RESUMO
During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades , Proteínas com Domínio T , Animais , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Botões de Extremidades/metabolismo , Botões de Extremidades/embriologia , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Gli3 com Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Regulação para Cima/genética , Padronização Corporal/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Mesoderma/metabolismo , Mesoderma/embriologiaRESUMO
North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy.
Assuntos
Distrofias Hereditárias da Córnea , Tomografia de Coerência Óptica , Adulto , Animais , Humanos , Linhagem , Retina/metabolismo , Xenopus laevis/genéticaRESUMO
Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox 3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.
Assuntos
Tecido Adiposo Marrom , Obesidade , Camundongos , Animais , Obesidade/genética , Obesidade/metabolismo , Tecido Adiposo Marrom/metabolismo , Aumento de Peso/genética , Tecido Adiposo Branco/metabolismo , Metabolismo Energético/genética , Fenótipo , Camundongos Endogâmicos C57BL , Dieta Hiperlipídica , Camundongos KnockoutRESUMO
Endometrial carcinoma (EC) is a rising concern among gynecological malignancies. Iroquois Homeobox 2 (IRX2), a member of the Iroquois homeobox gene family, demonstrates variable effects in different cancer types, emphasizing the need for extensive exploration of its involvement in EC progression. Utilizing TCGA and GEO databases, as well as performing immunohistochemistry (IHC) analysis on clinical samples, we assessed the expression levels of IRX2 and its promoter methylation in EC. To understand the functional roles of IRX2, we conducted various assays including in vitro CCK-8 assays, colony formation assays, cell invasion assays, and cell apoptosis assays. Moreover, we utilized in vivo subcutaneous xenograft mouse models. Additionally, we performed KEGG pathway and gene set enrichment analyses to gain insights into the underlying mechanisms. To validate the regulatory relationship between IRX2 and RUVBL1, we employed chromatin immunoprecipitation and luciferase reporter assays. Our results indicate significantly reduced levels of IRX2 expression in EC, correlating with higher histological grades, advanced clinical stages, and diminished overall survival. We observed that DNA methylation of the IRX2 promoter suppresses its expression in EC, with cg26333652 and cg11793269 playing critical roles as methylated sites. In contrast, ectopic overexpression of IRX2 substantially inhibits cell proliferation and invasion, and promotes cell apoptosis. Additionally, we discovered that IRX2 exerts negative regulation on the expression of RUVBL1, which is upregulated in EC and associated with a poorer prognosis. In conclusion, our findings indicate that decreased expression of IRX2 facilitates EC cell growth through the regulation of RUVBL1 expression, thereby contributing to the development of EC. Hence, targeting the IRX2-RUVBL1 axis holds promise as a potential therapeutic strategy for EC treatment.
Assuntos
Neoplasias do Endométrio , MicroRNAs , Feminino , Humanos , Animais , Camundongos , Transformação Celular Neoplásica/genética , Genes Homeobox , Apoptose/genética , Neoplasias do Endométrio/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismoRESUMO
Iroquois Homeobox 4 (IRX4) belongs to a family of homeobox TFs having roles in embryogenesis, cell specification, and organ development. Recently, large scale genome-wide association studies and epigenetic studies have highlighted the role of IRX4 and its associated variants in prostate cancer. No studies have investigated and characterized the structural aspect of the IRX4 homeodomain and its potential to bind to DNA. The current study uses sequence analysis, homology modeling, and molecular dynamics simulations to explore IRX4 homeodomain-DNA recognition mechanisms and the role of somatic mutations affecting these interactions. Using publicly available databases, gene expression of IRX4 was found in different tissues, including prostate, heart, skin, vagina, and the protein expression was found in cancer cell lines (HCT166, HEK293), B cells, ascitic fluid, and brain. Sequence conservation of the homeodomain shed light on the importance of N- and C-terminal residues involved in DNA binding. The specificity of IRX4 homodimer bound to consensus human DNA sequence was confirmed by molecular dynamics simulations, representing the role of conserved amino acids including R145, A194, N195, S190, R198, and R199 in binding to DNA. Additional N-terminal residues like T144 and G143 were also found to have specific interactions highlighting the importance of N-terminus of the homeodomain in DNA recognition. Additionally, the effects of somatic mutations, including the conserved Arginine (R145, R198, and R199) residues on DNA binding elucidated the importance of these residues in stabilizing the protein-DNA complex. Secondary structure and hydrogen bonding analysis showed the roles of specific residues (R145, T191, A194, N195, R198, and R199) in maintaining the homogeneity of the structure and its interaction with DNA. The differences in relative binding free energies of all the mutants shed light on the structural modularity of this protein and the dynamics behind protein-DNA interaction. We also have predicted that the C-terminal sequence of the IRX4 homeodomain could act as a potential cell-penetrating peptide, emphasizing the role these small peptides could play in targeting homeobox TFs.
Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição , Masculino , Humanos , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Estudo de Associação Genômica Ampla , Células HEK293RESUMO
Proper function of the vertebrate skeleton requires the development of distinct articulating embryonic cartilages. Irx transcription factors are arranged in co-regulated clusters that are expressed in the developing skeletons of the face and appendages. IrxB cluster genes are required for the separation of toes in mice and formation of the hyoid joint in zebrafish, yet whether Irx genes have broader roles in skeletal development remains unclear. Here, we perform a comprehensive loss-of-function analysis of all 11 Irx genes in zebrafish. We uncover conserved requirements for IrxB genes in formation of the fish and mouse scapula. In the face, we find a requirement for IrxAb genes and irx7 in formation of anterior neural crest precursors of the jaw, and for IrxBa genes in formation of endodermal pouches and gill cartilages. We also observe extensive joint loss and cartilage fusions in animals with combinatorial losses of Irx clusters, with in vivo imaging revealing that at least some of these fusions arise through inappropriate chondrogenesis. Our analysis reveals diverse roles for Irx genes in the formation and later segmentation of the facial skeleton.
Assuntos
Cartilagem/embriologia , Condrogênese/genética , Proteínas de Homeodomínio/metabolismo , Família Multigênica , Proteínas Mutantes/metabolismo , Crânio/embriologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Alelos , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Mutação , Crista Neural/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Hypoplastic left heart syndrome (HLHS) is a severe congenital cardiovascular malformation characterized by hypoplasia of the left ventricle, aorta, and other structures on the left side of the heart. The pathologic definition includes atresia or stenosis of both the aortic and mitral valves. Despite considerable progress in clinical and surgical management of HLHS, mortality and morbidity remain concerns. One barrier to progress in HLHS management is poor understanding of its cause. Several lines of evidence point to genetic origins of HLHS. First, some HLHS cases have been associated with cytogenetic abnormalities (e.g., Turner syndrome). Second, studies of family clustering of HLHS and related cardiovascular malformations have determined HLHS is heritable. Third, genomic regions that encode genes influencing the inheritance of HLHS have been identified. Taken together, these diverse studies provide strong evidence for genetic origins of HLHS and related cardiac phenotypes. However, using simple Mendelian inheritance models, identification of single genetic variants that "cause" HLHS has remained elusive, and in most cases, the genetic cause remains unknown. These results suggest that HLHS inheritance is complex rather than simple. The implication of this conclusion is that researchers must move beyond the expectation that a single disease-causing variant can be found. Utilization of complex models to analyze high-throughput genetic data requires careful consideration of study design.
Assuntos
Síndrome do Coração Esquerdo Hipoplásico , Humanos , Predisposição Genética para Doença/genética , Síndrome do Coração Esquerdo Hipoplásico/genética , FenótipoRESUMO
Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.
Assuntos
Comunicação Interventricular , Humanos , Aberrações Cromossômicas , Variações do Número de Cópias de DNA/genética , Predisposição Genética para Doença/genética , Comunicação Interventricular/genética , Mutação , Fatores de Transcrição/genéticaRESUMO
Iroquois homeobox (Irx) genes are TALE-class homeobox genes that are evolutionarily conserved across species and have multiple critical cellular functions in fundamental tissue development processes. Previous studies have shown that Irxs genes are expressed during tooth development. However, the precise roles of genes in teeth remain unclear. Here, we demonstrated for the first time that Irx3 is an essential molecule for the proliferation and differentiation of odontoblasts. Using cDNA synthesized from postnatal day 1 (P1) tooth germs, we examined the expression of all Irx genes (Irx1-Irx6) by RT-PCR and found that all genes except Irx4 were expressed in the tooth tissue. Irx1-Irx3 a were expressed in the dental epithelial cell line M3H1 cells, while Irx3 and Irx5 were expressed in the dental mesenchymal cell line mDP cells. Only Irx3 was expressed in both undifferentiated cell lines. Immunostaining also revealed the presence of IRX3 in the dental epithelial cells and mesenchymal condensation. Inhibition of endogenous Irx3 by siRNA blocks the proliferation and differentiation of mDP cells. Wnt3a, Wnt5a, and Bmp4 are factors involved in odontoblast differentiation and were highly expressed in mDP cells by quantitative PCR analysis. Interestingly, the expression of Wnt5a (but not Wnt3a or Bmp4) was suppressed by Irx3 siRNA. These results suggest that Irx3 plays an essential role in part through the regulation of Wnt5a expression during odontoblast proliferation and differentiation.
Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Odontoblastos/metabolismo , Genes Homeobox , Diferenciação Celular , Proliferação de CélulasRESUMO
Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14Cre /Pitx2flox/flox (Pitx2cKO ) and Rosa26CreERT/Pitx2flox/flox mice that loss of Pitx2 delays epithelial invagination, and decreases progenitor cell proliferation and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2+ labial cervical loop (LaCL) stem cell niche in concert with two signaling centers: the initiation knot and enamel knot. The loss of Pitx2 disrupted the patterning of these two signaling centers, resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2, Sox2, Lef1 and Irx1.
Assuntos
Células Epiteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Esmalte Dentário/metabolismo , Embrião de Mamíferos/metabolismo , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Camundongos Knockout , Odontogênese , Fatores de Transcrição SOXB1/genética , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Dente/citologia , Dente/crescimento & desenvolvimento , Dente/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas de Sinalização YAP , Proteína Homeobox PITX2RESUMO
Piao chicken, a Chinese indigenous rumpless chicken breed, lacks pygostyle, caudal vertebra, uropygial gland and tail feathers. The rumplessness in Piao chicken presents an autosomal dominant inheritance pattern. However, the molecular genetic mechanisms underlying the rumplessness in Piao chicken remains unclear. In this study, whole-genome resequencing was performed for 146 individuals from 10 chicken breeds, including 9 tailed chicken breeds and Piao rumpless breed. Tailbone CT scan for Piao chickens and WL chickens, revealed that some Piao chicken tails were normal in number, and for a few Piao chickens tail length and tail bone numbers were between the rumpless and the normal tailed chickens. The results showed that the rumpless phenotype has not been completely fixed in Piao chicken breed. Using selection signature analysis and structural variation detection, we found a 4174 bp deletion located in the upstream region of IRX1 gene on chromosome 2 related to rumpless phenotype. Structural variation genotyping showed that the deletion was present in all 32 rumpless Piao chickens (del/del, wild/del) and absent from all 112 tailed chickens included in the dataset for the other 9 breeds and 2 tailed Piao chickens (wild/wild). In summary, all rumpless Piao chickens tested here carry this deletion mutation, to show a complete linkage association with rumplessness trait. We suggested that the 4174 bp deletion could be causative for rumpless phenotype in Piao chicken since this is the only mutation to show the complete linkage disequilibrium with rumplessness on whole genome level across all of 146 chickens from the 10 breeds. This study could facilitate a better understanding of the genetic characteristics of Piao chicken.
Assuntos
Galinhas , Animais , Galinhas/genéticaRESUMO
Previous studies have established that transmural gradients of the fast transient outward K+ current (Ito,f) correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high Ito,f expression in the epimyocardium (EPI) being associated with short APs and low contractility and vice versa. Herein, we investigated the effects of altering the Ito,f gradients on transmural contractile properties using mice lacking Irx5 (Irx5-KO) or lacking Kcnd2 (KV4.2-KO) or both. Irx5-KO mice exhibited decreased global LV contractility in association with elevated Ito,f, as well as reduced cell shortening and Ca2+ transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of Irx5 ablation on ECC-related genes was to increase Ito,f gene expression (i.e., Kcnd2 and Kcnip2) in the ENDO, but not the EPI. By contrast, KV4.2-KO mice showed selective increases in cell shortening and Ca2+ transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both Irx5 and Kcnd2 displayed elevated ventricular contractility, comparable to KV4.2-KO mice, demonstrating a dominant role of Irx5-dependent modulation of Ito,f in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the Irx5-dependent Ito,f gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart.NEW & NOTEWORTHY Irx5 is a vital transcription factor that establishes the transmural heterogeneity of ventricular myocyte contractility, thereby ensuring proper contractile function in the healthy heart. Regional differences in excitation-contraction coupling in the ventricular myocardium are primarily mediated through the inverse relationship between Irx5 and the fast transient outward K+ current (Ito,f) across the ventricular wall.
