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Myocardial infarction, a coronary heart disease, is a serious hazard to human health. Cardiomyocyte oxidative stress and apoptosis have been considered as the main causes of myocardial infarction. Here, we aimed to investigate the role of miR-615-3p in oxidative stress and apoptosis of human cardiomyocytes. Reverse transcription-quantitative polymerase chain reaction was performed to determine miR-615-3p or MEF2A expression in human cardiomyocytes. Apoptosis and viability of human cardiomyocytes were assessed by flow cytometry analysis and CCK-8 assay. In addition, the contents of malondialdehyde, reactive oxygen species, and superoxide dismutase were detected by corresponding commercial kits. The binding of miR-615-3p and MEF2A in human cardiomyocytes was examined by luciferase reporterassay. Hypoxia/reoxygenation treatment downregulated the expression level ofmiR-615-3p in human cardiomyocytes. Overexpressing miR-615-3p increased human cardiomyocyte viability and decreased human cardiomyocyte apoptosis. Moreover, miR- 615-3p mimics suppressed oxidative stress in hypoxia/reoxygenation-stimulated human cardiomyocytes. MEF2A was confirmed as a target gene of miR-615-3p and was highly expressed in hypoxia/reoxygenation-stimulated human cardiomyocytes, and its upregu-lation partially reversed the influence of miR-615-3p mimics on oxidative stress and apop-tosis of human cardiomyocytes. Moreover, miR-615-3p inactivated the P13K/Akt pathway by inhibiting MEF2A. Overexpression of miR-615-3p protects human cardiomyocytes from oxida-tive stress injury by targeting MEF2A via the PI3K/Akt signaling.

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.

Transforming growth factor-β (TGF-β) signaling plays a key role in excessive fibrosis. As a class IIa family histone deacetylase (HDAC), HDAC5 shows a close relationship with TGF-β signaling and fibrosis. However, the effect and regulatory mechanism of HDAC5 in hypertrophic scar (HS) formation remain elusive. We show that HDAC5 was overexpressed in HS tissues and depletion of HDAC5 attenuated HS formation and inhibited fibroblast activation . HDAC5 knockdown (KD) significantly downregulated TGF-β1 induced Smad2/3 phosphorylation and increased Smad7 expression. Meanwhile, Smad7 KD rescued the Smad2/3 phosphorylation downregulation and scar hyperplasia inhibition mediated by HDAC5 deficiency. Luciferase reporter assays and ChIP-qPCR assays revealed that HDAC5 interacts with myocyte enhancer factor 2A (MEF2A) suppressing MEF2A binding to the Smad7 promoter region, which results in Smad7 promoter activity repression. HDAC4/5 inhibitor, LMK235, significantly alleviated hypertrophic scar formation. Our study provides clues for the development of HDAC5 targeting strategies for the therapy or prophylaxis of fibrotic diseases.

Angiogenesis is a pivotal mechanism driving tumor proliferation, and the epigenetic regulation of angiogenesis represents a cutting-edge area of current research in multiple myeloma (MM). High-throughput sequencing was carried out to detect the cargos of exosomes from clinical serum and U266 cells, then GSE108824 database was analyzed for the finding of differentially expressed genes (DEGs). The intersect set was made based on the three gene sets. The clinical features of Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1)were verified through GEO and clinicopathological data analyses. Cell viability, tube formation assay, level of MALAT1 and VEGFA were used to evaluate the effect of U266 exosome pretreated with or without paeoniflorin (PF) on angiogenesis in HUVEC cells. Subcutaneous tumor-bearing mice were established by injection of U266 cells and exosomes derived from U266 cells which pretreated with or without PF. Tumor size, HE staining, analysis of MALAT1 and VEGFA levels, as well as IHC staining for VEGFA, CD31, and Ki67 were performed to evaluate the in vivo effects of PF. The interactions between MALAT1, VEGFA, and microRNAs were demonstrated by TargetScan, MiRanda databases and Luciferase reporter assay. Furthermore, network pharmacology and RROMO, Genecards, AnimalTFDB, JASPAR databases were combinedto predict transcription factors (TF) associated with MALAT1 and analyze the binding sites between PF and these transcription factors. The validation of PF effect on TF was conducted by WB and PCR. Clinical studies indicated a notable positive correlation between MALAT1 level and VEGFA, CD31 expression, moreover, the high MALAT1 level is closely related to poor prognosis of MM. We demonstrated that MALAT1 was the highest expression linear RNA in U266 exosomes and could be transported to HUVEC cells through exosomes, promoting HUVEC cells differentiation and angiogenesis by stimulating VEGFA expression. The tube formation could be blocked if we knockdown the MALAT1 in U266 exosome. It was also proved that this pathological process can be blocked by PF in vitro and in vivo experiments. The ceRNA mechanism in MALAT1/miR-17/VEGFA was predicted and then confirmed by luciferase reporter assay. 2548 PF target genes were retrieved from databases, and the intersections with MALAT1-related differentially expressed proteins, mRNA and TF gene were identified Venn diagaram. MEF2A binding sites were predicted JASPAR, finally molecular docking showed strong affinity between PF and MEF2A (-16.5 kcal/mol).Then the effect of PF on MEF2A/MALAT1 was confirmed by WB or PCR test. To summarize, our study revealed that myeloma cells can increase angiogenesis by releasing exosome to influence the endothelial cells. The MALAT1 from myeloma cells is the crucial factor in this pathological process. PF can obstruct this process by intervening in the MEF2A/MALAT1 in myeloma cells.

