the pathogenesis of premature CAD/MI. To better understand this association, further in vitro and in vivo studies are required.

While MEF2 transcription factors are well known to cooperate in orchestrating cell fate and adaptive responses during development and adult life, additional studies over the last decade have identified a wide spectrum of genetic alterations of MEF2 in different cancers. The consequences of these alterations, including triggering and maintaining the tumorigenic process, are not entirely clear. A deeper knowledge of the molecular pathways that regulate MEF2 expression and function, as well as the nature and consequences of MEF2 mutations are necessary to fully understand the many roles of MEF2 in malignant cells. This review discusses the current knowledge of MEF2 transcription factors in cancer.

Prenatal stress (PS) induces molecular changes that alter neural connectivity, increasing the risk for neuropsychiatric disorders. Here we analyzed —in the hippocampus of adult rats exposed to PS— the epigenetic signature mediating the PS-induced neuroplasticity changes. Furthermore, using cultured hippocampal neurons, we investigated the effects on neuroplasticity of an epigenetic modulator. PS induced significant modifications in the mRNA levels of stress-related transcription factor MEF2A, SUV39H1 histone methyltransferase, and TET1 hydroxylase, indicating that PS modifies gene expression through chromatin remodeling. In in vitro analysis, histone acetylation inhibition with apicidin increased filopodium density, suggesting that the external regulation of acetylation levels might modulate neuronal morphology. These results offer a way to enhance neural connectivity that could be considered to revert PS effects. Fil: Monteleone, Melisa Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Pallares, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina Fil: Billi, Silvia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Antonelli, Marta Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina Fil: Brocco, Marcela Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Improving the efficacy of neovascularization is a promising strategy to restore perfusion of ischemic tissues in patients with peripheral arterial disease. The 14q32 microRNA cluster is highly involved in neovascularization. The Mef2a transcription factor has been shown to induce transcription of the microRNAs within this cluster. We inhibited expression of Mef2a using gene-silencing oligonucleotides (GSOs) in an in vivo hind limb ischemia model. Treatment with GSO-Mef2a clearly improved blood flow recovery within 3 days (44% recovery versus 25% recovery in control) and persisted until 14 days after ischemia induction (80% recovery versus 60% recovery in control). Animals treated with GSO-Mef2a showed increased arteriogenesis and angiogenesis in the relevant muscle tissues. Inhibition of Mef2a decreased expression of 14q32 microRNAs miR-329 (p = 0.026) and miR-494 (trend, p = 0.06), but not of other 14q32 microRNAs, nor of 14q32 microRNA precursors. Because Mef2a did not influence 14q32 microRNA transcription, we hypothesized it functions as an RNA-binding protein that influences processing of 14q32 microRNA miR-329 and miR-494. Mef2A immunoprecipitation followed by RNA isolation and rt/qPCR confirmed direct binding of MEF2A to pri-miR-494, supporting this hypothesis. Our study demonstrates a novel function for Mef2a in post-ischemic neovascularization via post-transcriptional regulation of 14q32 microRNAs miR-329 and miR-494., Graphical Abstract

Houttuynia cordata Thunb ( H cordata), a medicinal plant, has anticancer activity, as it inhibits cell growth and induces cell apoptosis in cancer. However, the potential anti-cancer activity and mechanism of H cordata for human liver cancer cells is not well understood. Recently, we identified hypoxia-inducible factor (HIF)-1A, Forkhead box (FOX)O3, and MEF2A as proapoptotic factors induced by H cordata, suggesting that HIF-1A, FOXO3, and MEF2A contribute to the apoptosis of HepG2 hepatocellular carcinoma cells. FOXO3 transcription factors regulate target genes involved in apoptosis. H cordata significantly increased the mRNA and protein expression of HIF-1A and FOXO3 and stimulated MEF2A expression in addition to increased apoptosis in HepG2 cells within 24 hours. Therefore, we determined the potential role of FOXO3 on apoptosis and on H cordata–induced MEF2A in HepG2 cells. HIF-1A silencing by siRNA attenuated MEF2A and H cordata–mediated FOXO3 upregulation in HepG2 cells. Furthermore, H cordata–mediated MEF2A expression enhanced caspase-3 and caspase-7, which were abolished on silencing FOXO3 with siRNA. In addition, H cordata inhibited growth of human hepatocellular carcinoma xenografts in nude mice. Taken together, our results demonstrate that H cordata enhances HIF-1A/FOXO3 signaling, leading to MEF2A upregulation in HepG2 cells, and in parallel, it disturbs the expression of Bcl-2 family proteins (Bax, Bcl-2, and Bcl-xL), which results in apoptosis. Taken together, these findings demonstrate that H cordata promotes the activation of HIF-1A–FOXO3 and MEF2A pathways to induce apoptosis in human HepG2 hepatocellular carcinoma cells and is, therefore, a promising candidate for antitumor drug development.

Metabolic adaptations are emerging as common traits of cancer cells and tumor progression. In vitro transformation of NIH 3T3 cells allows the analysis of the metabolic changes triggered by a single oncogene. In this work, we have compared the metabolic changes induced by H-RAS and by the nuclear resident mutant of histone deacetylase 4 (HDAC4). RAS-transformed cells exhibit a dominant aerobic glycolytic phenotype characterized by up-regulation of glycolytic enzymes, reduced oxygen consumption and a defect in complex I activity. In this model of transformation, glycolysis is strictly required for sustaining the ATP levels and the robust cellular proliferation. By contrast, in HDAC4/TM transformed cells, glycolysis is only modestly up-regulated, lactate secretion is not augmented and, instead, mitochondrial oxygen consumption is increased. Our results demonstrate that cellular transformation can be accomplished through different metabolic adaptations and HDAC4/TM cells can represent a useful model to investigate oncogene-driven metabolic changes besides the Warburg effect.

SummaryMef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart.

SummarySkeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve different cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is activated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and β-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Premature expression of Nfix activates fetal and suppresses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.

The function of nuclear receptor corepressor 1 (NCoR1) in cardiomyocytes is unclear, and its physiological and pathological implications are unknown. Here, we found that cardiomyocyte‐specific NCoR1 knockout (CMNKO) mice manifested cardiac hypertrophy at baseline and had more severe cardiac hypertrophy and dysfunction after pressure overload. Knockdown of NCoR1 exacerbated whereas overexpression mitigated phenylephrine‐induced cardiomyocyte hypertrophy. Mechanistic studies revealed that myocyte enhancer factor 2a (MEF2a) and MEF2d mediated the effects of NCoR1 on cardiomyocyte hypertrophy. The receptor interaction domains (RIDs) of NCoR1 interacted with MEF2a to repress its transcriptional activity. Furthermore, NCoR1 formed a complex with MEF2a and class IIa histone deacetylases (HDACs) to suppress hypertrophy‐related genes. Finally, overexpression of RIDs of NCoR1 in the heart attenuated cardiac hypertrophy and dysfunction induced by pressure overload. In conclusion, NCoR1 cooperates with MEF2 and HDACs to repress cardiac hypertrophy. Targeting NCoR1 and the MEF2/HDACs complex may be an attractive therapeutic strategy to tackle pathological cardiac hypertrophy.