RESUMO
The extracellular matrix (ECM) undergoes substantial changes during prostate cancer (PCa) progression, thereby regulating PCa growth and invasion. Herein, a meta-analysis of multiple PCa cohorts is performed which revealed that downregulation or genomic loss of ITGA1 and ITGA2 integrin genes is associated with tumor progression and worse prognosis. Genomic deletion of both ITGA1 and ITGA2 activated epithelial-to-mesenchymal transition (EMT) in benign prostate epithelial cells, thereby enhancing their invasive potential in vitro and converting them into tumorigenic cells in vivo. Mechanistically, EMT is induced by enhanced secretion and autocrine activation of TGFß1 and nuclear targeting of YAP1. An unbiased genome-wide co-expression analysis of large PCa cohort datasets identified the transcription factor TEAD1 as a key regulator of ITGA1 and ITGA2 expression in PCa cells while TEAD1 loss phenocopied the dual loss of α1- and α2-integrins in vitro and in vivo. Remarkably, clinical data analysis revealed that TEAD1 downregulation or genomic loss is associated with aggressive PCa and together with low ITGA1 and ITGA2 expression synergistically impacted PCa prognosis and progression. This study thus demonstrated that loss of α1- and α2-integrins, either via deletion/inactivation of the ITGA1/ITGA2 locus or via loss of TEAD1, contributes to PCa progression by inducing TGFß1-driven EMT.
Assuntos
Próstata , Neoplasias da Próstata , Masculino , Humanos , Próstata/metabolismo , Próstata/patologia , Linhagem Celular Tumoral , Neoplasias da Próstata/genética , Transdução de Sinais/genética , Integrina alfa2/genética , Integrina alfa2/metabolismo , Fatores de Transcrição de Domínio TEARESUMO
Transposable elements (TE) are repetitive genomic elements that harbor binding sites for human transcription factors (TF). A regulatory role for TEs has been suggested in embryonal development and diseases such as cancer but systematic investigation of their functions has been limited by their widespread silencing in the genome. Here, we utilize unbiased massively parallel reporter assay data using a whole human genome library to identify TEs with functional enhancer activity in two human cancer types of endodermal lineage, colorectal and liver cancers. We show that the identified TE enhancers are characterized by genomic features associated with active enhancers, such as epigenetic marks and TF binding. Importantly, we identify distinct TE subfamilies that function as tissue-specific enhancers, namely MER11- and LTR12-elements in colon and liver cancers, respectively. These elements are bound by distinct TFs in each cell type, and they have predicted associations to differentially expressed genes. In conclusion, these data demonstrate how different cancer types can utilize distinct TEs as tissue-specific enhancers, paving the way for comprehensive understanding of the role of TEs as bona fide enhancers in the cancer genomes.
Assuntos
Elementos de DNA Transponíveis , Neoplasias Hepáticas , Humanos , Elementos de DNA Transponíveis/genética , Neoplasias Hepáticas/genética , Sequências Reguladoras de Ácido Nucleico , Sítios de Ligação , Bioensaio , Fatores de Transcrição/genéticaRESUMO
Anti-inflammatory therapies have the potential to become an effective treatment for obesity-related diseases. However, the huge gap of immune system between human and rodent leads to limitations of drug discovery. This work aims at constructing a transgenic pig model with higher risk of metabolic diseases and outlining the immune responses at the early stage of metaflammation by transcriptomic strategy. We used CRISPR/Cas9 techniques to targeted knock-in three humanized disease risk genes, GIPRdn , hIAPP and PNPLA3I148M . Transgenic effect increased the risk of metabolic disorders. Triple-transgenic pigs with short-term diet intervention showed early symptoms of type 2 diabetes, including glucose intolerance, pancreatic lipid infiltration, islet hypertrophy, hepatic lobular inflammation and adipose tissue inflammation. Molecular pathways related to CD8+ T cell function were significantly activated in the liver and visceral adipose samples from triple-transgenic pigs, including antigen processing and presentation, T-cell receptor signaling, co-stimulation, cytotoxicity, and cytokine and chemokine secretion. The similar pro-inflammatory signaling in liver and visceral adipose tissue indicated that there might be a potential immune crosstalk between the two tissues. Moreover, genes that functionally related to liver antioxidant activity, mitochondrial function and extracellular matrix showed distinct expression between the two groups, indicating metabolic stress in transgenic pigs' liver samples. We confirmed that triple-transgenic pigs had high coincidence with human metabolic diseases, especially in the scope of inflammatory signaling at early stage metaflammation. Taken together, this study provides a valuable large animal model for the clinical study of metaflammation and metabolic diseases.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 2/imunologia , Gordura Intra-Abdominal/imunologia , Fígado/imunologia , Ativação Linfocitária , Hepatopatia Gordurosa não Alcoólica/imunologia , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Geneticamente Modificados , Glicemia/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Lipase/genética , Lipídeos/sangue , Fígado/metabolismo , Fígado/patologia , Masculino , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores dos Hormônios Gastrointestinais/genética , Suínos/genética , TranscriptomaRESUMO
Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.