Assuntos
Ventrículos do Coração , Miocárdio , Potenciais de Ação/fisiologia , Animais , Ventrículos do Coração/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular compartments with discrete physiological identities. Here, from a forward genetic screen using zebrafish, we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that activates the terminal differentiation program of distal segments in the pronephros. We found that tfap2a acts downstream of Iroquois homeobox 3b (irx3b), a distal lineage transcription factor, to operate a circuit consisting of tfap2b, irx1a and genes encoding solute transporters that dictate the specialized metabolic functions of distal nephron segments. Interestingly, this regulatory node is distinct from other checkpoints of differentiation, such as polarity establishment and ciliogenesis. Thus, our studies reveal insights into the genetic control of differentiation, where tfap2a is essential for regulating a suite of segment transporter traits at the final tier of zebrafish pronephros ontogeny. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to facilitate drug discovery and regenerative therapies.
Assuntos
Rim/embriologia , Néfrons/embriologia , Organogênese/genética , Fator de Transcrição AP-2/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Diferenciação Celular/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes de Troca/fisiologia , Rim/metabolismo , Néfrons/metabolismo , Pronefro/embriologia , Pronefro/crescimento & desenvolvimento , Pronefro/metabolismo , Fator de Transcrição AP-2/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Sepsis-induced acute kidney injury is a general critical complication having high relevance to kidney inflammation. In spite of advances in clinical and critical care, the specific and effective therapies for acute kidney injury are still insufficient. The present study aimed to investigate the protective effect of Iroquois homeobox genes (IRX) on sepsis-induced kidney dysfunction in mice. METHODS: In order to gain insight into sepsis-related actions in acute kidney injury, the cecal puncture-induced kidney injury animal model was established. The hematoxylin and eosin staining was used to measure the pathology of kidney tissues. The kidney function-related biomarkers, including neutrophil gelatinase-associated lipocalin, creatinine, kidney injury molecule-1, blood urea nitrogen, and inflammatory cytokines, which included tumor necrosis factor α, interleukin 1ß (IL-1ß), IL-6, and monocyte chemotactic protein 1, were detected by automated biochemical analyzer or their corresponding test kits. The protein expression was measured using Western blot analysis, and the apoptotic rate of kidney tissue was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: The present study revealed the protective ability of IRX1 in sepsis-induced acute kidney injury. This study also determined the potential mechanism of IRX1 on sepsis-induced inflammatory response and cell apoptosis. Finally, it highlighted that IRX1 exerted a protective influence on CLP-induced acute kidney injury by suppressing the activation of chemokine (C-X-C motif) ligand 14 (CXCL14). CONCLUSION: To conclude, the results suggest that overexpression of IRX1 could promote survival rate and suppress the CLP-induced apoptosis, inflammatory response, and kidney dysfunction through the activation of CXCL14. IRX1 and CXCL14 are essential to elucidate the mechanism of acute kidney injury. These findings may help to identify the promising targets for clinical sepsis therapy.
Assuntos
Injúria Renal Aguda , Sepse , Camundongos , Animais , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Sepse/complicações , Sepse/tratamento farmacológico , Sepse/patologia , Fator de Necrose Tumoral alfa , Modelos Animais de Doenças , Apoptose , Rim/metabolismo , Rim/patologia , Quimiocinas CXC/farmacologia , Quimiocinas CXC/uso terapêuticoRESUMO
BACKGROUND: This study aimed to explore role of miR-646 in breast IDC. METHODS: miR-646, TET1, IRX1, and HIST2H2BE expression was detected by RT-qPCR and/or Western blot analysis. The methylation status of IRX1 promoter region was evaluated by methylation specific PCR. ChIP assay was used to determine the enrichment of TET1 at IRX1 promoter region. Loss- and gain-of functions were performed to determine the roles of miR-646, TET1, IRX1, and HIST2H2BE in cell proliferation, migration, invasion, and apoptosis. The tumor growth, volume, weight, and apoptosis status were measured. RESULTS: miR-646 was upregulated while TET1 was downregulated in IDC tissues. miR-646 targeted TET1. Downregulated TET1 impairs demethylation of IRX1 promoter region resulting in reduced expression of IRX1, which subsequently leads to upregulation of HIST2H2BE in IDC. Consequently, elevated HIST2H2BE promotes progression of IDC. CONCLUSION: Our study has demonstrated that miR-646 facilitates the tumorigenesis of IDC via regulating TET1/IRX1/HIST2H2BE axis.
