RESUMO
The proportions of the various muscle fiber types are important in the regulation of skeletal muscle metabolism, as well as animal meat production. Four-and-a-half LIM domain protein 3 (FHL3) is highly expressed in fast glycolytic muscle fibers and differentially regulates the expression of myosin heavy chain (MyHC) isoforms at the cellular level. Whether FHL3 regulates the transformation of muscle fiber types in vivo and the regulatory mechanism is unclear. In this study, muscle-specific FHL3 transgenic mice were generated by random integration, and lentivirus-mediated gene knockdown or overexpression in muscles of mice or pigs was conducted. Functional analysis showed that overexpression of FHL3 in muscles significantly increased the proportion of fast-twitch myofibers and muscle mass but decreased muscle succinate dehydrogenase (SDH) activity and whole-body oxygen consumption. Lentivirus-mediated FHL3 knockdown in muscles significantly decreased muscle mass and the proportion of fast-twitch myofibers. Mechanistically, FHL3 directly interacted with the Yin yang 1 (YY1) DNA-binding domain, repressed the binding of YY1 to the fast glycolytic MyHC2b gene regulatory region, and thereby promoted MyHC2b expression. FHL3 also competed with EZH2 to bind the repression domain of YY1 and reduced H3K27me3 enrichment in the MyHC2b regulatory region. Moreover, FHL3 overexpression reduced glucose tolerance by affecting muscle glycolytic metabolism, and its mRNA expression in muscle was positively associated with hemoglobin A1c (HbA1c) in patients with type 2 diabetes. Therefore, FHL3 is a novel potential target gene for the treatment of muscle metabolism-related diseases and improvement of animal meat production.
Assuntos
Diabetes Mellitus Tipo 2 , Camundongos , Suínos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Glicólise/genética , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismoRESUMO
Our previous studies showed that SYISL is a negative regulator of muscle growth and regeneration in mice, pigs and humans. SYISL knockout resulted in an increase in the density of muscle fibers and muscle growth. However, it is unclear whether there are natural mutations in pig SYNPO2 intron sense-overlapping lncRNA (pSYISL) that affect the expression of pSYISL and muscle growth traits. In this study, three SNPs in exons and six SNPs within the promoter of pSYISL were identified. Association analysis showed that the two SNPs in exons are significantly associated with loin muscle area (p < 0.05); the six SNPs in the promoter that show complete linkage are significantly associated with live backfat thickness and live loin muscle area in American Large White pigs. Bioinformatics and luciferase reporter assays as well as in vitro binding experiments indicated that the mutation of SNP rs702045770 (g.539G>A) leads to the loss of YY1 binding to the promoter, thus affecting the expression level of pSYISL, and we found that Jiangshan Black pigs with genotype GG have a higher expression level of pSYISL than genotype AA individuals, but the muscle fiber density was significantly lower than in genotype AA individuals. Furthermore, the association analysis showed that the carcass backfat thickness of genotype GG of SNP rs702045770 was significantly higher than that of other genotypes in (Pietrain × Duroc) × (Landrace × Yorkshire) crossbred pigs (p < 0.05). The glycolytic potential of genotype GG was significantly higher than that of other genotypes (p < 0.05). These results provide novel insight into the identification of functional SNPs in non-coding genomic regions.
