RESUMO
BACKGROUND: Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood. METHODS: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins. RESULTS: Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown. CONCLUSIONS: These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.
Assuntos
Hemangioblastos , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Hemangioblastos/metabolismo , Hematopoese/genética , Mamíferos , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
AGGF1 is an angiogenic factor with G-Patch and FHA domains 1 described by our group. Gain-of-function mutations in AGGF1 cause Klippel-Trenaunay syndrome, whereas somatic loss-of-function mutations cause cancer. Paraspeckles are small membraneless subnuclear structures with a diameter of 0.5-1 µm, and composed of lncRNA NEAT1 as the scaffold and three core RNA-binding proteins NONO, PSPC1, and PSF. Here, we show that AGGF1 is a key regulatory and structural component of paraspeckles that induces paraspeckle formation, forms an outside rim of paraspeckles, wraps around the NONO/PSF/PSPC1/NEAT1 core, and regulates the size and number of paraspeckles. AGGF1-paraspeckles are larger (>1 µm) than conventional paraspeckles. RNA-FISH in combination with immunostaining shows that AGGF1, NONO, and NEAT1_2 co-localize in 20.58% of NEAT1_2-positive paraspeckles. Mechanistically, AGGF1 interacts with NONO, PSF, and HNRNPK, and upregulates NEAT1_2, a longer, 23 kb NEAT1 transcript with a key role in regulation of paraspeckle size and number. RNA-immunoprecipitation shows that AGGF1 interacts with NEAT1, which may be another possible mechanism underlying the formation of AGGF1-paraspeckles. NEAT1_2 knockdown reduces the number and size of AGGF1-paraspeckles. Functionally, AGGF1 regulates alternative RNA splicing as it decreases the exon skipping/inclusion ratio in a CD44 model. AGGF1 is also localized in some nuclear foci without NEAT1 or NONO, suggesting that AGGF1 is an important liquid-liquid phase separation (LLPS) driver for other types of AGGF1-positive nuclear condensates (referred to as AGGF1-bodies). Our results identify a special type of AGGF1-coated paraspeckles and provide important insights into the formation, structure, and function of paraspeckles.
Assuntos
Paraspeckles , RNA Longo não Codificante , Núcleo Celular/metabolismo , Domínios Proteicos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Atrial fibrillation (AF) is the most common form of sustained clinical arrhythmia. We previously mapped an AF locus to chromosome 5p13 in an AF family with sudden death in early childhood. Here we show that the specific AF gene underlying this linkage is NUP155, which encodes a member of the nucleoporins, the components of the nuclear pore complex (NPC). We have identified a homozygous mutation, R391H, in NUP155 that cosegregates with AF, affects nuclear localization of NUP155, and reduces nuclear envelope permeability. Homozygous NUP155(-/-) knockout mice die before E8.5, but heterozygous NUP155(+/-) mice show the AF phenotype. The R391H mutation and reduction of NUP155 are associated with inhibition of both export of Hsp70 mRNA and nuclear import of Hsp70 protein. These human and mouse studies indicate that loss of NUP155 function causes AF by altering mRNA and protein transport and link the NPC to cardiovascular disease.
Assuntos
Fibrilação Atrial/genética , Morte Súbita Cardíaca , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Linhagem , Alinhamento de SequênciaRESUMO
PURPOSE: Osteoarthritis (OA) is the most common inflammatory disease associated with pain and cartilage destruction. Interleukin (IL)-1ß is widely used to induce inflammatory response in OA models. This study aimed to explore the role of Danshensu (DSS) in IL-1ß-induced inflammatory responses in OA. METHODS: IL-1ß was used to induce chondrocyte inflammation. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay. IL-6, COX-2, TNF-α, and iNOS mRNA levels were detected by qRT-PCR. MMP3, MMP13, ADAMTS4, ADAMTS5, Aggrecan, Collagen, p-IκBα, and p-p65 protein levels were detected by Western blot. An OA mouse model was established by surgical destabilization of the medial meniscus (DMM), and the Osteoarthritis Research Society International (OARSI) score was evaluated by H&E staining. RESULTS: DSS did not affect the levels of inflammatory indicators including IL-6, COX-2, TNF-α, iNOS, PEG2, and NO but suppressed COX-2 and iNOS protein expression in IL-1ß treated chondrocytes. In addition, DSS downregulated IL-1ß-enhanced expression of MMP3, MMP13, ADAMTS4, and ADAMTS5 and upregulated aggrecan and collagen expression. Moreover, DSS significantly inhibited IL-1ß-induced phosphorylation of p-IκBα and p-p65 in a dose-dependent manner in chondrocytes, suggesting it plays a role in the NF-κB signaling pathway. Furthermore, DSS significantly reduced DMM-induced cartilage OARSI score in mice, further demonstrating its protective role in OA progression in vivo. CONCLUSIONS: Our study revealed the protective role of DSS in OA, suggesting that DSS might act as a potential treatment for OA.