Background The high mutation rate throughout the entire melanoma genome presents a major challenge in stratifying true driver events from the background mutations. Numerous recurrent non-coding alterations, such as those in enhancers, can shape tumor evolution, thereby emphasizing the importance in systematically deciphering enhancer disruptions in melanoma. Results Here, we leveraged 297 melanoma whole-genome sequencing samples to prioritize highly recurrent regions. By performing a genome-scale CRISPR interference (CRISPRi) screen on highly recurrent region-associated enhancers in melanoma cells, we identified 66 significant hits which could have tumor-suppressive roles. These functional enhancers show unique mutational patterns independent of classical significantly mutated genes in melanoma. Target gene analysis for the essential enhancers reveal many known and hidden mechanisms underlying melanoma growth. Utilizing extensive functional validation experiments, we demonstrate that a super enhancer element could modulate melanoma cell proliferation by targeting MEF2A , and another distal enhancer is able to sustain PTEN tumor-suppressive potential via long-range interactions. Conclusions Our study establishes a catalogue of crucial enhancers and their target genes in melanoma growth and progression, and illuminates the identification of novel mechanisms of dysregulation for melanoma driver genes and new therapeutic targeting strategies.

Transcriptional regulators encoded by the M yocyte E nhancer F actor 2 (MEF2) gene family play a fundamental role in cardiac development, homeostasis and pathology. Previous studies indicate that MEF2A protein-protein interactions serve as a network hub in several cardiomyocyte cellular processes. Based on the idea that interactions with regulatory protein partners underly the diverse roles of MEF2A in cardiomyocyte gene expression, we undertook a systematic unbiased screen of the MEF2A protein interactome in primary cardiomyocytes using an affinity purification-based quantitative mass spectrometry approach. Bioinformatic processing of the MEF2A interactome revealed protein networks involved in the regulation of programmed cell death, inflammatory responses, actin dynamics and stress signaling in primary cardiomyocytes. Further biochemical and functional confirmation of specific protein-protein interactions documented a dynamic interaction between MEF2A and STAT3 proteins. Integration of transcriptome level data from MEF2A and STAT3-depleted cardiomyocytes reveals that the balance between MEF2A and STAT3 activity exerts a level of executive control over the inflammatory response and cardiomyocyte cell survival and experimentally ameliorates Phenylephrine induced cardiomyocyte hypertrophy. Lastly, we identified several MEF2A/STAT3 co-regulated genes, including the MMP9 gene. Herein, we document the cardiomyocyte MEF2A interactome, which furthers our understanding of protein networks involved in the hierarchical control of normal and pathophysiological cardiomyocyte gene expression in the mammalian heart.

Exercise plays an important role in cardiac health and enhances the transport of glucose in cardiac muscle by increasing the glucose transporter-4 (GLUT4) content at the cell membrane. The GLUT4 gene is a target of myocyte enhancer transcription factor 2A (MEF2A). Several transcription factors are regulated by microRNAs (miRs), small non-coding RNAs that control gene expression at the posttranscriptional level. In this study we tested the hypothesis that exercise regulates the expression of miR-223 and that MEF2A is a direct target of miR-223. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that GLUT4 gene expression and protein abundance increased by 30 and 23%, respectively, in the microsomal fraction immediately after exercise, and had returned to control levels after 18 h. In contrast, the increase in GLUT4 in the membrane fraction was delayed. Exercise also increased the protein abundance of transcription factors involved in GLUT4 expression. Immediately after exercise, the protein abundance of MEF2A, nuclear respiratory factor 1 (NRF1), and forkhead box O1 (FOXO1) increased by 18, 30, and 40%, respectively. qRT-PCR experiments showed that miR-223-3p and miR-223-5p expression decreased immediately after exercise by 60 and 30%, respectively, and luciferase assays indicated that MEF2A is a target of the 5p strand of miR-223. Overexpression of miR-223-5p in H9c2 cells decreased the protein abundance of MEF2A. Our results suggest that the exercise-induced increase in GLUT4 content in cardiac muscle is partly due to the posttranscriptional increase in MEF2A protein abundance caused by the decrease in miR-223-5p expression. The exercise-induced decrease in miR-223-3p expression likely contributes to the increases in NRF1 and FOXO1 abundance and GLUT4 content.