Assuntos
Fibras Musculares Esqueléticas , Polimorfismo de Nucleotídeo Único , Humanos , Suínos , Animais , Camundongos , Fenótipo , Genótipo , Regiões Promotoras GenéticasRESUMO
Long noncoding RNAs (lncRNAs) play important roles in the spatial and temporal regulation of muscle development and regeneration. Nevertheless, the determination of their biological functions and mechanisms underlying muscle regeneration remains challenging. Here, we identified a lncRNA named lncMREF (lncRNA muscle regeneration enhancement factor) as a conserved positive regulator of muscle regeneration among mice, pigs and humans. Functional studies demonstrated that lncMREF, which is mainly expressed in differentiated muscle satellite cells, promotes myogenic differentiation and muscle regeneration. Mechanistically, lncMREF interacts with Smarca5 to promote chromatin accessibility when muscle satellite cells are activated and start to differentiate, thereby facilitating genomic binding of p300/CBP/H3K27ac to upregulate the expression of myogenic regulators, such as MyoD and cell differentiation. Our results unravel a novel temporal-specific epigenetic regulation during muscle regeneration and reveal that lncMREF/Smarca5-mediated epigenetic programming is responsible for muscle cell differentiation, which provides new insights into the regulatory mechanism of muscle regeneration.
Assuntos
RNA Longo não Codificante , Adenosina Trifosfatases , Animais , Diferenciação Celular , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Humanos , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Regeneração , SuínosRESUMO
BACKGROUND: Diabetic retinopathy (DR), one of the most common microvascular complications of diabetes mellitus (DM), is a major contributor of vision impairment and blindness worldwide. Studies have shown that air pollution exposure is adversely associated with DM. However, evidence is scarce regarding how air pollution exposure affects DR. This study aimed to investigate the association between ambient air pollution exposure and DR risk. METHODS: The study population was based on the Fujian Eye Study (FJES), an ophthalmologic, epidemiologic survey investigating the eye health condition of residents in Fujian Province from 2018 to 2019. Daily average concentrations of ambient air pollutants (PM2.5, PM10, SO2, NO2, and O3) were acquired from a high-resolution air quality dataset in China from 2013 to 2018. We used a logistic regression model to examine the associations between DR risk and long-term air pollution at various exposure windows. RESULTS: A total of 2405 out of the 8211 participants were diagnosed with diabetes, among whom 183 had DR. Ambient air pollution, especially particulate matter (i.e., PM2.5 and PM10) and NO2 were positively associated with DR prevalence among all the study subjects. Ambient SO2 and O3 concentrations were not associated with DR prevalence. PM2.5 and NO2 seemed to be borderline significantly associated with increased prevalence of DR in subjects with DM, especially under the model adjusted for sex, age, BMI, SBP, and DBP. CONCLUSIONS: These findings showed that long-term exposure to ambient particulate matter and NO2 was associated with a high DR risk in Fujian province, where ambient air pollution is relatively low.
Assuntos
Poluentes Atmosféricos , Poluição do Ar , Retinopatia Diabética , Exposição Ambiental , Material Particulado , Humanos , Retinopatia Diabética/epidemiologia , Masculino , Poluição do Ar/efeitos adversos , Poluição do Ar/estatística & dados numéricos , China/epidemiologia , Feminino , Pessoa de Meia-Idade , Poluentes Atmosféricos/análise , Exposição Ambiental/estatística & dados numéricos , Exposição Ambiental/efeitos adversos , Idoso , Material Particulado/análise , Adulto , PrevalênciaRESUMO
The Brother of Regulator of Imprinted Sites (BORIS, gene symbol CTCFL) has previously been shown to promote colorectal cancer cell proliferation, inhibit cancer cell apoptosis, and resist chemotherapy. However, it is unknown whether Boris plays a role in the progression of in situ colorectal cancer. Here Boris knockout (KO) mice were constructed. The function loss of the cloned Boris mutation that was retained in KO mice was verified by testing its activities in colorectal cell lines compared with the Boris wild-type gene. Boris knockout reduced the incidence and severity of azoxymethane/dextran sulfate-sodium (AOM/DSS)-induced colon cancer. The importance of Boris is emphasized in the progression of in situ colorectal cancer. Boris knockout significantly promoted the phosphorylation of γH2AX and the DNA damage in colorectal cancer tissues and suppressed Wnt and MAPK pathways that are responsible for the callback of DNA damage repair. This indicates the strong inhibition of colorectal cancer in Boris KO mice. By considering that the DSS-promoted inflammation contributes to tumorigenesis, Boris KO mice were also studied in DSS-induced colitis. Our data showed that Boris knockout alleviated DSS-induced colitis and that Boris knockdown inhibited the NF-κB signaling pathway in RAW264.7 cells. Therefore Boris knockout eliminates colorectal cancer generation by inhibiting DNA damage repair in cancer cells and relieving inflammation in macrophages. Our findings demonstrate the importance of Boris in the development of in situ colorectal cancer and provide evidence for the feasibility of colorectal cancer therapy on Boris.
