MEF2A Research Articles

MEF2A restrains cisplatin resistance in gastric cancer cells by modulating NFKBIA/NF-κB signaling pathway.

Cisplatin (DDP) resistance represents a pivotal contributing factor to chemotherapy failure and adverse patient outcomes in gastric cancer (GC). The objective of the present study was to investigate the roles and underlying mechanisms of myocyte enhancer factor 2A (MEF2A) in DDP resistance in GC. GC cell line AGS and MKN-45 cells were applied to construct DDP-resistant cells. CCK-8, colony formation, and flow cytometry methods were validated for determining the IC50 value of DDP and cell survival of GC cells. qRT-PCR and western blotting analysis quantified the molecular levels at mRNA and protein, respectively. Chromatin immunoprecipitation and dual-luciferase assays validated the molecular relationship between MEF2A and NF-κB inhibitor alpha (NFKBIA). Roles of MEF2A in in vivo were performed employing a xenograft model. The results showed that NFKBIA was greatly decreased in DDP-resistant AGS and MKN-45 cells compared to their respective parental cells. Increasing NFKBIA expression impaired the IC50 value of DDP and cell survival in DDP-resistant cells, while these alterations were rescued upon TNF-α treatment. Mechanistically, MEF2A acts as a transcriptional activator of NFKBIA, which led to the reduction of phosphorylation of p65 and cytoplasmic retention. Moreover, MEF2A overexpression promoted the sensitivity of GC cells to DDP and tumor growth, whereas these effects were partially reversed by NFKBIA silence. Collectively, MEF2A mitigated the DDP resistance in GC cells by modulatory actions on the NFKBIA/NF-κB signaling, shedding light on MEF2A/NFKBIA might be a promising intervention target for improving DDP resistance in GC.

MEF2A, a gene associated with mitochondrial biogenesis, promotes drug resistance in gastric cancer

Chemotherapeutic resistance is a major obstacle to the effectiveness of cisplatin-based chemotherapy for gastric cancer (GC), leading to treatment failure and poor survival rates. However, the underlying mechanisms are not fully understood. Our study demonstrated that the transcription factor myocyte enhancer factor 2A (MEF2A) plays a role in chemotherapeutic drug resistance by regulating the transcription of PGC1α and KEAP1, promoting mitochondrial biogenesis. It was found that increased MEF2A expression is linked with poor prognosis, cisplatin insensitivity, and mitochondrial function in GC. MEF2A overexpression significantly decreases GC cell sensitivity in vitro and in vivo, while MEF2A knockdown enhances the sensitivity to cisplatin. Mechanistically, MEF2A activates the transcription of PGC1α, leading to increased mitochondrial biogenesis. In addition, MEF2A inhibits KEAP1 transcription, reduces NRF2 ubiquitination degradation, and activates the KEAP1/NRF2 signaling pathway, which modulates the reactive oxygen species level. The present study identifies MEF2A as a new critical oncogene involved in GC chemoresistance, suggesting a novel therapeutic target for GC.

Genome-wide mapping of the binding sites of myocyte enhancer factor 2A in chicken primary myoblasts

Myocyte enhancer factor 2A (MEF2A) is a transcription factor that plays a critical role in cell proliferation, differentiation and apoptosis. In contrast to the wide characterization of its regulation mechanism in mammalian skeletal muscle, its role in chickens is limited. Especially, its wide target genes remain to be identified. Therefore, we utilized Cleavage Under Targets and Tagmentation (CUT&Tag) technology to reveal the genome-wide binding profile of MEF2A in chicken primary myoblasts thus gaining insights into its potential role in muscle development. Our results revealed that MEF2A binding sites were primarily distributed in intergenic and intronic regions. Within the promoter region, although only 8.87% of MEF2A binding sites were found, these binding sites were concentrated around the transcription start site (TSS). Following peak annotation, a total of 1903 genes were identified as potential targets of MEF2A. Gene Ontology (GO) enrichment analysis further revealed that MEF2A target genes may be involved in the regulation of embryonic development in multiple organ systems, including muscle development, gland development, and visual system development. Moreover, a comparison of the MEF2A target genes identified in chicken primary myoblasts with those in mouse C2C12 cells revealed 388 target genes are conserved across species, 1515 target genes are chicken specific. Among these conserved genes, ankyrin repeat and SOCS box containing 5 (ASB5), transmembrane protein 182 (TMEM182), myomesin 2 (MYOM2), leucyl and cystinyl aminopeptidase (LNPEP), actinin alpha 2 (ACTN2), sorbin and SH3 domain containing 1 (SORBS1), ankyrin 3 (ANK3), sarcoglycan delta (SGCD), and ORAI calcium release-activated calcium modulator 1 (ORAI1) exhibited consistent expression patterns with MEF2A during embryonic muscle development. Finally, TMEM182, as an important negative regulator of muscle development, has been validated to be regulated by MEF2A by dual-luciferase and quantitative real-time PCR (qPCR) assays. In summary, our study for the first time provides a wide landscape of MEF2A target genes in chicken primary myoblasts, which supports the active role of MEF2A in chicken muscle development.