Assuntos
Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Interleucina-1beta/metabolismo , Lactatos/farmacologia , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Suscetibilidade a Doenças , Matriz Extracelular/metabolismo , Mediadores da Inflamação/metabolismo , Lactatos/administração & dosagem , Lactatos/química , Camundongos , Osteoartrite/tratamento farmacológico , Osteoartrite/etiologia , Osteoartrite/patologiaRESUMO
Aim: This experimental design was based on DHRS12 to explore its biological effects on osteosarcoma (OS). Materials & methods: The expression level of endogenous DHRS12 was analyzed by immunohistochemical analysis. DHRS12 was overexpressed in MG-63 and HOS cells by plasmid transfection. Cell proliferation, invasion, migration, apoptosis and western blot were used in the experiment. Results: The expression of DHRS12 was significantly reduced in OS. Overexpression of DHRS12 inhibited the proliferation, migration and invasion of MG-63 and HOS cells and induced apoptosis of OS cells. Overexpression of DHRS12 upregulated Bax, Caspase 9 and Caspase 3. Overexpression of DHRS12 resulted in inactivation of the Wnt3a/ß-catenin signaling pathway. Conclusion: Overexpression of DHRS12 inhibited the progression of OS via the Wnt3a/ß-catenin pathway.
Assuntos
Osteossarcoma/patologia , Redutases-Desidrogenases de Cadeia Curta/metabolismo , Via de Sinalização Wnt , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/mortalidade , Redutases-Desidrogenases de Cadeia Curta/genética , Taxa de Sobrevida , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMO
Atrial fibrillation (AF) is the most common cardiac arrhythmia. Here, we show the identification and functional characterization of one AF-associated mutation p.Arg399Cys in lamin A/C. Co-immunoprecipitation and GST pull-down assays demonstrate that lamin A/C interacts with NUP155, which is a nucleoporin and causes AF when mutated. Lamin A/C mutation p.Arg399Cys impairs the interaction between lamin A/C and NUP155, and increases extractability of NUP155 from the nuclear envelope (NE). Mutation p.Arg399Cys leads to aggregation of lamin A/C in the nucleus, although it does not impair the integrity of NE upon cellular stress. Mutation p.Arg399Cys inhibits the export of HSP70 mRNA and the nuclear import of HSP70 protein. Electrophysiological studies show that mutation p.Arg399Cys decreases the peak cardiac sodium current by decreasing the cell surface expression level of cardiac sodium channel Nav 1.5, but does not affect IKr potassium current. In conclusion, our results indicate that lamin A/C mutation p.Arg399Cys weakens the interaction between nuclear lamina (lamin A/C) and the nuclear pore complex (NUP155), leading to the development of AF. The findings provide a novel molecular mechanism for the pathogenesis of AF.
Assuntos
Fibrilação Atrial/genética , Lamina Tipo A/genética , Mutação/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Sequência de Bases , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos , Ativação do Canal Iônico , Carioferinas/metabolismo , Lamina Tipo A/química , Camundongos , Membrana Nuclear/metabolismo , Ligação Proteica , Transporte Proteico , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais de Sódio/metabolismo , Estresse FisiológicoRESUMO
Atrial fibrillation (AF) affects 33.5 million individuals worldwide. It accounts for 15% of strokes and increases risk of heart failure and sudden death. The voltage-gated cardiac sodium channel complex is responsible for the generation and conduction of the cardiac action potential, and composed of the main pore-forming α-subunit Nav 1.5 (encoded by the SCN5A gene) and one or more auxiliary ß-subunits, including Nav ß1 to Nav ß4 encoded by SCN1B to SCN4B, respectively. We and others identified loss-of-function mutations in SCN1B and SCN2B and dominant-negative mutations in SCN3B in patients with AF. Three missense variants in SCN4B were identified in sporadic AF patients and small nuclear families; however, the association between SCN4B variants and AF remains to be further defined. In this study, we performed mutational analysis in SCN4B using a panel of 477 AF patients, and identified one nonsynonymous genomic variant p.Gly8Ser in four patients. To assess the association between the p.Gly8Ser variant and AF, we carried out case-control association studies with two independent populations (944 AF patients vs. 9,81 non-AF controls in the first discovery population and 732 cases and 1,291 controls in the second replication population). Significant association was identified in the two independent populations and in the combined population (p = 4.16 × 10-4 , odds ratio [OR] = 3.14) between p.Gly8Ser and common AF as well as lone AF (p = 0.018, OR = 2.85). These data suggest that rare variant p.Gly8Ser of SCN4B confers a significant risk of AF, and SCN4B is a candidate susceptibility gene for AF.