Assuntos
Carcinoma Ductal , MicroRNAs , Linhagem Celular Tumoral , Metilação de DNA , Desmetilação , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The H&E stromal tumor-infiltrating lymphocyte (sTIL) score and programmed death ligand 1 (PD-L1) SP142 immunohistochemistry assay are prognostic and predictive in early-stage breast cancer, but are operator-dependent and may have insufficient precision to characterize dynamic changes in sTILs/PD-L1 in the context of clinical research. We illustrate how multiplex immunofluorescence (mIF) combined with statistical modeling can be used to precisely estimate dynamic changes in sTIL score, PD-L1 expression, and other immune variables from a single paraffin-embedded slide, thus enabling comprehensive characterization of activity of novel immunotherapy agents. METHODS: Serial tissue was obtained from a recent clinical trial evaluating loco-regional cytokine delivery as a strategy to promote immune cell infiltration and activation in breast tumors. Pre-treatment biopsies and post-treatment tumor resections were analyzed by mIF (PerkinElmer Vectra) using an antibody panel that characterized tumor cells (cytokeratin-positive), immune cells (CD3, CD8, CD163, FoxP3), and PD-L1 expression. mIF estimates of sTIL score and PD-L1 expression were compared to the H&E/SP142 clinical assays. Hierarchical linear modeling was utilized to compare pre- and post-treatment immune cell expression, account for correlation of time-dependent measurement, variation across high-powered magnification views within each subject, and variation between subjects. Simulation methods (Monte Carlo, bootstrapping) were used to evaluate the impact of model and tissue sample size on statistical power. RESULTS: mIF estimates of sTIL and PD-L1 expression were strongly correlated with their respective clinical assays (p < .001). Hierarchical linear modeling resulted in more precise estimates of treatment-related increases in sTIL, PD-L1, and other metrics such as CD8+ tumor nest infiltration. Statistical precision was dependent on adequate tissue sampling, with at least 15 high-powered fields recommended per specimen. Compared to conventional t-testing of means, hierarchical linear modeling was associated with substantial reductions in enrollment size required (n = 25ân = 13) to detect the observed increases in sTIL/PD-L1. CONCLUSION: mIF is useful for quantifying treatment-related dynamic changes in sTILs/PD-L1 and is concordant with clinical assays, but with greater precision. Hierarchical linear modeling can mitigate the effects of intratumoral heterogeneity on immune cell count estimations, allowing for more efficient detection of treatment-related pharmocodynamic effects in the context of clinical trials. TRIAL REGISTRATION: NCT02950259 .