Assuntos
Sistemas CRISPR-Cas , Metilação de DNA , Edição de Genes/métodos , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Neoplasias da Próstata/genética , Animais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Locos de Características Quantitativas/genética , Elementos Reguladores de Transcrição/genética , Fatores de RiscoRESUMO
We studied a family with severe primary osteoporosis carrying a heterozygous p.Arg8Phefs*14 deletion in COL1A2, leading to haploinsufficiency. Three affected individuals carried the mutation and presented nearly identical spinal fractures but lacked other typical features of either osteogenesis imperfecta or Ehlers-Danlos syndrome. Although mutations leading to haploinsufficiency in COL1A2 are rare, mutations in COL1A1 that lead to less protein typically result in a milder phenotype. We hypothesized that other genetic factors may contribute to the severe phenotype in this family. We performed whole-exome sequencing in five family members and identified in all three affected individuals a rare nonsense variant (c.1282C > T/p.Arg428*, rs150257846) in ZNF528. We studied the effect of the variant using qPCR and Western blot and its subcellular localization with immunofluorescence. Our results indicate production of a truncated ZNF528 protein that locates in the cell nucleus as per the wild-type protein. ChIP and RNA sequencing analyses on ZNF528 and ZNF528-c.1282C > T indicated that ZNF528 binding sites are linked to pathways and genes regulating bone morphology. Compared with the wild type, ZNF528-c.1282C > T showed a global shift in genomic binding profile and pathway enrichment, possibly contributing to the pathophysiology of primary osteoporosis. We identified five putative target genes for ZNF528 and showed that the expression of these genes is altered in patient cells. In conclusion, the variant leads to expression of truncated ZNF528 and a global change of its genomic occupancy, which in turn may lead to altered expression of target genes. ZNF528 is a novel candidate gene for bone disorders and may function as a transcriptional regulator in pathways affecting bone morphology and contribute to the phenotype of primary osteoporosis in this family together with the COL1A2 deletion. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Assuntos
Osteogênese Imperfeita , Osteoporose , Fatores de Transcrição/genética , Colágeno Tipo I/genética , Exoma/genética , Humanos , Mutação , Osteogênese Imperfeita/genética , Osteoporose/genética , Fenótipo , Deleção de Sequência , Sequenciamento do ExomaRESUMO
Spatiotemporal patterns of gene expression depend on enhancer elements and other factors during individual development and disease progression. The rapid progress of high-throughput techniques has led to well-defined enhancer chromatin properties. Various genome-wide methods have revealed a large number of enhancers and the discovery of three-dimensional (3D) genome architecture showing the distant interacting mechanisms of enhancers that loop to target gene promoters. Whole genome sequencing projects directed at cancer have led to the discovery of substantial enhancer dysfunction in misregulating gene expression and in tumor initiation and progression. Results from genome-wide association studies (GWAS) combined with functional genomics analyses have elucidated the functional impacts of many cancer risk-associated variants that are enriched within the enhancer regions of chromatin. Risk variants dysregulate the expression of enhancer variant-associated genes via 3D genomic interactions. Moreover, these enhancer variants often alter the chromatin binding affinity for cancer-relevant transcription factors, which in turn leads to aberrant expression of the genes associated with cancer susceptibility. In this review, we investigate the extent to which these genetic regulatory circuits affect cancer predisposition and how the recent development of genome-editing methods have enabled the determination of the impacts of genomic variation and alteration on cancer phenotype, which will eventually lead to better management plans and treatment responses to human cancer in the clinic.