A Study of transcription factor MEF2A gene polymorphisms in patients with coronary artery disease.

MEF2A (myocyte enhancer factor-2A) is a transcription factor of the MEF2 family. It has been recognized as the cause of coronary artery disease in the absence of any other clinical characteristic. It is involved in vascular development and is most commonly found in the coronary artery endothelium. The goal of this case-control study was to see if there was a link between polymorphisms in the MEF2A gene and coronary artery disease. A case-control study was carried out to look into the possible significance of MEF2A polymorphisms as a risk factor for coronary artery disease. This research included 225 patients and 225 healthy controls. A biochemical examination was carried out to evaluate the risk factors for developing this condition. The polymorphisms of Mef2A (1250 C > T in exon 8 and 452 G > T, 481 A > G in exon 11) were found using the PCR-RFLP technique. All identified risk variables, such as hypercholesterolemia, diabetes mellitus, and hypertriglyceridemia, were shown to be statistically significant in the current study for coronary artery disease occurrence. The most polymorphisms were found in MEF2A 1250 C > T, MEF2A 452 G > T, and MEF2A 481 A > G. The genotyping results for MEF2A 1250, MEF2A 452, and MEF2A 481 were (X2 = 2.985; P = 0.235), (X2 = 4.371; P = 0.112), and (X2 = 4.025; P = 0.134), respectively. In conclusion, we identified a much higher incidence of MEF2A in people with coronary artery disease, and MEF2A may play a crucial role in cardiovascular pathophysiology. Patients and controls had considerably different genetics and frequency of alleles.

GRK5 promoted renal fibrosis via HDAC5/Smad3 signaling pathway.

Renal fibrosis is a common pathological feature of chronic kidney diseases (CKD), poses a significant burden in the aging population, and is a major cause of end-stage renal disease (ESRD). In this study, we investigated the role of G protein-coupled receptor kinases (GRKs) 5 in the pathogenesis of renal fibrosis. GRK5 is a serine/threonine kinase that regulates G protein-coupled receptor (GPCR) signaling. GRK5 has been shown to play a role in various diseases including cardiac disorders and cancer. However, the role of GRK5 in renal fibrosis remains largely unknown. Our finding revealed that GRK5 was significantly overexpressed in renal fibrosis. Specifically, GRK5 was transferred into the nucleus via its nuclear localization sequence to regulate histone deacetylases (HDAC) 5 expression under renal fibrosis. GRK5 acted as an upstream regulator of HDAC5/Smad3 signaling pathway. HDAC5 regulated and prevented the transcriptional activity of myocyte enhancer factor 2A (MEF2A) to repress the transcription of Smad7 which leading to the activation of Smad3. These findings first revealed that GRK5 may be a potential therapeutic target for the treatment of renal fibrosis. Inhibition of GRK5 activity may be a promising strategy to attenuate the progression of renal fibrosis.

Microbiome analysis reveals the intestinal microbiota characteristics and potential impact of Procambarus clarkii

The intestinal microbiota interacts with the host and plays an important role in the immune response, digestive physiology, and regulation of body functions. In addition, it is also well documented that the intestinal microbiota of aquatic animals are closely related to their growth rate. However, whether it resulted in different sizes of crayfish in the rice-crayfish coculture model remained vague. Here, we analyzed the intestinal microbiota characteristics of crayfish of three sizes in the same typical rice-crayfish coculture field by high-throughput sequencing technology combined with quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme activity, investigating the relationship between intestinal microbiota in crayfish and water and sediments. The results showed that the dominant intestinal microbiota of crayfish was significantly different between the large size group (BS), normal size group (NS), and small size group (SS), where Bacteroides and Candidatus_Bacilloplasma contributed to the growth of crayfish by facilitating food digestion through cellulolysis, which might be one of the potential factors affecting the difference in sizes. Follow-up experiments confirmed that the activity of lipase (LPS) and protease was higher in BS, and the relative expression of development-related genes, including alpha-amylase (α-AMY), myocyte-specific enhancer factor 2a (MEF2a), glutathione reductase (GR), chitinase (CHI), and ecdysone receptor (EcR), in BS was significantly higher than that in SS. These findings revealed the intestinal microbiota characteristics of crayfish of different sizes and their potential impact on growth, which is valuable for managing and manipulating the intestinal microbiota in crayfish to achieve high productivity in practice.Key points• Significant differences in the dominant microflora of BS, NS, and SS in crayfish.• Cellulolysis might be a potential factor affecting different sizes in crayfish.• Adding Bacteroides and Candidatus_Bacilloplasma helped the growth of crayfish.