Assuntos
Antígeno B7-H1/metabolismo , Biomarcadores Tumorais , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Antígeno B7-H1/genética , Análise de Dados , Feminino , Imunofluorescência/métodos , Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/patologia , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologiaRESUMO
BACKGROUND: The roles of FTO gene and the level of serum 25-OH-vitamin D in obesity are frequently reported. This study aimed to investigate the interactions of serum 25-OH-vitamin D level, FTO and IRX3 genes expression, and FTO genotype in obese and overweight boys. METHODS: This study was carried out on the 120 male adolescents with overweight in Tehran, Iran. Blood samples were collected from the participants in order to evaluate the serum level of 25-OH-vitamin D, the expression level of FTO and IRX3 genes, and FTO genotype for rs9930506 at baseline and after 18 weeks of the study. RESULTS: In general, no significant association was found between serum 25-OH-vitamin D level and IRX3 and FTO genes expression. The results of linear regression on the relationship between 25-OH-vitamin D serum level and FTO and IRX3 genes expression based on FTO genotypes for rs9930506 indicated that in AA/AG genotype carriers, serum 25-OH-vitamin D level was positively associated with FTO gene expression (B = 0.07, p = 0.02) and inversely associated with IRX3 gene expression (B = - 0.07, p = 0.03). In GG carriers, serum 25-OH-vitamin D level was not associated with expression of IRX3 and FTO genes. CONCLUSION: There are significant interactions between 25-OH-vitamin D and the expression of FTO and IRX3 genes in the subset of obese patients with specific genotypes for FTO rs9930506. There was no association between serum 25-OH-vitamin D levels and the expression of FTO and IRX genes in individuals with a homozygous genotype for the risk allele of the FTO gene polymorphism.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Polimorfismo de Nucleotídeo Único , Adolescente , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Expressão Gênica , Genótipo , Proteínas de Homeodomínio/genética , Humanos , Irã (Geográfico) , Masculino , Obesidade/genética , Sobrepeso , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Vitamina DRESUMO
Obesity has become a genuine global pandemic due to lifestyle and environmental modifications, and is associated with chronic lethal comorbidities. Various environmental factors such as lack of physical activity due to modernization and higher intake of energy-rich diets are primary obesogenic factors in pathogenesis of obesity. Genome-wide association study has identified the crucial role of FTO (fat mass and obesity) in human obesity. A bunch of SNPs in the first intron of FTO has been identified and subsequently correlated to body mass index and body composition. Findings of in silico, in vitro, and in vivo studies have manifested the robust role of FTO in regulation of energy expenditure and food consumption. Numerous studies have highlighted the mechanistic pathways behind the concomitant functions of FTO in adipogenesis and body size. Current investigation has also revealed the link of FTO neighbouring genes i.e., RPGRIP1L, IRX3 and IRX5 and epigenetic factors with obesity phenotypes. The motive behind this review is to cite the consequences of FTO on obesity vulnerability.
Assuntos
Estudo de Associação Genômica Ampla , Obesidade , Índice de Massa Corporal , Metabolismo Energético , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
As a member of the Iroquois homeobox gene family, the IRX3 gene plays an important role in regulating the growth, development and fat deposition of chordates. In the present study, we found, using real-time PCR, that the bovine IRX3 gene was highly expressed in lung, kidney, heart, subcutaneous fat and longissimus dorsi muscle. We cloned the full-length sequence of the bovine IRX3 gene promoter and constructed eight series of 5' deletion promoter plasmid luciferase reporter assays and then transfected them to 3T3-L1 and C2C12 cell lines to detect its core promoter regions. The results showed that the core promoter of bovine IRX3 was located within a -292/-42 bp region relative to the transcriptional start site. Furthermore, sequence analysis identified eight CpG islands in the promoter region. A chromatin immunoprecipitation assay in combination with site-directed mutation and siRNA interference demonstrated that SREBF2 and PPARG binding occurs in region -292/-42 and is essential in bovine IRX3 transcription. These results lay an important theoretical foundation for exploring the molecular regulation mechanism of the IRX3 gene in bovine fat deposition.
Assuntos
Proteínas de Homeodomínio/genética , PPAR gama/metabolismo , Regiões Promotoras Genéticas/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Fatores de Transcrição/genética , Células 3T3-L1 , Animais , Sítios de Ligação , Bovinos , Imunoprecipitação da Cromatina , Ilhas de CpG/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Rim/metabolismo , Pulmão/metabolismo , Camundongos , PPAR gama/genética , Interferência de RNA , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica , TransfecçãoRESUMO
The Iroquois homeobox gene 5 (IRX5), one of the members of the Iroquois homeobox family, has been identified to correlate with worse prognosis in many cancers, including colorectal cancer (CRC). In this study, upregulation of IRX5 revealed a great reduction in the proliferation of CRC colorectal cancer cell line SW480 and DLD-1, which was accompanied by G1/S arrest, increased expression in cyclin E1, P21, and P53 and a decrease in cyclin A2, B1, and D1. Furthermore, IRX5-mediated an increase expression of RH2A protein, the biomarker of DNA damage. Consequently, the SA-ß-gal level is higher in IRX5-overexpression cells compared to control ones, which showed elevated DNA damage triggered cellular senescence. Recapitulating the above findings, IRX5 exhibited higher levels of genomic instability. IRX5 may be a perspective target for cancer therapy and it deserves further investigation.