Assuntos
Genoma Humano/genética , Neoplasias/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
The 19q13 allele rs11672691 has been reproducibly found in association with aggressive form of prostate cancer, yet the underlying mechanism remains totally unknown. We have recently uncovered a mechanism by which rs11672691 influenced a novel oncogenic regulatory circuit, including HOXA2, PCAT19 and CEACAM21, thereby contributing to prostate cancer aggressiveness.
RESUMO
Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic.
Assuntos
Neoplasias da Próstata/genética , RNA Longo não Codificante/genética , RNA não Traduzido/genética , Adulto , Alelos , Linhagem Celular Tumoral , Cromossomos Humanos Par 19/genética , Estudos de Coortes , Regulação Neoplásica da Expressão Gênica/genética , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Genótipo , Proteínas de Homeodomínio , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , PrognósticoRESUMO
Functional characterization of disease-causing variants at risk loci has been a significant challenge. Here we report a high-throughput single-nucleotide polymorphisms sequencing (SNPs-seq) technology to simultaneously screen hundreds to thousands of SNPs for their allele-dependent protein-binding differences. This technology takes advantage of higher retention rate of protein-bound DNA oligos in protein purification column to quantitatively sequence these SNP-containing oligos. We apply this technology to test prostate cancer-risk loci and observe differential allelic protein binding in a significant number of selected SNPs. We also test a unique application of self-transcribing active regulatory region sequencing (STARR-seq) in characterizing allele-dependent transcriptional regulation and provide detailed functional analysis at two risk loci (RGS17 and ASCL2). Together, we introduce a powerful high-throughput pipeline for large-scale screening of functional SNPs at disease risk loci.
Assuntos
Predisposição Genética para Doença , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/diagnóstico , Locos de Características Quantitativas , Alelos , Conjuntos de Dados como Assunto , Detecção Precoce de Câncer/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas Nucleares/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Ligação Proteica , RiscoRESUMO
Genome-wide association studies have identified more than 90 susceptibility loci for breast cancer. However, the missing heritability is evident, and the contributions of coding variants to breast cancer susceptibility have not yet been systematically evaluated. Here, we present a large-scale whole-exome association study for breast cancer consisting of 24,162 individuals (10,055 cases and 14,107 controls). In addition to replicating known susceptibility loci (e.g., ESR1, FGFR2, and TOX3), we identify two novel missense variants in C21orf58 (rs13047478, Pmeta = 4.52 × 10-8) and ZNF526 (rs3810151, Pmeta = 7.60 × 10-9) and one new noncoding variant at 7q21.11 (P < 5 × 10-8). C21orf58 and ZNF526 possessed functional roles in the control of breast cancer cell growth, and the two coding variants were found to be the eQTL for several nearby genes. rs13047478 was significantly (P < 5.00 × 10-8) associated with the expression of genes MCM3AP and YBEY in breast mammary tissues. rs3810151 was found to be significantly associated with the expression of genes PAFAH1B3 (P = 8.39 × 10-8) and CNFN (P = 3.77 × 10-4) in human blood samples. C21orf58 and ZNF526, together with these eQTL genes, were differentially expressed in breast tumors versus normal breast. Our study reveals additional loci and novel genes for genetic predisposition to breast cancer and highlights a polygenic basis of disease development.Significance: Large-scale genetic screening identifies novel missense variants and a noncoding variant as predisposing factors for breast cancer. Cancer Res; 78(11); 3087-97. ©2018 AACR.