Twist-related protein 1 promotes transforming growth factor β receptor 1 in keloid fibroblasts via regulating the stability of myocyte enhancer factor 2A.

Keloid scarring is caused by a fibroproliferative disorder due to abnormal activation of genes, the underlying mechanism of which is still unclear. The basic helix-loop-helix transcription factor Twist-related protein 1 (TWIST1) controls cell proliferation and differentiation in tissue development and disease processes. In this study, we aimed to clarify the essential role of TWIST1 in the pathogenesis of keloids. Immunohistochemistry, cell counting kit-8 assays, western blotting, PCR, matrigel invasion assays and immunofluorescence assays were applied to demonstrate the effects and mechanisms of TWIST1 in fibroblasts derived from normal skin and keloids. Mass spectrometry, ubiquitination assays, chromatin immunoprecipitation and dual luciferase reporter assay were applied to explore the interaction of TWIST1 with downstream molecules. In the present study, we confirmed that TWIST1 was upregulated in keloid tissue of patients and in keloid-derived fibroblasts (KFBs). In vitro, TWIST1 inhibition prevented KFB proliferation, invasion and activation. We also discovered a link between TWIST1 and the transforming growth factor β (TGF-β) signaling related molecules TGF-β receptor 1 (TΒR1), SMAD family member 2 (Smad2) and Smad3, and the fibrosis markers α-smooth muscle actin, collagen type I and collagen type III in KFBs. Mechanistically, we uncovered a brand-new mechanism by which TWIST1 interacts with myocyte enhancer factor 2A (MEF2A) and suppresses its ubiquitination and degradation. Using chromatin immunoprecipitation and dual-luciferase reporter assay, TΒR1 was identified as a novel downstream target of MEF2A, which directly binds to its promoter. Overexpression of TWIST1 promoted the recruitment of MEF2A to the TΒR1 promoter and restored TΒR1 functional expression. Our research highlights a significant function of TWIST1 in the development of keloid and its related fibroblasts, partially facilitated by elevated MEF2A-dependent TΒR1 expression. Blocking the expression of TWIST1 in KFBs could potentially pave a novel therapeutic avenue for keloid treatment.

Effect of MEF2A and SLC22A3-LPAL2-LPA gene polymorphisms on warfarin sensitivity and responsiveness in Jordanian cardiovascular patients.

This study aims to investigate the influence of MEF2A and SLC22A3-LPAL2-LPA polymorphisms on cardiovascular disease susceptibility and responsiveness to warfarin medication in Jordanian patients, during the initiation and maintenance phases of treatment. Several candidate genes have been reported to be involved in warfarin metabolism and studying such genes may help in finding an accurate way to determine the needed warfarin dose to lower the risk of adverse drug effects, resulting in more safe anticoagulant therapy. The study population included 212 cardiovascular patients and 213 healthy controls. Genotyping of MEF2A and SLC22A3-LPAL2-LPA polymorphisms was conducted to examine their effects on warfarin efficiency and cardiovascular disease susceptibility using PCR-based methods. One SNP (SLC22A3-LPAL2-LPA rs10455872) has been associated with cardiovascular disease in the Jordanian population, whereas the other SNPs in the MEF2A gene and SLC22A3-LPAL2-LPA gene cluster did not have any significant differences between cardiovascular patients and healthy individuals. Moreover, SLC22A3-LPAL2-LPA rs10455872 was correlated with moderate warfarin sensitivity, the other SNPs examined in the current study have not shown any significant associations with warfarin sensitivity and responsiveness. Our data refer to a lack of correlation between the MEF2A polymorphism and the efficacy of warfarin treatment in both phases of treatment, the initiation, and maintenance phases. However, only rs10455872 SNP was associated with sensitivity to warfarin during the initiation phase. Furthermore, rs3125050 has been found to be associated with the international normalized number treatment outcomes in the maintenance phase.