Assuntos
Colite , Neoplasias Colorretais , Animais , Masculino , Camundongos , Azoximetano/toxicidade , Colite/induzido quimicamente , Colite/genética , Colite/complicações , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/genética , Neoplasias Colorretais/tratamento farmacológico , Sulfato de Dextrana/toxicidade , Sulfato de Dextrana/uso terapêutico , Modelos Animais de Doenças , Dano ao DNA/genética , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle-specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer-binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca2+ in PPARγ pigs. Raising of Ca2+ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle-specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.
Assuntos
DNA Mitocondrial/metabolismo , Músculo Esquelético/metabolismo , PPAR gama/metabolismo , Adipócitos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Animais , Southern Blotting , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Variações do Número de Cópias de DNA/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Oxirredução , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Perilipina-1/genética , Perilipina-1/metabolismo , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , SuínosRESUMO
PURPOSE: G-protein coupled receptor (GPR 34) has been found to play important roles in some cancers and regulates the proliferation, apoptosis, and migration of these cancer cells. However, the mechanisms underlying how GPR34 functions to regulate growth and proliferation of colorectal cancer cells remains to be clarified. METHODS: We employed stable GPR34 knockdown LS174T cell models, GPR34 Mab blocking, a CCK-8 kit, and a colony formation assay to characterize the effect of GPR34 on the proliferation of LS174T in vitro and xenograft tumor growth in vivo. The mRNA level of GPR34 was detected by RT-PCR in tumor tissues and adjacent normal tissues from 34 CRC patients. RESULTS: Based on RT-PCR results, GPR34 exhibited high level in tumor samples compared with adjacent normal samples. Increased expression of GPR34 is more associated with poor prognosis of CRC as shown in The Cancer Genome Atlas (TCGA) dataset by Kaplan-Meier survival analysis. Furthermore, we showed that GPR34 knockdown inhibited the proliferation of LS174T colon cancer cells and related xenograft tumor growth. Searching for the distinct molecular mechanism, we identified several contributors to proliferation of LS174T colon cancer cells: PI3K subunits/PTEN, PDK1/AKT, and Src/Raf/Ras/ERK. GPR34 knockdown inhibited the proliferation of LS174T cells by upregulating expression of PTEN, and downregulating expression of PI3K subunits p110-beta. CONCLUSION: Our findings provide direct evidence that GPR34 regulates the proliferation of LS174T cells and the growth of LS174T tumor xenografts by regulating different pathways. High expression of GPR34 mRNA could then be used to predict poor prognosis of CRC.
Assuntos
Neoplasias Colorretais , Fosfatidilinositol 3-Quinases , Receptores de Lisofosfolipídeos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisofosfolipídeos/genética , Receptores de Lisofosfolipídeos/metabolismo , Transdução de Sinais/genéticaRESUMO
Although many long noncoding RNAs (lncRNAs) have been identified in muscle, their physiological function and regulatory mechanisms remain largely unexplored. In this study, we systematically characterized the expression profiles of lncRNAs during C2C12 myoblast differentiation and identified an intronic lncRNA, SYISL (SYNPO2 intron sense-overlapping lncRNA), that is highly expressed in muscle. Functionally, SYISL promotes myoblast proliferation and fusion but inhibits myogenic differentiation. SYISL knockout in mice results in significantly increased muscle fiber density and muscle mass. Mechanistically, SYISL recruits the enhancer of zeste homolog 2 (EZH2) protein, the core component of polycomb repressive complex 2 (PRC2), to the promoters of the cell-cycle inhibitor gene p21 and muscle-specific genes such as myogenin (MyoG), muscle creatine kinase (MCK), and myosin heavy chain 4 (Myh4), leading to H3K27 trimethylation and epigenetic silencing of target genes. Taken together, our results reveal that SYISL is a repressor of muscle development and plays a vital role in PRC2-mediated myogenesis.