Assuntos
Povo Asiático/genética , Neoplasias da Mama/genética , Exoma/genética , Predisposição Genética para Doença/genética , Locos de Características Quantitativas/genética , Adulto , Estudos de Casos e Controles , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Glucocorticoids are associated with lipid metabolism and their abnormal expression has an important function in the development of metabolic syndrome. The 11ßhydroxysteroid dehydrogenase type 1 (11ßHSD1) is a metabolic enzyme of glucocorticoids and may be a potential drug target for the treatment of metabolic syndrome. However, the association between the systemic expression of 11ßHSD1 and metabolic syndrome remains to be elucidated. The present study used a cytomegalovirus promoter to obtain mice that systemically overexpressed the 11ßHSD1 gene. The transgenic mice and negative control groups received a highfat diet at the age of 10 weeks in order to induce metabolic syndrome and this diet was continued for 12 weeks. Several indicators, including body weight, blood glucose, glucose tolerance and insulin resistance, were monitored in vivo. In addition, the protein expression levels of 11ßHSD1 and DNA damage inducible transcript 3 were detected and the histopathology of important tissues for metabolic syndrome were analyzed. The current findings revealed that the body weights of transgenic mice were significantly higher compared with the control group before and during the periods of high fat diet induction. Transgenic mice also exhibited significantly impaired glucose tolerance, insulin resistance, endoplasmic reticulum stress and increased metabolic syndromeassociated biochemical indicators in the blood and severely impaired liver and kidney functions. The present study successfully established a 11ßHSD1 systemic overexpression mouse model that exhibited typical characteristics of metabolic syndrome and may be useful for future studies of metabolic syndrome.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Estresse do Retículo Endoplasmático/genética , Efeito Fundador , Resistência à Insulina/genética , Síndrome Metabólica/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Glicemia/metabolismo , Peso Corporal , Citomegalovirus/genética , Citomegalovirus/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Insulina/metabolismo , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Síndrome Metabólica/enzimologia , Síndrome Metabólica/etiologia , Síndrome Metabólica/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Suínos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , TransgenesRESUMO
The RTK/ERK signaling pathway has been implicated in prostate cancer progression. However, the genetic relevance of this pathway to aggressive prostate cancer at the SNP level remains undefined. Here we performed a SNP and gene-based association analysis of the RTK/ERK pathway with aggressive prostate cancer in a cohort comprising 956 aggressive and 347 non-aggressive cases. We identified several loci including rs3217869/CCND2 within the pathway shown to be significantly associated with aggressive prostate cancer. Our functional analysis revealed a statistically significant relationship between rs3217869 risk genotype and decreased CCND2 expression levels in a collection of 119 prostate cancer patient samples. Reduced expression of CCND2 promoted cell proliferation and its overexpression inhibited cell growth of prostate cancer. Strikingly, CCND2 downregulation was consistently observed in the advanced prostate cancer in 18 available clinical data sets with a total amount of 1,095 prostate samples. Furthermore, the lower expression levels of CCND2 markedly correlated with prostate tumor progression to high Gleason score and elevated PSA levels, and served as an independent predictor of biochemical relapse and overall survival in a large cohort of prostate cancer patients. Together, we have identified an association of genetic variants and genes in the RTK/ERK pathway with prostate cancer aggressiveness, and highlighted the potential importance of CCND2 in prostate cancer susceptibility and tumor progression to metastasis.
Assuntos
Ciclina D2/genética , Ciclina D2/metabolismo , Variação Genética , Sistema de Sinalização das MAP Quinases , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Humanos , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Polimorfismo de Nucleotídeo Único , Prognóstico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Análise de SobrevidaRESUMO
N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.