Assuntos
Alelos , Substituição de Aminoácidos , Fibrilação Atrial/genética , Variação Genética , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/genética , Idoso , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Estudos de Casos e Controles , Biologia Computacional/métodos , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Polimorfismo de Nucleotídeo Único , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/metabolismoRESUMO
BACKGROUND: Iatrogenic supraclavicular nerve injury is frequent during surgical repair of clavicle fractures through a transverse incision. The use of an oblique incision may be a potential approach to avoiding this complication. This study compared the clinical effectiveness of oblique and transverse incisions in the treatment of fractures in the middle and outer thirds of the clavicle. METHODS: This prospective observational study included patients with fracture of the mid-to-outer third of the clavicle between August 2011 and August 2016. We allocated the patients into 2 groups based on their choice of treatment: oblique incision (n = 62) and transverse incision (n = 64). We compared the following parameters between the 2 groups: operative time, intraoperative blood loss, postoperative fracture healing time, incision size, clinical complications, postoperative subjective satisfaction, and shoulder function. RESULTS: Operative time, postoperative fracture healing time, postoperative shoulder function (Constant-Murley and disabilities of the arm, shoulder and hand [DASH] scores), and clinical complications did not differ significantly between groups (all P > .05). The oblique incision group had less intraoperative blood loss (41.4 ± 16.4 vs. 65.3 ± 10.4 mL, P < .001) and smaller surgical incisions (3.6 ± 1.6 vs. 10.3 ± 2.6 cm, P < .001). The oblique incision group showed better outcomes for postoperative satisfaction (85.5% vs. 64.1%, P = .015), absence of shoulder numbness at the last follow-up (89.3% vs. 70.3%, P = .010), and satisfaction with the scar (90.3% vs. 3.1%, P < .001). CONCLUSION: Oblique incisions have several advantages over transverse incisions: less bleeding, smaller incisions, less iatrogenic injury to supraclavicular nerves, and higher patient satisfaction. These 2 approaches have equivalent effects on recovery of shoulder joint function.
Assuntos
Clavícula/cirurgia , Fixação Interna de Fraturas/efeitos adversos , Fixação Interna de Fraturas/métodos , Fraturas Ósseas/cirurgia , Traumatismos dos Nervos Periféricos/prevenção & controle , Adulto , Perda Sanguínea Cirúrgica , Clavícula/lesões , Feminino , Consolidação da Fratura , Humanos , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Satisfação do Paciente , Traumatismos dos Nervos Periféricos/etiologia , Complicações Pós-Operatórias/etiologia , Período Pós-Operatório , Estudos Prospectivos , Ombro/fisiopatologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Atrial fibrillation (AF) is the most common cardiac arrhythmia at the clinic. Recent GWAS identified several variants associated with AF, but they account for <10% of heritability. Gene-gene interaction is assumed to account for a significant portion of missing heritability. Among GWAS loci for AF, only three were replicated in the Chinese Han population, including SNP rs2106261 (G/A substitution) in ZFHX3, rs2200733 (C/T substitution) near PITX2c, and rs3807989 (A/G substitution) in CAV1. Thus, we analyzed the interaction among these three AF loci. We demonstrated significant interaction between rs2106261 and rs2200733 in three independent populations and combined population with 2,020 cases/5,315 controls. Compared to non-risk genotype GGCC, two-locus risk genotype AATT showed the highest odds ratio in three independent populations and the combined population (OR=5.36 (95% CI 3.87-7.43), P=8.00×10-24). The OR of 5.36 for AATT was significantly higher than the combined OR of 3.31 for both GGTT and AACC, suggesting a synergistic interaction between rs2106261 and rs2200733. Relative excess risk due to interaction (RERI) analysis also revealed significant interaction between rs2106261 and rs2200733 when exposed two copies of risk alleles (RERI=2.87, P<1.00×10-4) or exposed to one additional copy of risk allele (RERI=1.29, P<1.00×10-4). The INTERSNP program identified significant genotypic interaction between rs2106261 and rs2200733 under an additive by additive model (OR=0.85, 95% CI: 0.74-0.97, P=0.02). Mechanistically, PITX2c negatively regulates expression of miR-1, which negatively regulates expression of ZFHX3, resulting in a positive regulation of ZFHX3 by PITX2c; ZFHX3 positively regulates expression of PITX2C, resulting in a cyclic loop of cross-regulation between ZFHX3 and PITX2c. Both ZFHX3 and PITX2c regulate expression of NPPA, TBX5 and NKX2.5. These results suggest that cyclic cross-regulation of gene expression is a molecular basis for gene-gene interactions involved in genetics of complex disease traits.