Assuntos
Epigênese Genética , Desenvolvimento Muscular/fisiologia , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Inativação Gênica , Camundongos , Camundongos Knockout , Complexo Repressor Polycomb 2/genética , Regiões Promotoras Genéticas , RNA Longo não Codificante/genéticaRESUMO
In order to better understand the key regulatory mechanisms of PGC1α in muscle fiber type transition, the RNA-seq was used to compare the change of gene expression in gastrocnemius muscles between wild type pigs and transgenic pigs with overexpression of PGC1α gene in muscle. 371 differentially expressed genes (P ≤ 0.05 and Ratio ≥ 2), including 184 up-regulated genes and 187 down-regulated genes, were identified. Five main signaling pathways including metabolic pathways, ECM-receptor interaction, PPAR signaling pathway, adipocytokine signaling pathway and insulin signaling pathway, were authenticated using KEGG pathway analysis. Our results indicate that the fat metabolism pathway plays an important role in the transformation of muscle fiber types regulated by PGC1α.
Assuntos
Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Suínos/genética , Animais , Animais Geneticamente Modificados , Metabolismo dos Lipídeos , Carne/análise , Redes e Vias Metabólicas , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA/metabolismo , Transdução de Sinais , TranscriptomaRESUMO
BACKGROUND: The protective effect of Neuregulin-1 (NRG-1) on heart failure is well established. In this study, we assessed whether NRG-1 could protect the heart by mimicking the cardioprotective effects of ischaemic postconditioning (IP). METHODS: We used a myocardial reperfusion injury rat model in vivo to compare the cardioprotective effects of NRG-1(3 µg/kg, iv. at the onset of reperfusion) and IP. In Langendorff isolated heart perfusion experiments, we used the erythroblastic leukaemia viral oncogene homolog 4 (ErbB4) inhibitor AG1478, a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and a mitogen-activated protein/extracellular signal regulated kinase (MEK) inhibitor PD98059 to clarify whether the protective effects of NRG-1and IP depend on the NRG-1/ErbB4 signals and the reperfusion injury salvage kinase (RISK) pathway. Infarct size was detected by Evans blue and TTC. Apoptosis was detected by TUNEL assays. The expression of NRG-1/ErbB4 and downstream ERK1/2, AKT, AMPK and p70s6K were detected by western blotting. Hematoxylin/eosin (H&E) staining was used for histological analysis. RESULTS: We found that NRG-1 and IP had similar effects on reducing myocardial infarct size and apoptosis in vivo. NRG-1 heart protein levels were upregulated in the IP group. Phosphorylation of AKT, ERK1/2 and ErbB4 were also increased in both the IP and NRG-1 groups. Furthermore, in Langendorff analyses, the ErbB4 inhibitor AG1478 suppressed the phosphorylation of ErbB4 and the RISK pathway and aggravated myocardial edema and fiber fracture, thereby inhibited the cardioprotective effects in both the IP and NRG-1 groups. For assessment of downstream signals, the PI3K inhibitor LY294002 and the MEK inhibitor PD98059 suppressed the phosphorylation of AKT and ERK1/2 respectively and abolished the cardioprotective effects induced by IP and NRG-1. CONCLUSION: In conclusion, both IP and NRG-1 could reduce infarct size and apoptosis through ErbB4-dependent activation of the RISK pathway in the same model; these results indicated the therapeutic potential of NRG-1 as a pharmacological postconditioning agent against myocardial reperfusion injury.