Assuntos
Etilnitrosoureia/metabolismo , Estudos de Associação Genética/métodos , Mutagênese , Mutagênicos/metabolismo , Suínos/genética , Animais , China , Projetos PilotoRESUMO
Today, obesity and nonalcoholic steatohepatitis are a worldwide epidemic, although how these syndromes are regulated with respect to lncRNAs remains largely unknown. Our previous studies have revealed important pathological features and molecular characteristics of nonalcoholic steatohepatitis in the minipig model, and in this study, we analyze the features of lncRNAs and their potential target genes. Minipig samples only from liver were analyzed using next-generation deep sequencing. In total, we obtained 585 million raw reads approximately 70.4 Gb of high quality data. After a strict five-step filtering process, 1,179 lncRNAs were identified, including 89 differentially expressed lncRNAs (P < 0.05) in the experiment group relative to the control group. The cis and trans analysis identified target genes that were enriched for specific GO terms (P < 0.01), including immune processes, chemokine activity, cytokine activity, and G-protein coupled receptor binding, which are closely related to nonalcoholic steatohepatitis. The predicted protein-coding targets of the differentially expressed lncRNAs were further analyzed, such as PPAR, FADS2, DGAT2, ACAA2, CYP2E1, ADH4, and Fos. This study reveals a wealth of candidate lncRNAs involved in NASH and their regulated pathways, which should facilitate further research into the molecular mechanisms of this disorder.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Perfilação da Expressão Gênica , Hepatopatia Gordurosa não Alcoólica/patologia , RNA Longo não Codificante/análise , Animais , Modelos Animais de Doenças , Sequenciamento de Nucleotídeos em Larga Escala , Fígado/patologia , RNA Longo não Codificante/genética , Suínos , Porco MiniaturaRESUMO
Skeletal muscle is as an important regulator of blood glucose and glycolipid metabolism and is closely related to motor ability. The underlying mechanisms by which dietary ectopic lipids in skeletal muscle prevents muscle growth remain elusive. We utilized miniature Bama swine as a model to mimic human obesity using prolonged dietary induction. After 23 months on a high-fat, high-sucrose diet, metabolic disorders were induced in the animals, which exhibited increased body weight, extensive lipid deposition in the skeletal muscle and amyotrophy. Microarray profiles demonstrated the up-regulation of genes related to fat deposition and muscle growth inhibition. We outline a clear potential pathway that in combination with increased 11ß-hydroxysteroid dehydrogenase type 1, promotes expression of a major inhibitor, myostatin, by converting corticosterone to cortisol, which leads to the growth inhibition of skeletal muscle. This research provides new insights into the treatment of muscle diseases induced by obesity.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Síndrome Metabólica/complicações , Atrofia Muscular/etiologia , Miostatina/genética , Sacarose/efeitos adversos , Regulação para Cima , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/genética , Atrofia Muscular/genética , Análise de Sequência com Séries de Oligonucleotídeos , Sacarose/administração & dosagem , Suínos , Porco MiniaturaRESUMO
There are currently no multitransgenic minipig models of diabetes for the regulation of multiple genes involved in its pathogenesis. The foot and mouth disease virus 2A (F2A)mediated polycistronic system possesses several advantages, and the present study developed a novel multitransgenic minipig model associated with diabetes using this system. The tissuespecific polycistronic system used in the present study consisted of two expression cassettes, separated by an insulator: (i) 11ßhydroxysteroid dehydrogenase 1 (11ßHSD1), driven by the porcine liverspecific apolipoprotein E promoter; (ii) human islet amyloid polypeptide (hIAPP) and C/EBP homologous protein (CHOP), linked to the furin digested site and F2A, driven by the porcine pancreasspecific insulin promoter. In the present study, porcine fetal fibroblasts were transfected with this vector. Following somatic cell nuclear transfer using 10 cell clones and the transplantation of 1,459 embryos in total, three Landrace x Yorkshire surrogates became pregnant and delivered three Wuzhishan piglets. Genomic polymerase chain reaction (PCR) demonstrated that the piglets were multitransgenic. Reverse transcriptionquantitative PCR confirmed that 11ßHSD1 transcription was upregulated in the targeted liver. Similarly, hIAPP and CHOP were expressed at high levels, compared with the control (P<0.05 and P<0.01) in the pancreas, consistent with the western blotting and immunohistochemistry results. The primary results also showed that overexpression of 11ßHSD1 in the liver increased the liver fat lipid parameters; and the levels of hIAPP and CHOP in the pancreatic islet cells, leading to delayed ßcell development and apoptosis. This novel tissuespecific polycistronic system offers a promising starting point for efficiently mimicking multigenic metabolic disease.