Gene Regulatory Networks Controlling Sinusoidal Hematopoietic Niche Function

The bone marrow sinusoidal vascular niche is important for HSC engraftment and regeneration of hematopoiesis after transplantation, but the epigenetic factors that control these biological functions are undefined. Here, we show that sinusoidal hematopoietic niche functions are controlled by gene regulatory networks (GRNs), involving transcription factors (TFs) that bind to enhancers to regulate corresponding target genes. To characterize sinusoidal niche specific transcriptional and chromatin accessibility states, we performed multimodal single nuclei RNA and ATAC-seq analysis on unsorted zebrafish embryos. We identified 4 niche clusters, including mesenchymal stem cells, fibroblasts, osteoblasts and sinusoidal endothelial cells. Differential RNA transcriptome analysis confirmed the identity of each niche cluster with canonical gene markers for each niche cluster. To examine differential transcriptional accessibility in zebrafish niche clusters, we aggregated co-accessible regions in cells from these clusters. Each cluster demonstrated a unique chromatin accessibility profile with differentially accessible regions. Differential motif activity based on chromatin accessibility analysis further exhibited unique TF motifs activity in sinusoidal endothelial cells. To identify potential regulatory TFs in sinusoidal vascular niche, we predicted TFs based on the TF binding sites accessibility and TF expression. Thus, we identified 3 specific expressed and enriched TFs for sinusoidal vascular niche, including Fli1 (Friend leukemia integration 1), Mef2a (Myocyte enhancer factor 2A) and Mef2c. Next, to investigate genomic regulatory regions that interact with TFs, in which control the transcription of their target genes, we performed random forests and regression trees machine learning methods to infer GRNs. We observed multiple active GRNs in sinusoidal vascular niche, which were predicted to essential for regulating hematopoietic niche development and HSC maintenance, including klf12, egfl7, ebf3 and ldb2. Finally, we examined whether these putative gene regulatory networks were conserved across species and tissue types. We used same approach (single-cell multiome sequencing) on E4-HUVEC cells, a transduced human umbilical vein endothelial cells (HUVEC) with the adenovirus E4ORF1 gene, which was a well-defined model of the human sinusoidal vascular niche. We likewise observed regulatory networks in HUVEC cells. As in zebrafish, GRNs in human sinusoidal cluster consisted of FLI1 and MEF2 family, regulatory regions and their target genes. Our findings reveal that unique sinusoidal vascular niche specific gene regulatory networks, including TFs, regulatory regions and target genes indicate the treatments targeting GRNs may help to facilitate reprogramming of sinusoidal endothelial niche to support HSC homing and regeneration after transplantation.

Anomalous right coronary artery originating from the aorta: a series of nine pediatric cases

BackgroundTo investigate the clinical manifestations, prognosis, and possibly related genes of anomalous right coronary artery originating from the aorta (ARCA-L) in children.MethodsThis case series study included pediatric patients diagnosed with ARCA-L at the Department of Cardiology in Beijing Children’s Hospital affiliated to Capital Medical University, between January 2017 and December 2019.ResultsNine pediatric patients (aged 3 months to 12 years, 4 boys) were included. Two cases presented with cardiac insufficiency as their primary manifestation, while the remaining seven had post-infection or post-exercise symptoms such as chest pain, chest tightness, long exhalation, lack of strength, and dizziness. Six patients displayed varying degrees of ST-T changes on the electrocardiograph, while two patients had a reduced left ventricular ejection fraction (LVEF) of 20-32% according to echocardiography. Multislice computed tomographic angiography confirmed the presence of ARCA-L in all patients. One patient underwent the unroofing technique. The remaining eight received conservative treatment. After a follow-up of 2–64 months, eight children had a good prognosis and survived. One child experienced sudden death due to aggravated heart failure. Whole exome sequencing revealed that one child tested negative, one had mutations in the RYR2 and LDB3 genes, and the remaining four patients had a mutation in the GDF1, LRP6, MEF2A, and KALRN genes, respectively.ConclusionsARCA-L in children might have a wide variation in clinical manifestations and a risk of sudden death. The occurrence of the disease might be associated with genetic defects.