Assuntos
Fibrilação Atrial/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas , Fibrilação Atrial/metabolismo , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Sequência de Bases , Sítios de Ligação , Estudos de Casos e Controles , Caveolina 1/genética , Caveolina 1/metabolismo , Epistasia Genética , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Humanos , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Interferência de RNA , Fatores de Transcrição/metabolismo , Proteína Homeobox PITX2RESUMO
Coronary artery disease (CAD) is the leading cause of death worldwide. GWAS have identified >50 genomic loci for CAD, including ADTRP and MIA3/TANGO1. However, it is important to determine whether the GWAS genes form a molecular network. In this study, we have uncovered a novel molecular network between ADTRP and MIA3/TANGO1 for the pathogenesis of CAD. We showed that knockdown of ADTRP expression markedly down-regulated expression of MIA3/TANGO1. Mechanistically, ADTRP positively regulates expression of PIK3R3 encoding the regulatory subunit 3 of PI3K, which leads to activation of AKT, resulting in up-regulation of MIA3/TANGO1. Both ADTRP and MIA3/TANGO1 are involved in endothelial cell (EC) functions relevant to atherosclerosis. Knockdown of ADTRP expression by siRNA promoted oxidized-LDL-mediated monocyte adhesion to ECs and transendothelial migration of monocytes, inhibited EC proliferation and migration, and increased apoptosis, which was reversed by expression of constitutively active AKT1 and MIA3/TANGO1 overexpression, while the over-expression of ADTRP in ECs blunted these processes. Knockdown of MIA3/TANGO1 expression also promoted monocyte adhesion to ECs and transendothelial migration of monocytes, and vice versa for overexpression of MIA3/TANGO1. We found that ADTRP negatively regulates the levels of collagen VII and ApoB in HepG2 and endothelial cells, which are downstream regulatory targets of MIA3/TANGOI. In conclusion, we have uncovered a novel molecular signaling pathway for the pathogenesis of CAD, which involves a novel gene-gene regulatory network. We show that ADTRP positively regulates PIK3R3 expression, which leads to activation of AKT and up-regulation of MIA3/TANGO1, thereby regulating endothelial cell functions directly relevant to atherosclerosis.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/biossíntese , Aterosclerose/metabolismo , Doença da Artéria Coronariana/metabolismo , Redes Reguladoras de Genes , Predisposição Genética para Doença , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Aterosclerose/genética , Aterosclerose/patologia , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Regulação da Expressão Gênica/genética , Estudo de Associação Genômica Ampla , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas de Membrana/genética , Fosfatidilinositol 3-Quinases/biossíntese , Fosfatidilinositol 3-Quinases/genéticaRESUMO
BACKGROUND: This study aimed to evaluate the application of 3D printing in assisting preoperative plan of pedicle screw placement for treating middle-upper thoracic trauma. METHODS: A preoperative plan was implemented in seven patients suffering from middle-upper thoracic (T3-T7) trauma between March 2013 and February 2016. In the 3D printing models, entry points of 56 pedicle screws (Magerl method) and 4 important parameters of the pedicle screws were measured, including optimal diameter (Ï, mm), length (L, mm), inclined angle (α), head-tilting angle (+ß), and tail-tilting angle (-ß). In the surgery, bare-hands fixation of pedicle screws was performed using 3D printing models and the measured parameters as guidance. RESULTS: A total of seven patients were enrolled, including five men and two women, with the age of 21-62 years (mean age of 37.7 years). The position of the pedicle screw was evaluated postoperatively using a computerized tomography scan. Totally, 56 pedicle screws were placed, including 33 pieces of level 0, 18 pieces of level 1, 4 pieces of level 2 (pierced lateral wall), and 1 piece of level 3 (pierced lateral wall, no adverse consequences), with a fine rate of 91.0%. CONCLUSIONS: 3D printing technique is an intuitive and effective assistive technology to pedicle screw fixation for treating middle-upper thoracic vertebrae, which improve the accuracy of bare-hands screw placement and reduce empirical errors. TRIAL REGISTRATION: The trial was approved by the Ethics Committee of the Fujian Provincial Hospital. It was registered on March 1st, 2013, and the registration number was K2013-03-001.