Assuntos
Cardiotônicos/farmacologia , Pós-Condicionamento Isquêmico , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Neuregulina-1/farmacologia , Receptor ErbB-4/metabolismo , Animais , Apoptose/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Infarto do Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
BACKGROUND: We aimed to explore whether monocyte to lymphocyte ratio (MLR) provides predictive value of the lesion severity in patients with coronary artery disease (CAD). METHODS: Five hundred forty-three patients undergoing coronary angiography were analyzed in this retrospective study. Patients with coronary stenosis were divided into three groups on the basis of Syntax score. The control group consisted of patients with normal coronary arteries. MLR was calculated by dividing monocytes count by lymphocytes count obtained from routine blood examination. Multivariate logistic analysis was used to assess risk factors of CAD. Ordinal logistic regression analysis was used to assess the relationship between MLR and the lesion severity of coronary arteries. RESULTS: MLR was found to be an independent risk factor of the presence of CAD (OR: 3.94, 95% CI: 1.20-12.95) and a predictor of the lesion severity (OR: 2.05, 95% CI: 1.15-3.66). Besides, MLR was positively correlated with Syntax score(r = 0.437, p < 0.001). In the receiver-operating characteristic (ROC) curve analysis, MLR, with an optimal cut-off value of 0.25, predicted the severe coronary lesion with a sensitivity of 60.26% and specificity of 78.49%. CONCLUSIONS: MLR was an independent risk factor of the presence of CAD, and a predictor of the lesion severity. Compared to neutrophil to lymphocyte ratio (NLR), MLR has better performance to reflect the severity of coronary lesion.
Assuntos
Angiografia Coronária , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico por imagem , Estenose Coronária/sangue , Estenose Coronária/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Linfócitos , Monócitos , Idoso , Área Sob a Curva , Feminino , Humanos , Modelos Logísticos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Valor Preditivo dos Testes , Prognóstico , Curva ROC , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
The peroxisome proliferator-activated receptor gamma, co-activator 1 alpha(PGC1α) effectively induced the biosynthesis of the mitochondria and the energy metabolism, and also regulated the muscle fiber-type shift. Overexpression of PGC1α gene in mice led to higher oxidative muscle fiber composition in muscle. However, no researches about the significant differences of muscle fiber phenotype in pigs after PGC1α overexpression had been reported. The composition of muscle fiber-types which were distinguished by four myosin heavy chain(MYHC) isoforms, can significantly affect the muscle functions. In our study, we generated the transgenic pigs to investigate the effect of overexpression of PGC1α gene on muscle fiber-type conversion. The results showed that the number of oxidative muscle fiber(type1 muscle fiber) was increased and the number of glycolytic muscle fiber(type2b muscle fiber) was decreased in the transgenic pigs. Furthermore, we found that PGC1α overexpression up-regulated the expression of MYHC1 and MYHC2a and down-regulated the expression of MYHC2b.The analysis of genes expression demonstrated the main differentially expressed genes were MSTN, Myog and FOXO1. In conclusion, the overexpression of PGC1α gene can promote the glycolytic muscle fiber transform to the oxidative muscle fiber in pigs.
Assuntos
Diferenciação Celular/fisiologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Animais Geneticamente Modificados , Células Cultivadas , Fibras Musculares Esqueléticas/classificação , Cadeias Pesadas de Miosina/classificação , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Suínos/genética , Regulação para Cima/genéticaRESUMO
Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of â¼22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at both mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 2'-O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis.