Assuntos
Apoptose , Resistência à Insulina , Fígado/patologia , Pâncreas/patologia , Porco Miniatura/genética , Animais , Animais Geneticamente Modificados , Embrião de Mamíferos/metabolismo , Feminino , Feto/citologia , Fibroblastos/metabolismo , Dosagem de Genes , Técnicas de Genotipagem , Imuno-Histoquímica , Técnicas de Transferência Nuclear , Fenótipo , Gravidez , Suínos , TransgenesRESUMO
Metabolic syndrome can induce chronic renal injury in humans. In the present study, Bama minipigs were fed a high-fat/high-sucrose diet (HFHSD) for 23 months, which caused them to develop the pathological characteristics of metabolic syndrome, including obesity, hyperinsulinemia, and hyperlipidemia, and resulted in kidney tissue damage. In the HFHSD group, the ratio of the glomus areas to the glomerulus area and the glomerular density inside the renal cortex both decreased. Lipid deposition in the renal tubules was detected in the HFHSD group, and up-regulated expression levels of SREBP-1, FABP3 and LEPR promoted lipid deposition. The decreased levels of SOD, T-AOC and GSH-PX indicated that the antioxidant capacity of the renal tissues was diminished in the HFHSD group compared with MDA, which increased. The renal tissue in the HFHSD group exhibited clear signs of inflammation as well as significantly elevated expression of key genes associated with inflammation, including tumor necrosis factor-α (TNF-α) and macrophage migration inhibitory factor (MIF), compared with the control group. The tubular epithelial cells in the HFHSD group displayed significantly greater numbers of apoptotic cells, and the expression of proliferating cell nuclear antigen (PCNA) in the renal tubules decreased. Caspase-3 expression increased significantly, and the transcription factor nuclear factor κB (NF-κB) was activated and translocated into the nucleus. In conclusion, long-term HFHSDs cause metabolic syndrome and chronic renal tissue injury in Bama minipigs. These findings provide a foundation for further studies investigating metabolic syndrome and nephropathy.
Assuntos
Apoptose , Dieta Hiperlipídica/efeitos adversos , Sacarose Alimentar/efeitos adversos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Metabolismo dos Lipídeos , Animais , Antioxidantes/metabolismo , Caspase 3/metabolismo , Regulação da Expressão Gênica , Hipertrofia , Inflamação/complicações , Inflamação/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Síndrome Metabólica/complicações , NF-kappa B/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Coloração e Rotulagem , Suínos , Porco Miniatura , Fatores de TempoRESUMO
The minipig can serve as a good pharmacological model for human subjects. However, the long-term pathogenesis of high-calorie diet-induced metabolic syndromes, including NASH, has not been well described in minipigs. We examined the development of metabolic syndromes in Bama minipigs that were fed a high-fat, high-sucrose diet (HFHSD) for 23 months, by using histology and serum biochemistry and by profiling the gene expression patterns in the livers of HFHSD pigs compared to controls. The pathology findings revealed microvesicular steatosis, iron overload, arachidonic acid synthesis, lipid peroxidation, reduced antioxidant capacity, increased cellular damage, and inflammation in the liver. RNA-seq analysis revealed that 164 genes were differentially expressed between the livers of the HFHSD and control groups. The pathogenesis of early-stage NASH was characterized by hyperinsulinemia and by de novo synthesis of fatty acids and nascent triglycerides, which were deposited as lipid droplets in hepatocytes. Hyperinsulinemia shifted the energy supply from glucose to ketone bodies, and the high ketone body concentration induced the overexpression of cytochrome P450 2E1 (CYP2E1). The iron overload, CYP2E1 and alcohol dehydrogenase 4 overexpression promoted reactive oxygen species (ROS) production, which resulted in arachidonic and linoleic acid peroxidation and, in turn, led to malondialdehyde production and a cellular response to ROS-mediated DNA damage.