Assuntos
Fraturas por Compressão/cirurgia , Parafusos Pediculares , Cuidados Pré-Operatórios/métodos , Impressão Tridimensional , Fraturas da Coluna Vertebral/cirurgia , Vértebras Torácicas/cirurgia , Adulto , Estudos de Coortes , Feminino , Seguimentos , Fraturas por Compressão/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Cuidados Pré-Operatórios/instrumentação , Fraturas da Coluna Vertebral/diagnóstico por imagem , Cirurgia Assistida por Computador/instrumentação , Cirurgia Assistida por Computador/métodos , Vértebras Torácicas/diagnóstico por imagem , Vértebras Torácicas/lesõesRESUMO
BACKGROUND: Aortic aneurysms and/or dissection (AADs) in the aorta are a leading cause of human morbidity and mortality. To date, data on non-syndromic thoracic AADs (TAADs) have been mainly derived from Caucasians, and the genetic basis of TAADs remains to be elucidated. In this study, we assessed gene mutations in a Chinese population with TAADs. METHODS: A cohort of 68 non-syndromic familial TAAD Chinese patients was screened for the most common TAAD-causing genes (ACTA2, MYH11, TGFBR1, TGFBR2, and SMAD3) using high-resolution melting (HRM) analysis. Thereafter, 142 unrelated non-syndromic sporadic cases were recruited and further analyzed using HRM analysis to estimate the prevalence of disease-causing mutations in these candidate genes. RESULTS: Two novel ACTA2 mutations (N117I and L348R) were identified in each familial TAAD proband separately, and an additional novel ACTA2 mutation (Y168N) was identified in one patient with sporadic TAADs. In contrast, none of the three mutations occurred in 480 control subjects. Also, no other gene mutations were identified in this cohort of Chinese TAAD patients. CONCLUSIONS: The current study identified three novel ACTA2 mutations in Chinese TAAD patients, and these mutations represented the most predominant genes responsible for non-syndromic TAADs. In addition, HRM analysis was shown to be a sensitive and high-throughput method for screening gene mutations.
Assuntos
Actinas/genética , Aneurisma da Aorta Torácica/genética , Povo Asiático/genética , Adulto , Idoso , Sequência de Aminoácidos , Animais , Aneurisma da Aorta Torácica/patologia , Estudos de Casos e Controles , Criança , China , Estudos de Coortes , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Transição de Fase , Polimorfismo de Nucleotídeo Único , Alinhamento de SequênciaRESUMO
HSF4 mutations lead to both congenital and age-related cataract. The purpose of this study was to explore the mechanism of cataract formation caused by HSF4 mutations. The degradation of nuclear DNA is essential for the lens fiber differentiation. DNase 2ß (DLAD) is highly expressed in lens cells, and mice with deficiencies in the DLAD gene develop nuclear cataracts. In this study, we found that HSF4 promoted the expression and DNase activity of DLAD by directly binding to the DLAD promoter. In contrast, HSF4 cataract causative mutations failed to bind to the DLAD promoter, abrogating the expression and DNase activity of DLAD. These results were confirmed by HSF4 knockdown in zebrafish, which led to incomplete de-nucleation of the lens and decreased expression and activity of DLAD. Together, our results suggest that HSF4 exerts its function on lens differentiation via positive regulation of DLAD expression and activity, thus facilitating de-nucleation of lens fiber cells. Our demonstration that HSF4 cataract causative mutations abrogate the induction of DLAD expression reveals a novel molecular mechanism regarding how HSF4 mutations cause cataractogenesis.