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.

Background The myocyte enhancer factor-2 (MEF2) family genes were involved in the carcinogenesis and prognosis of multiple human tumors. The impact of MEF2s on the occurrences, progression, and clinical outcome of pancreatic cancer (PAAD) remains unknown. Methods This study used the CCLE, HPA, EMBL-EBI, and GEPIA2 databases to study MEF2s expression in PAAD patients. We also investigated the relationship between MEF2s expression and methylation through the DiseaseMeth database, and used MEXPRESS to verify the association. Then we utilized the Kaplan–Meier Plotter and GEPIA2 databases to evaluate the prognostic value of MEF2s in PAAD. The cBioPortal database was used to explore the alteration features of MEF2s in PAAD. We then investigated the association between MEF2s expression, immune cells infiltration, and immune infiltration markers using the TIMER database. Finally, Metascape, STRING, and Cytoscape tools were used for functional enrichment analysis. Results MEF2A, MEF2C, and MEF2D were found to be highly expressed in PAAD patients’ tissues compared to normal tissues, whereas MEF2B expression did not show significant differential expression. In addition, the protein expression of MEF2A, MEF2C, and MEF2D was higher in PAAD tissues. Negative correlations were observed between the expression level of MEF2A, MEF2C, and MEF2D and the methylation levels in multiple sites. High expression of MEF2A was related to poor overall survival ( p  = 0.0071) and relapse-free survival (RFS) ( p  = 0.0089) of PAAD. High expression of MEF2C was associated with worse RFS of PAAD ( p  = 0.043). MEF2A was a Truncating mutation, and it was shown that the “G27Wfs*8” mutation point was distributed in the SRF-TF domain. Both MEF2C and MEF2D were a Missense mutation. MEF2A, MEF2C, and MEF2D expression was positively corresponded with five immune cells infiltration (CD8 + T cells, B-cells, neutrophils, macrophages, and dendritic cells), especially for CD8 + T cells and macrophages. Among the 20 pathways, hsa05140 (Leishmania infection), hsa04022 (cGMP-PKG signaling pathway), hsa05145 (Toxoplasmosis), hsa04371 (Apelin signaling pathway), and hsa04064 (NF-kappa B signaling pathway), were closely connected with the occurrence and development of PAAD. Conclusions Our results indicated that the overexpression of MEF2A, MEF2C, and MEF2D in patients with PAAD. MEF2A could be used as a prognostic biomarker for PAAD, MEF2C might be a potential oncogene for PAAD, and MEF2D had potential biological significance.

Background Thrombosis or embolism causes blood flow stoppage, resulting in ischemic stroke (IS), the most frequent clinical cerebrovascular disorder. It accounts for more than 80% of all strokes and has a significant disability and mortality rate. Regulating critical targets to stop the ischemia pathophysiological cascade and preserve brain cells is essential for treating IS. Methods We included a deCODE analysis encompassing data on cis-acting variations for 4719 blood proteins. The IS summary statistics consist of 11,929 cases and 472,192 controls. We identified blood proteins associated with IS susceptibility using the Mendelian randomization (MR) method. We comprehensively analyzed the GSE58294 dataset, including differential expression gene identification, functional enrichment analysis, weighted gene co-expression network analysis, least absolute shrinkage and selection operator analysis, and transcription factor identification. The expression patterns of key genes were identified using single-cell sequencing data. The oxygen-glucose deprivation/reperfusion (OGD/R) model was established, and myocyte enhancer factor 2 A ( MEF2A ) was overexpressed in Neuro-2a/PC12 cells using an overexpression vector. We used CCK-8 and western blotting to observe the cell viability, oxidative stress level, and p-PI3K and p-Akt protein expression. Results We discovered 73 blood proteins related to IS, and the sensitivity analysis findings supported the causal associations. The MR results and bioinformatics underwent a thorough examination, revealing MEF2A as a transcription factor involved in the development of IS. Single-cell sequencing results revealed that MEF2A was mostly expressed in microglia and endothelial cells. Additional in vitro experiments showed that overexpression of MEF2A could significantly mitigate OGD/R-induced Neuro-2a/PC12 cell injury and oxidative stress, potentially associated with increased PI3K/Akt protein production. Conclusion This study, using MR analysis, transcriptomics, and in vitro experiments, identified the transcription factor MEF2A as a potential target for treatment. This gives preclinical evidence for drug development. Clinical trial number Not applicable.