Assuntos
Diferenciação Celular/fisiologia , MicroRNAs/fisiologia , Mioblastos/citologia , Fatores de Regulação Miogênica/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Mioblastos/metabolismo , Fatores de Regulação Miogênica/genética , Ligação ProteicaRESUMO
Fat-induced transcript 1 (FIT1/FITM1) gene is a member of the conserved gene family important for triglyceride-rich lipid droplet accumulation. FIT1 gene displays a similar muscle-specific expression across pigs, mice, and humans. Thus pigs can act as a useful model of many human diseases resulting from misexpression of FIT1 gene. Triglyceride content in skeletal muscle plays a key role in pork meat quality and flavors. An insertion/deletion mutation in porcine FIT1 coding region shows a high correlation with a series of fat traits. To gain better knowledge of the potential role of FIT1 gene in human diseases and the correlations with pork meat quality, our attention is given to the region upstream of the porcine FIT1 coding sequence. We cloned ~1 kb of the 5'-flanking region of porcine FIT1 gene to define the role of this sequence in modulating the myogenic expression. A canonical E-box element that activated porcine FIT1 promoter activity during myogenesis was identified. Further analysis demonstrated that promoter activity was induced by overexpression of MyoD1, which bound to this canonical E-box during C2C12 differentiation. This is the first evidence that FIT1 as the direct novel target of MyoD is involved in muscle development.
Assuntos
Elementos E-Box/fisiologia , Proteínas de Membrana/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Elementos E-Box/genética , Proteínas de Membrana/genética , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Suínos , Ativação TranscricionalRESUMO
Brown adipose tissue (BAT) is specialized to dissipate energy as heat, therefore reducing fat deposition and counteracting obesity. Brown adipocytes arise from myoblastic progenitors during embryonic development by the action of transcription regulator PRDM16 binding to PPARγ, which promotes BAT-like phenotype in white adipose tissue. To investigate the capability of converting white adipose tissue to BAT or browning by PPARγ in vivo, we generated transgenic mice with over-expressed PPARγ2. The transgenic mice showed strong brown fat features in subcutaneous fat in morphology and histology. To provide molecular evidences on browning characteristics of the adipose tissue, we employed quantitative real-time PCR to determine BAT-specific gene expressions. The transgenic mice had remarkably elevated mRNA level of UCP1, Elovl3, PGC1α and Cebpα in subcutaneous fat. Compared with wild-type mice, UCP1 protein levels were increased significantly in transgenic mice. ATP concentration was slightly decreased in the subcutaneous fat of transgenic mice. Western blotting analysis also confirmed that phosphorylated AMPK and ACC proteins were significantly (P<0.01) increased in the transgenic mice. Therefore, this study demonstrated that over-expression of PPARγ2 in skeletal muscle can promote conversion of subcutaneous fat to brown fat formation, which can have beneficial effects on increasing energy metabolisms and combating obesity.
Assuntos
Tecido Adiposo Marrom/crescimento & desenvolvimento , PPAR gama/genética , Regulação para Cima , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Trifosfato de Adenosina/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , PPAR gama/metabolismo , RNA Mensageiro/genética , Gordura Subcutânea/crescimento & desenvolvimento , Gordura Subcutânea/metabolismo , Transgenes , Proteína Desacopladora 1RESUMO
The microsomal enzyme 1, 2-acyl CoA: diacylglyceroltransferase-1 (DGAT1) plays an important role in triglyceride storage in adipose tissue and expresses in skeletal muscle as well. The primary goal of the present study was to investigate the effect of porcine DGAT1 on intramuscular fat (IMF) content of transgenic mice produced by pronuclear microinjection with muscle specific promoter of porcine muscle creatine kinase (MCK). In normal chow-fed diet, 4 month-old male transgenic mice expressed more DGAT1, ACC1, UCP1, and FABP4 mRNAs and proteins in skeletal muscle than control mice by real-time PCR and western blot. No significant changes were detected for ACC2, CD36, ADRP, PPAR gamma and LPL. Triacylglycerol assay and soleus muscle sections showed overexpression of porcine DGAT1 in skeletal muscle increased intramyocellular triglyceride and percent of the total cell surface covered by lipid droplets. Thus, upregulation of porcine DGAT1 in skeletal muscle increases IMF content. The present study may further serve to develop transgenic pigs with higher IMF content and improved meat quality.