Assuntos
Catarata/fisiopatologia , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/biossíntese , Cristalino/fisiologia , Fatores de Transcrição/metabolismo , Animais , Catarata/genética , Catarata/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Células Cultivadas , DNA/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Fatores de Transcrição de Choque Térmico , Humanos , Cristalino/metabolismo , Mutação , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Peixe-ZebraRESUMO
BACKGROUND AND PURPOSE: ANRIL has long been considered as the strongest candidate gene at the 9p21 locus, robustly associated with stroke and coronary artery disease. However, the underlying molecular mechanism remains unknown. The present study works to elucidate such a mechanism. METHODS: Using expression quantitative loci analysis, we identified potential genes whose expression may be influenced by genetic variation in ANRIL. To verify the identified gene(s), knockdown and overexpression of ANRIL were evaluated in human umbilical vein endothelial cells and HepG2 cells. Ischemic stroke and coronary artery disease risk were then evaluated in the gene(s) demonstrated to be mediated by ANRIL in 3 populations of Chinese Han ancestry: 2 ischemic stroke populations consisting of the Central China cohort (903 cases and 873 controls) and the Northern China cohort (816 cases and 879 controls) and 1 coronary artery disease cohort consisting of 772 patients and 873 controls. RESULTS: Expression quantitative loci analysis identified CARD8 among others, with knockdown of ANRIL expression decreasing CARD8 expression and overexpression of ANRIL increasing CARD8 expression. The minor T allele of a previously identified CARD8 variant (rs2043211) was found to be significantly associated with a protective effect of ischemic stroke under the recessive model in 2 independent stroke cohorts. No significant association was found between rs2043211 and coronary artery disease. CONCLUSIONS: CARD8 is a downstream target gene regulated by ANRIL. Single nucleotide polymorphism rs2043211 in CARD8 is significantly associated with ischemic stroke. ANRIL may increase the risk of ischemic stroke through regulation of the CARD8 pathway.
Assuntos
Isquemia Encefálica/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Neoplasias/genética , RNA Longo não Codificante/genética , Acidente Vascular Cerebral/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Povo Asiático , Isquemia Encefálica/epidemiologia , China/epidemiologia , Doença da Artéria Coronariana/epidemiologia , Doença da Artéria Coronariana/genética , Feminino , Expressão Gênica/fisiologia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Reação em Cadeia da Polimerase em Tempo Real , Acidente Vascular Cerebral/epidemiologia , TransfecçãoRESUMO
A single nucleotide polymorphism (SNP) rs1122608 on chromosome 19p13.2 and in the BRG1/SMARCA4 gene was previously associated with coronary artery disease (CAD). CAD and ischemic stroke are both associated with atherosclerosis. Thus, we tested the hypothesis that rs1122608 is associated with ischemic stroke. Further studies were used to identify the most likely mechanism by which rs1122608 regulates atherosclerosis. For case-control association studies, two independent Chinese Han GeneID cohorts were used, including a Central cohort with 1,075 cases and 2,685 controls and the Northern cohort with 1,208 cases and 824 controls. eQTL and real-time RT-PCR analyses were used to identify the potential candidate gene(s) affected by rs1122608. The minor allele T of SNP rs1122608 showed significant association with a decreased risk of ischemic stroke in the Central GeneID cohort (adjusted P adj = 2.1 × 10(-4), OR 0.61). The association was replicated in an independent Northern GeneID cohort (P adj = 6.00 × 10(-3), OR 0.69). The association became more significant in the combined population (P adj = 7.86 × 10(-5), OR 0.73). Allele T of SNP rs1122608 also showed significant association with a decreased total cholesterol level (P adj = 0.013). Allele T of rs1122608 was associated with an increased expression level of SFRS3 encoding an mRNA splicing regulator, but not with the expression of BRG1/SMARCA4 or LDLR (located 36 kb from rs1122608). Increased expression of SFSR3 may decrease IL-1ß expression and secretion, resulting in reduced risk of atherosclerosis and stroke. This is the first study that demonstrates that rs1122608 confers protection against ischemic stroke and implicates splicing factor SFSR3 in the disease process.