Brusatol enhances MEF2A expression to inhibit RCC progression through the Wnt signalling pathway in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most aggressive subtype of kidney tumour with a poor prognosis and an increasing incidence rate worldwide. Brusatol, an essential active ingredient derived from Brucea javanica, exhibits potent antitumour properties. Our study aims to explore a novel treatment strategy for RCC patients. We predicted 37 molecular targets of brusatol based on the structure of brusatol, and MEF2A (Myocyte Enhancer Factor 2A) was selected as our object through bioinformatic analyses. We employed various experimental techniques, including RT-PCR, western blot, CCK8, colony formation, immunofluorescence, wound healing, flow cytometry, Transwell assays and xenograft mouse models, to investigate the impact of MEF2A on RCC. MEF2A expression was found to be reduced in patients with RCC, indicating a close correlation with MEF2A deubiquitylation. Additionally, the protective effects of brusatol on MEF2A were observed. The overexpression of MEF2A inhibits RCC cell proliferation, invasion and migration. In xenograft mice, MEF2A overexpression in RCC cells led to reduced tumour size compared to the control group. The underlying mechanism involves the inhibition of RCC cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) through the modulation of Wnt/β-catenin signalling. Altogether, we found that MEF2A overexpression inhibits RCC progression by Wnt/β-catenin signalling, providing novel insight into diagnosis, treatment and prognosis for RCC patients.

Serine deficiency exacerbates psoriatic skin inflammation by regulating S-adenosyl methionine-dependent DNA methylation and NF-κB signalling activation in keratinocytes.

Serine metabolism is crucial for tumour oncogenesis and immune responses. S-adenosyl methionine (SAM), a methyl donor, is typically derived from serine-driven one-carbon metabolism. However, the involvement of serine metabolism in psoriatic skin inflammation remains unclear. To investigate the association between serine metabolism and psoriatic skin inflammation. Clinical samples were collected from patients with psoriasis and the expression of serine biosynthesis enzymes was evaluated. The HaCaT human keratinocyte cell line was transfected with small interfering RNA (siRNA) of key enzyme or treated with inhibitors. RNA sequencing and DNA methylation assays were performed to elucidate the mechanisms underlying serine metabolism-regulated psoriatic keratinocyte inflammation. An imiquimod (IMQ)-induced psoriasis mouse model was established to determine the effect of the SAM administration on psoriatic skin inflammation. The expression of serine synthesis pathway enzymes, including the first rate-limiting enzyme in serine biosynthesis, phosphoglycerate dehydrogenase (PHGDH), was downregulated in the epidermal lesions of patients with psoriasis compared with that in healthy controls. Suppressing PHGDH in keratinocytes promoted the production of proinflammatory cytokines and enrichment of psoriatic-related signalling pathways, including the tumour necrosis factor-alpha (TNF-α) signalling pathway, interleukin (IL)-17 signalling pathway and NF-κB signalling pathway. In particular, PHGDH inhibition markedly promoted the secretion of IL-6 in keratinocytes with or without IL-17A, IL-22, IL-1α, oncostatin M and TNF-α (mix) stimulation. Mechanistically, PHGDH inhibition upregulated the expression of IL-6 by inhibiting SAM-dependent DNA methylation at the promoter and increasing the binding of myocyte enhancer factor 2A. Furthermore, PHGDH inhibition increased the secretion of IL-6 by increasing the activation of NF-κB via SAM inhibition. SAM treatment effectively alleviated IMQ-induced psoriasis-like skin inflammation in mice. Our study revealed the crucial role of PHGDH in antagonising psoriatic skin inflammation and indicated that targeting serine metabolism may represent a novel therapeutic strategy for treating psoriasis.

Evolutionary conserved circular MEF2A RNAs regulate myogenic differentiation and skeletal muscle development.

Circular RNAs (circRNAs) have been recognized as critical regulators of skeletal muscle development. Myocyte enhancer factor 2A (MEF2A) is an evolutionarily conserved transcriptional factor that regulates myogenesis. However, it remains unclear whether MEF2A produces functional circRNAs. In this study, we identified two evolutionarily conserved circular MEF2A RNAs (circMEF2As), namely circMEF2A1 and circMEF2A2, in chicken and mouse muscle stem cells. Our findings revealed that circMEF2A1 promotes myogenesis by regulating the miR-30a-3p/PPP3CA/NFATC1 axis, whereas circMEF2A2 facilitates myogenic differentiation by targeting the miR-148a-5p/SLIT3/ROBO2/β-catenin signaling pathway. Furthermore, in vivo experiments demonstrated that circMEF2As both promote skeletal muscle growth. We also discovered that the linear MEF2A mRNA-derived MEF2A protein binds to its own promoter region, accelerating the transcription of MEF2A and upregulating the expression of both linear MEF2A and circMEF2As, forming a MEF2A autoregulated positive feedback loop. Moreover, circMEF2As positively regulate the expression of linear MEF2A by adsorbing miR-30a-3p and miR-148a-5p, which directly contribute to the MEF2A autoregulated feedback loop. Importantly, we found that mouse circMEF2As are essential for the myogenic differentiation of C2C12 cells. Collectively, our results demonstrated the evolution, function, and underlying mechanisms of circMEF2As in animal myogenesis, which may provide novel insight for both the farm animal meat industry and human medicine.