Assuntos
Diacilglicerol O-Aciltransferase , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Triglicerídeos , Animais , Diacilglicerol O-Aciltransferase/biossíntese , Diacilglicerol O-Aciltransferase/genética , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Suínos/genética , Triglicerídeos/biossíntese , Triglicerídeos/genética , Triglicerídeos/metabolismoRESUMO
Titin immunoglobulin domain protein (TTID) is localized to the Z-line and binds to alpha-actinin, gamma-filamin. It plays an indispensable role in stabilization and anchorage of thin filaments. In this study, the full-length cDNA sequence was isolated by the reverse transcription-polymerase chain reaction (RT-PCR) and the rapid amplification of cDNA ends (RACE). The TTID sequence was deposited into the Genbank under the accession no. DQ157551. The deduced protein of 499 amino acids showed 93 % identity to the corresponding human and rat sequence. Semi-quantitative RT-PCR revealed porcine TTID gene was expressed highest level in skeletal muscle, at second-highest level in the heart, but only low expression in the fat was detected. Bioinformatics analysis shows the molecular weight of the TTID protein is 55.747 kD with a PI of 9.26. It contains the protein function site of two potential Ig-like domain profiles, six N-myristoylation sites, six potential Casein kinase II phosphorylation sites, eight protein kinase C phosphorylation sites, three N-glycosylation sites, a tyrosine kinase phosphorylation site and a cell attachment sequence site. No putative base substitution was detected in the coding region by comparing sequences of Large White, Landrace and Meishan pig breeds. A T978C single nucleotide polymorphism in the intron 6 of porcine TTID gene was detected by a HinfI PCR-restriction fragment length polymorphism. Study showed allele frequency differences among four purebreds. Association of the genotypes with meat quality traits showed that different genotypes of porcine TTID gene were significantly associated with meat pH (m.Biceps Femoris) (P < 0.05), meat color value (m.longissimus Dorsi) (P < 0.05) and Water Moisture (m.longissimus Dorsi) (P < 0.05).
Assuntos
Proteínas do Citoesqueleto/genética , Expressão Gênica , Carne/normas , Proteínas Musculares/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Feminino , Qualidade dos Alimentos , Frequência do Gene , Estudos de Associação Genética , Genótipo , Íntrons , Masculino , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Especificidade de Órgãos , Filogenia , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sus scrofa/genéticaRESUMO
Background: Remote ischemic postconditioning (RIPostC) induced by brief episodes of the limb ischemia is a potential therapeutic strategy for myocardial ischemia/reperfusion injury, achieved by reducing cardiomyocyte death, inflammation and so on. The actual mechanisms underlying cardioprotection conferred by RIPostC remain unclear. Exploring gene expression profiles in myocardium at transcriptional level is helpful to deepen the understanding on the cardioprotective mechanisms of RIPostC. This study aims to investigate the effect of RIPostC on gene expressions in rat myocardium using transcriptome sequencing. Methods: Rat myocardium samples from the RIPostC group, the control group (myocardial ischemia/reperfusion group) and the sham group were performed transcriptome analysis using RNA sequencing. The levels of cardiac IL-1ß, IL-6, IL-10 and TNFα were analyzed by Elisa. The expression levels of candidate genes were verified by qRT-PCR technique. Infarct size was measured by Evans blue and TTC staining. Apoptosis was assessed by TUNEL assays and caspase-3 levels were detected using western blotting. Results: RIPostC can markedly decrease infarct size and reduce the levels of cardiac IL-1ß, IL-6 and increase the level of cardiac IL-10. This transcriptome analysis showed that 2 genes were up-regulated (Prodh1 and ADAMTS15) and 5 genes (Caspase-6, Claudin-5, Sccpdh, Robo4 and AABR07011951.1) were down-regulated in the RIPostC group. Go annotation analysis showed that Go terms mainly included cellular process, metabolic process, cell part, organelle, catalytic activity and binding. The KEGG annotation analysis of DEGs found only one pathway, amino acid metabolism, was up-regulated. The relative mRNA expression levels of ADAMTS15, Caspase-6, Claudin-5 and Prodh1 were verified by qRT-PCR, which were consistent with the RNA-seq results. In addition, the relative expression of ADAMTS15 was negatively correlated with the level of cardiac IL-1ß (r = -0.748, P = 0.005) and positively correlated with the level of cardiac IL-10 (r = 0.698, P = 0.012). A negative correlation statistical trend was found between the relative expression of ADAMTS15 and the level of cardiac IL-6 (r = -0.545, P = 0.067). Conclusions: ADAMTS15 may be a potential inflammation-related gene in regulation of cardioprotection conferred by remote ischemic postconditioning and a possible therapeutic target for myocardial ischemia reperfusion injury in the future.