Assuntos
Glucose/metabolismo , Hiperinsulinismo/complicações , Hiperinsulinismo/metabolismo , Corpos Cetônicos/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Antioxidantes/metabolismo , Peso Corporal , Colesterol/sangue , Colesterol/metabolismo , Dano ao DNA , Dieta Hiperlipídica , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Fibrose , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Hiperplasia , Resistência à Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Metabolismo dos Lipídeos , Peroxidação de Lipídeos , Fígado/metabolismo , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo , Fenótipo , Suínos , Transcriptoma , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
A long-term high-energy diet affects human health and leads to obesity and metabolic syndrome in addition to cardiac steatosis and hypertrophy. Ectopic fat accumulation in the heart has been demonstrated to be a risk factor for heart disorders, but the molecular mechanism of heart disease remains largely unknown. Bama miniature pigs were fed a high-fat, high-sucrose diet (HFHSD) for 23 months. These pigs developed symptoms of metabolic syndrome and showed cardiac steatosis and hypertrophy with a greatly increased body weight (2.73-fold, P<0.01), insulin level (4.60-fold, P<0.01), heart weight (1.82-fold, P<0.05) and heart volume (1.60-fold, P<0.05) compared with the control pigs. To understand the molecular mechanisms of cardiac steatosis and hypertrophy, nine pig heart cRNA samples were hybridized to porcine GeneChips. Microarray analyses revealed that 1,022 genes were significantly differentially expressed (P<0.05, ≥1.5-fold change), including 591 up-regulated and 431 down-regulated genes in the HFHSD group relative to the control group. KEGG analysis indicated that the observed heart disorder involved the signal transduction-related MAPK, cytokine, and PPAR signaling pathways, energy metabolism-related fatty acid and oxidative phosphorylation signaling pathways, heart function signaling-related focal adhesion, axon guidance, hypertrophic cardiomyopathy and actin cytoskeleton signaling pathways, inflammation and apoptosis pathways, and others. Quantitative RT-PCR assays identified several important differentially expressed heart-related genes, including STAT3, ACSL4, ATF4, FADD, PPP3CA, CD74, SLA-8, VCL, ACTN2 and FGFR1, which may be targets of further research. This study shows that a long-term, high-energy diet induces obesity, cardiac steatosis, and hypertrophy and provides insights into the molecular mechanisms of hypertrophy and fatty heart to facilitate further research.
Assuntos
Cardiomegalia/genética , Dieta Hiperlipídica/efeitos adversos , Perfilação da Expressão Gênica , Miocárdio/patologia , Porco Miniatura/genética , Animais , Cardiomegalia/complicações , Análise por Conglomerados , Dislipidemias/complicações , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Obesidade/complicações , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Coloração e Rotulagem , Suínos , Fatores de TempoRESUMO
OBJECTIVES: The effect of a long-term high-fat, high-caloric diet on the dysfunction of pancreas has not been clarified. We investigated the pancreatic histopathology and ß-cell apoptosis in Bama minipigs after 23 months on a high-fat high-sucrose diet (HFHSD). METHODS: Bama minipigs were randomly assigned to control (n = 6) and HFHSD groups (n = 6) for 23 months, and biochemical parameters were measured. Pancreata were subjected to histological analysis, followed by assessment with transmission electron microscopy. Lipid peroxidation was determined by the malondialdehyde concentration and antioxidant enzyme activity. Β-cell apoptosis was measured by an immunohistochemical method. RESULTS: In the HFHSD group, the islets were enlarged, and the pancreatic tissue had observed significant fatty infiltration. Moreover, the feeding program damaged the normal pancreatic tissue structure. The level of lipid peroxidation was increased, and the activities of pancreatic antioxidant enzymes were significantly decreased. The expression levels of caspase-3, Bax, and insulin were significantly increased (P < 0.05), and the expression levels of proliferating cell nuclear antigen and Bcl-2 were decreased (P < 0.05). CONCLUSIONS: The long-term HFHSD promotes pancreatic steatosis and oxidative stress, which increases ß-cell apoptosis as indicated by the activation of caspase-3 through the mitochondrial pathway (Bcl-2/Bax).