HDAC5 inhibition attenuates ventricular remodeling and cardiac dysfunction

BackgroundThis study aimed to investigate the role of histone deacetylase 5 (HDAC5) in ventricular remodeling and explore the therapeutic potential of the HDAC5 inhibitor LMK235.MethodsA transverse aortic constriction (TAC) mouse model and angiotensin II (Ang II)-treated H9C2 cells were used to evaluate the effects of HDAC5 inhibition with LMK235 on ventricular remodeling and cardiac dysfunction. Additionally, the involvement of the extracellular signal-regulated kinase (ERK)/early growth response protein 1 (EGR1) signaling pathway in regulating myocyte enhancer factor 2 A (MEF2A) expression was assessed.ResultsHDAC5 was upregulated in TAC mice and Ang II-treated H9C2 cells, suggesting its involvement in ventricular remodeling and cardiac dysfunction. LMK235 treatment significantly improved cardiac function in TAC mice and attenuated TAC-induced ventricular remodeling and Ang II-induced H9C2 cell hypertrophy. Mechanically, HDAC5 inhibition activated the ERK/EGR1 signaling pathway.ConclusionsOur findings demonstrate that HDAC5 may suppress the activation of ERK/EGR1 signaling to regulate MEF2A expression and therefore participate in cardiac pathophysiology.

The effect of feeding with different protein levels on internal organ weight and gene expression of MEF2A and ATF3 in crossbred local chicken using RT-PCR

BackgroundMyogenic enhancer transcription factor 2A (MEF2A) is a transcription factor known for its role in controlling skeletal muscle regeneration and metabolic processes, while activating transcription factor 3 (ATF3) is a stress-induced transcription factor that plays a role in modulating metabolic processes, immunity, and oncogenesis. Environmental factors, such as dietary protein, can influence gene expression levels. Insufficient protein intake can negatively affect the metabolic performance of internal organs, leading to the abnormal weight of internal organs. A total of 192 non-sexing crossbred local chickens day-old-chick (DOC) with a completely randomized design (CRD) method of 3 treatments and 8 replicates. Real-time polymerase chain reaction (RT-PCR) is used to measure the gene expression levels. This study aimed to determine the effect of feeding with various protein levels on internal organ weight and gene expression of MEF2A and ATF3 in crossbred local chickens. ResultThe analysis of treatment revealed that the results were not significantly different (P > 0.05) on gizzard weight and spleen weight. However, it shows a significantly different result (P < 0.05) on heart weight and a highly significantly different result (P < 0.01) on pancreas weight. These findings suggest that protein levels in the diet had a significant impact on heart and pancreas weights. In terms of gene expression, the increased utilization of protein did not result in an elevation of MEF2A gene expression in both muscle tissue and liver tissue. Specifically, in muscle tissue, MEF2A gene expression was highly expressed at 18% protein feed for the starter phase and 16% for the finisher phase. Conversely, in liver tissue, MEF2A gene expression was highly expressed at 22% protein feed for the starter phase and 20% for the finisher phase. Moreover, ATF3 gene expression in muscle tissue exhibited a negative correlation with increasing feed protein levels. ConclusionThe results indicate that varying protein levels did not lead to abnormal weights in the liver, kidney, heart, and spleen organs. Additionally, the differential gene expression patterns of MEF2A and ATF3 in muscle tissue and liver tissue suggest that these genes respond differently to varying protein-feeding treatments. These findings provide insights into the complex regulatory mechanisms of MEF2A and ATF3 genes in relation to protein levels and organ-specific responses in crossbred local chickens.

Effect of Bovine MEF2A Gene Expression on Proliferation and Apoptosis of Myoblast Cells.

Myocyte enhancer factor 2A (MEF2A) is a member of the myocyte enhancer factor 2 family. MEF2A is widely distributed in various tissues and organs and participates in various physiological processes. This study aimed to investigate the effect of MEF2A expression on the proliferation and apoptosis of bovine myoblasts. CCK8, ELISA, cell cycle, and apoptosis analyses were conducted to assess cell status. In addition, the mRNA expression levels of genes associated with bovine myoblast proliferation and apoptosis were evaluated using RT-qPCR. The results showed that the upregulation of MEF2A mRNA promoted the proliferation rate of myoblasts, shortened the cycle process, and increased the anti-apoptotic rate. Furthermore, the RT-qPCR results showed that the upregulation of MEF2A mRNA significantly increased the cell proliferation factors MyoD1 and IGF1, cell cycle factors CDK2 and CCNA2, and the apoptotic factors Bcl2 and BAD (p < 0.01). These results show that the MEF2A gene can positively regulate myoblast proliferation and anti-apoptosis, providing a basis for the analysis of the regulatory mechanism of the MEF2A gene on bovine growth and development.