RESUMO
OBJECTIVE: Placental growth factor (PlGF), an important polypeptide hormone, plays an important regulatory role in various physiological processes. Observational studies have shown that PlGF is associated with the risk of coronary heart disease (CHD). However, the causal association between PlGF and CHD is unclear at present. This study aimed to investigate the causal association between genetically predicted PlGF levels and CHD. METHODS: Single nucleotide polymorphisms (SNPs) associated with PlGF were selected as instrumental variables (IVs) to evaluate the causal association between genetically predicted circulating PlGF levels and CHD risk by two-sample Mendelian randomization (MR). RESULTS: Inverse variance weighted (IVW) analysis showed that there was a suggestive causal association between genetically predicted PlGF level and the risk of CHD (OR = 0.79, 95% CI: 0.66-0.95, P = 0.011) overall. In addition, PlGF levels had a significant negative causal association with the risk of myocardial infarction (OR = 0.83, 95% CI: 0.72-0.95, P = 0.007). A negative correlation trend was found between PlGF level and the risk of angina pectoris (OR = 0.89, 95% CI: 0.79-1.01, P = 0.067). In addition, PlGF levels had a significant negative association with the risk of unstable angina pectoris (OR = 0.78, 95% CI: 0.64-0.94, P = 0.008). PlGF levels were negatively correlated with CHD events with suggestive significance (OR = 0.89, 95% CI: 0.80-0.99, P = 0.046). CONCLUSION: Genetically predicted circulating PlGF levels are causally associated with the risk of CHD, especially acute coronary syndrome, and PlGF is a potential therapeutic target for CHD.
Assuntos
Doença das Coronárias , Infarto do Miocárdio , Feminino , Humanos , Fator de Crescimento Placentário/genética , Análise da Randomização Mendeliana , Doença das Coronárias/genética , Angina Pectoris , Polimorfismo de Nucleotídeo Único , Estudo de Associação Genômica AmplaRESUMO
OBJECTIVE: This study was performed to investigate the dynamics of intracranial pressure (ICP) alterations and bilateral transverse-sigmoid sinus morphologies in patients with venous pulsatile tinnitus (PT). METHODS: This retrospective study involved 27 patients with venous PT associated with sigmoid sinus wall anomalies. ICP and ICP metrics were measured by cerebrospinal fluid manometry and internal jugular vein compression tests. Correlation analysis was performed to determine the statistical correlation between ICP and the morphological metrics. RESULTS: The mean ICP was 212.5 ± 47.3 mmH2O. The median ΔICPtotal was 130 (range, 55-150) mmH2O. The ΔICPtotal was linearly correlated with the open lumbar pressure, and a significant difference was found between patients with normal and elevated cerebrospinal fluid pressure. The ΔICPdifference was linearly correlated with the Lendifference and Voldifference. ΔICP was linearly correlated with Lendifference. CONCLUSIONS: Complete obstruction of flow patency should be avoided in patients with low ICP and large volumetric/patency differences in the bilateral transverse-sigmoid sinus systems.