Assuntos
Cromossomos Humanos Par 19 , DNA Helicases/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Acidente Vascular Cerebral/genética , Fatores de Transcrição/genética , Alelos , Sequência de Bases , Primers do DNA , Humanos , Lipídeos/sangue , Reação em Cadeia da Polimerase , Locos de Características Quantitativas , Fatores de Processamento de Serina-Arginina , Acidente Vascular Cerebral/prevenção & controleRESUMO
OBJECTIVE: The clinical effects of artificial dermis in treating skin and soft tissue defects accompanied by bone or tendon exposure were assessed. APPROACH: A retrospective analysis was conducted on the clinical data of 45 cases of skin and soft tissue defects accompanied by bone or tendon exposure admitted to the trauma surgery department of Fujian Provincial Hospital between February 2018 and August 2020. They were divided into the artificial dermis and control groups. The wound was assessed using the Vancouver Scar Scale (mVSS), and the postoperative visual analogue scale (VAS) scores were recorded at 3, 6, 9, and 12 months after surgery. At the 12-month after surgery, skin sensation recovery was evaluated using the criteria of the British Medical Research Council (BMRC). RESULTS: The cases included 26 males and 19 females, aged 50 to 85 years. All patients were followed up for an average of 13.8 months (range: 12-18 months). Compared with controls, the wound healing time of the observation group was longer (35.8 ± 10.6 vs. 28.5 ± 4.8, P = 0.007), without significant differences for the number of operations and length of hospitalization. The mVSS scores were not different between groups (Pgroup = 0.294), but the scores decreased with time (Ptime < 0.001), and the group×time interaction was significant (Pinteraction < 0.001). Similarly, the VAS scores were not different between groups (Pgroup = 0.667), but the scores decreased with time (Ptime < 0.001); the group×time interaction was not significant (Pinteraction = 0.274). At the 12-month mark following the operation, in the artificial dermis group, the MCRR score was S3+ in 23 patients, while it ranged from S0 to S3 in two patients; in the control group, S3+ was observed in 17 patients, and S0-S3 in three (P = 0.815). CONCLUSION: Artificial dermis treatment is considered a safe and effective alternative therapy for patients with skin and soft tissue defects accompanied by bone or tendon exposure who cannot tolerate or are unwilling to undergo autologous skin flap transplantation. It offers the advantages of minimal donor site trauma, simplicity in operation, and favorable postoperative functional recovery.
RESUMO
PURPOSE: The purpose of the present work was to assess the specific effects and underlying mechanisms of Daprodustat (GSK1278863) on skeletal muscle injury induced by ischemia reperfusion (I/R). METHODS: C57BL/6 mice were randomized into the skeletal muscle I/R injury (I/R), Daprodustat (GSK1278863) pretreatment and I/R (I/R + GSK) and sham operation (Sham) groups. The skeletal muscle I/R injury model was established by placing an orthodontic rubber band at the left hip joint for 3 h and releasing it for 3 h. H&E staining, wet weight/dry weight ratio assessment, TUNEL assay, ELISA, qRT-PCR and immunoblot were utilized to assess the effects of Daprodustat. RESULTS: Daprodustat pretreatment significantly ameliorated apoptosis in skeletal muscle cells, reduced oxidative damage and suppressed inflammatory cytokines. Mechanistically, Daprodustat positively affected NF-κB signaling activation. CONCLUSION: These data demonstrated that Daprodustat may provide a potential clinical approach for preventing or treating skeletal muscle injury induced by I/R.
Assuntos
Camundongos Endogâmicos C57BL , Músculo Esquelético , Traumatismo por Reperfusão , Animais , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/etiologia , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Masculino , Modelos Animais de Doenças , NF-kappa B/metabolismo , Distribuição AleatóriaRESUMO
Heat shock factor protein 4 (HSF4) is expressed exclusively in the ocular lens and plays a critical role in the lens formation and differentiation. Mutations in the HSF4 gene lead to congenital and senile cataract. However, the molecular mechanisms causing this disease have not been well characterized. DNA damage in lens is a crucial risk factor in senile cataract formation, and its timely repair is essential for maintaining the lens' transparency. Our study firstly found evidence that HSF4 contributes to the repair of DNA strand breaks. Yet, this does not occur with cataract causative mutations in HSF4. We verify that DNA damage repair is mediated by the binding of HSF4 to a heat shock element in the Rad51 promoter, a gene which assists in the homologous recombination (HR) repair of DNA strand breaks. HSF4 up-regulates Rad51 expression while mutations in HSF4 fail, and DNA does not get repaired. Camptothecin, which interrupts the regulation of Rad51 by HSF4, also affects DNA damage repair. Additionally, with HSF4 knockdown in the lens of Zebrafish, DNA damage was observed and the protein level of Rad51 was significantly lower. Our study presents the first evidence demonstrating that HSF4 plays a role in DNA damage repair and may contribute a better understanding of congenital cataract formation.