DIPG-46. TARGETING THE METABOLIC ERK5-PFKFB3 AXIS IN PEDIATRIC DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma is an aggressive brain tumor with a median age at diagnosis of 6.3 years and 5-year survival rate of 2.2%.The defining histone mutation H3K27M precludes docking of a protein responsible for epigenetic modification resulting in erroneously accessible chromatin and subsequent transcription of oncogenic pathways, including the RAS-MERK5-ERK5 signaling cascade. To determine which oncogenic processes are regulated by extracellular signal-regulated kinase 5 (ERK5), gene-set enrichment analysis of patient-derived datasets demonstrated gene networks involved in glycolysis to be enriched with ERK5 expression. In confirmation, loss of ERK5 via shRNA interference reduced cell proliferation and glycolysis in DIPG IV and SF8628 cells. Reintroduction of ERK5 wildtype (WT) into ERK5 knockdown lines rescued cell proliferation and glycolysis, while the addition of ERK5 kinase dead domain (KDD) only partially rescued these survival and metabolic defects. Targeted evaluation of glycolysis enzyme expression via qRT-PCR revealed a direct relationship between ERK5 and proglycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Mechanistically, ERK5 activation of PFKFB3 was mediated by activation of transcriptional factor myocyte enhancer factor 2A (MEF2A) as demonstrated by coimmunoprecipitation and luciferase promoter assays. Expression of PFKFB3 was elevated at both the mRNA and protein level in DMG patient-derived samples. Genetic knockdown of PFKFB3 via shRNA interference resulted in reduced proliferation and glycolysis in DIPG IV and SF8628 cells. Similarly, pharmacologic inhibition of PFKFB3 with small molecule inhibitor PFK-158 capitulated these results. This inhibitor demonstrated blood brain barrier penetrance in silico and extended survival of in vivo mouse models. Multitargeted drug therapy against both ERK5 and PFKFB3 produced a synergetic in vitro response with increased sensitivity of these cells to apoptosis compared to single treatment alone. In conclusion, these results support ERK5 regulation of glycolysis through the critical metabolic effector PFKFB3. Multitargeted drug therapy against this axis represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Myocyte Enhancer Factor 2A Contributes to the TGF-β1-Mediated Cholangiocyte Epithelial to Mesenchymal Transition and Senescence in Cholestatic Liver Fibrosis.

Cholangiocytes are primary targets in chronic cholestatic liver diseases. Myocyte enhancer factor 2A (MEF2A) is a transcription factor with a crucial role in some fibrogenic diseases. However, whether it contributes to cholestatic liver fibrosis is still obscure. A bile duct-ligated (BDL) mouse model was established to detect MEF2A expression during cholestatic liver fibrosis. In addition, human intrahepatic biliary epithelial cells (HIBECs) were transfected with lentivirus-expressing shMEF2A (LV-shMEF2A) to regulate the expression of MEF2A in vitro. Biomarkers of epithelial to mesenchymal transition (EMT), senescence, and fibrogenesis were evaluated using various assays: Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, senescence-associated β-galactosidase (SA-β-gal), and immunofluorescence. Furthermore, MEF2A expression and cytoplasm translocation induced by transforming growth factor β1 (TGF-β1) in HIBECs were determined by qRT-PCR, western blotting, and immunofluorescence. The expression of TGF-β1-induced MEF2A, EMT, senescence, and fibrosis markers inhibited by p38 MAPK signaling were evaluated by western blotting. Finally, the peripheral blood from primary biliary cholangitis (PBC) patients and healthy controls (HCs) was collected to analyze expression of MEF2A using Enzyme-linked immunosorbent assay (ELISA). We found that MEF2A expression increased in liver tissues of BDL mice, and positively related to the extent of fibrosis. Silencing MEF2A in HIBECs restrained TGF-β1-induced EMT, senescence, and fibrotic reaction. Moreover, TGF-β1 enhanced the expression of MEF2A and induced its cytoplasm translocation in a concentration- and time-dependent manner, partially through interacting with p38 MAPK. The expression of MEF2A was also higher in the serum of PBC patients than in HCs, and positively correlated with fibrosis degree. Our study demonstrates that MEF2A is a central mediator linking TGF-β1-induced EMT and senescence in HIBECs. We propose it as a novel biomarker of fibrogenesis in cholestatic liver fibrosis. We also suggest inhibiting MEF2A as a potential strategy in treating cholestatic liver fibrosis.

Differential protein expression studies in hypercholesterolemic rabbits using aqueous terminalia arjuna extract

Background and Aims : Atherosclerosis is the major cause of mortality worldwide. Much attention has been focused on the use of herbal products to cure this disease. Hence the present study was designed to evaluate the cardio-protective effect of aqueous Terminalia arjuna extract (TAE) on different proteins in hypercholesterolemic rabbits using proteomic approach.Methods: Five groups of rabbits were employed and fed normal chow-fed diet (Gp 1), high-fat diet (Gp 2), normal chow-fed diet plus TAE (Gp 3), high fat diet plus TAE (Gp 4) and high-fat diet plus atorvastatin (Gp 5) respectively for 6 months. Aortic lesions were excised and protein lysate was separated by Two-dimensional gel electrophoresis. Differentially expressed proteins were identified by MALDI-TOF in Linear Reflectron Mass Spectrometer.Results: 850 spots could be detected among which 79 spots (p<0.05) matched among all 5 groups and 58 individual proteins were identified from 79 spots. Proteins like Interstitial collagenase, GRB2-related adapter protein 2, Interleukin 2, Myocyte enhancer-factor 2A, Protein S100-A9 and HSP60 with >10 fold expression were found to be differentially and significantly upregulated in Gp 2. TAE and atorvastatin treatment reduced the expression of these proteins to almost basal level (<1 fold change). TAE also significantly (p<0.05) downregulated expression of 5-lipoxygenase-activating protein, 72 kDa type IV collagenase, HSP90-alpha, and Vimentin proteins. Compared to atorvastatin, TAE was significantly more effective in downregulating all these proteins implicated in atherosclerotic disease.Conclusions: This study reveals that Terminalia arjuna is a potent downregulator of various atherosclerosis-related proteins, hence should be explored in future clinical trials. Background and Aims : Atherosclerosis is the major cause of mortality worldwide. Much attention has been focused on the use of herbal products to cure this disease. Hence the present study was designed to evaluate the cardio-protective effect of aqueous Terminalia arjuna extract (TAE) on different proteins in hypercholesterolemic rabbits using proteomic approach. Methods: Five groups of rabbits were employed and fed normal chow-fed diet (Gp 1), high-fat diet (Gp 2), normal chow-fed diet plus TAE (Gp 3), high fat diet plus TAE (Gp 4) and high-fat diet plus atorvastatin (Gp 5) respectively for 6 months. Aortic lesions were excised and protein lysate was separated by Two-dimensional gel electrophoresis. Differentially expressed proteins were identified by MALDI-TOF in Linear Reflectron Mass Spectrometer. Results: 850 spots could be detected among which 79 spots (p<0.05) matched among all 5 groups and 58 individual proteins were identified from 79 spots. Proteins like Interstitial collagenase, GRB2-related adapter protein 2, Interleukin 2, Myocyte enhancer-factor 2A, Protein S100-A9 and HSP60 with >10 fold expression were found to be differentially and significantly upregulated in Gp 2. TAE and atorvastatin treatment reduced the expression of these proteins to almost basal level (<1 fold change). TAE also significantly (p<0.05) downregulated expression of 5-lipoxygenase-activating protein, 72 kDa type IV collagenase, HSP90-alpha, and Vimentin proteins. Compared to atorvastatin, TAE was significantly more effective in downregulating all these proteins implicated in atherosclerotic disease. Conclusions: This study reveals that Terminalia arjuna is a potent downregulator of various atherosclerosis-related proteins, hence should be explored in future clinical trials.

Tissue Expression Analysis, Cloning, and Characterization of the 5'-Regulatory Region of the Bovine LATS1 Gene.

As a member of the large tumor suppressor (LATS) gene family, LATS1 plays an important role in regulating muscle growth and development. In this study, we determined the distinct exhibit patterns of tissue expression of bovine LATS1. Further, we determined the functional proximal minimal promoter of bovine LATS1 and identified the key transcription factors in the core promoter region to elucidate its molecular regulation mechanism. The results showed that bovine LATS1 was highly expressed in the longissimus thoracis and upregulation in infancy muscle. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with site-directed mutation and small interfering RNA (siRNA) interference demonstrated that myogenic differentiation 1 (Myod1) and myocyte enhancer factor 2A (MEF2A) binding in the core promoter region (−298/−123 bp) play important roles in the transcriptional regulation of the bovine LATS1 promoter. Taken together, these interactions provide insight into the regulatory mechanisms of LATS1 transcription in mediating skeletal muscle growth in cattle.