Assuntos
Catarata/genética , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/genética , Cristalino/metabolismo , Rad51 Recombinase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Camptotecina/farmacologia , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/metabolismo , Humanos , Regiões Promotoras Genéticas , Rad51 Recombinase/genética , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Peixe-ZebraRESUMO
OBJECTIVE: Genetic variants in ninjurin-2 (NINJ2; nerve injury-induced protein 2) confer risk of ischemic strokes and coronary artery disease as well as endothelial activation and inflammation. However, little is known about NINJ2's in vivo functions and underlying mechanisms. METHODS: The phenotypes of NINJ2 knockout mice were analyzed, and mechanisms of NINJ2 that regulate body weight, insulin resistance, and glucose homeostasis and lipogenesis were investigated in vivo and in vitro. RESULTS: This study found that mice lacking NINJ2 showed impaired adipogenesis, increased insulin resistance, and abnormal glucose homeostasis, all of which are risk factors for strokes and coronary artery disease. Mechanistically, NINJ2 directly interacts with insulin receptor/insulin-like growth factor 1 receptor (INSR/IGF1R), and NINJ2 knockdown can block insulin-induced mitotic clonal expansion during preadipocyte differentiation by inhibiting protein kinase B/extracellular signal-regulated kinase (AKT/ERK) signaling and by decreasing the expression of key adipocyte transcriptional regulators CCAAT/enhancer-binding protein ß (C/EBP-ß), C/EBP-α, and peroxisome proliferator-activated receptor γ (PPAR-γ). Furthermore, the interaction between NINJ2 and INSR/IGF1R is needed for maintaining insulin sensitivity in adipocytes and muscle via AKT and glucose transporter type 4. Notably, adenovirus-mediated NINJ2 overexpression can ameliorate diet-induced insulin resistance in mice. CONCLUSIONS: In conclusion, these findings reveal NINJ2 as an important new facilitator of insulin receptors, and the authors propose a unique regulatory mechanism between insulin signaling, adipogenesis, and insulin resistance.
Assuntos
Moléculas de Adesão Celular Neuronais , Resistência à Insulina , Animais , Camundongos , Células 3T3-L1 , Adipogenia/genética , Diferenciação Celular/genética , Doença da Artéria Coronariana , Glucose/metabolismo , Insulina , Resistência à Insulina/genética , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Moléculas de Adesão Celular Neuronais/genéticaRESUMO
Our earlier studies identified MOG1 as a Nav1.5-binding protein that promotes Nav1.5 intracellular trafficking to plasma membranes. Genetic studies have identified MOG1 variants responsible for cardiac arrhythmias. However, the physiological functions of MOG1 in vivo remain incompletely characterized. In this study, we generated Mog1 knockout (Mog1-/-) mice. Mog1-/- mice did not develop spontaneous arrhythmias at the baseline, but exhibited a prolongation of QRS duration. Mog1-/- mice treated with isoproterenol (ISO), but not with flecainide, exhibited an increased risk of arrhythmias and even sudden death. Mog1-/- mice had normal cardiac morphology, however, LV systolic dysfunction was identified and associated with an increase in ventricular fibrosis. Whole-cell patch-clamping and Western blotting analysis clearly demonstrated the normal cardiac expression and function of Nav1.5 in Mog1-/- mice. Further RNA-seq and iTRAQ analysis identified critical pathways and genes, including extracellular matrix (Mmp2), gap junction (Gja1), and mitochondrial components that were dysregulated in Mog1-/- mice. RT-qPCR, Western blotting, and immunofluorescence assays revealed reduced cardiac expression of Gja1 in Mog1-/- mice. Dye transfer assays confirmed impairment of gap-junction function; Cx43 gap-junction enhancer ZP123 decreased arrhythmia inducibility in ISO-treated Mog1-/- mice. Transmission electron microscopy analysis revealed abnormal sarcomere ultrastructure and altered mitochondrial morphology in Mog1-/- mice. Mitochondrial dynamics was found to be disturbed, and associated with a trend toward increased mitochondrial fusion in Mog1-/- mice. Meanwhile, the level of ATP supply was increased in the hearts of Mog1-/- mice. These results indicate that MOG1 plays an important role in cardiac electrophysiology and cardiac contractile function.