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1.
Int Heart J ; 60(5): 1113-1122, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31484864

RESUMO

Occurring in about 1% of all live births, congenital heart defects (CHDs) represent the most frequent type of developmental abnormality and account for remarkably increased infant morbidity and mortality. Aggregating studies demonstrate that genetic components have a key role in the occurrence of CHDs. Nevertheless, due to pronounced genetic heterogeneity, the genetic causes of CHDs remain unclear in most patients. In this research, 114 unrelated patients affected with CHDs and 218 unrelated individuals without CHDs served as controls were recruited. The coding regions and splicing donors/acceptors of the ISL1 gene, which codes for a transcription factor required for proper cardiovascular development, were screened for mutations by sequencing in all study participants. The functional characteristics of an identified ISL1 mutation were delineated with a dual-luciferase reporter assay system. As a result, a new heterozygous ISL1 mutation, NM_002202.2: c.225C>G; p. (Tyr75*), was discovered in an index patient with double outlet right ventricle and ventricular septal defect. Analysis of the proband's family unveiled that the mutation co-segregated with the CHD phenotype. The nonsense mutation was absent in the 436 control chromosomes. Biological analysis showed that the mutant ISL1 protein had no transcriptional activity. Furthermore, the mutation nullified the synergistic activation between ISL1 and TBX20, another CHD-associated transcription factor. This research for the first time links an ISL1 loss-of-function mutation to double outlet right ventricle in humans, which adds insight to the molecular pathogenesis underpinning CHDs, suggesting potential implications for timely personalized management of CHD patients.


Assuntos
Dupla Via de Saída do Ventrículo Direito/genética , Genes Reporter/genética , Predisposição Genética para Doença/epidemiologia , Proteínas com Homeodomínio LIM/genética , Mutação com Perda de Função/genética , Fatores de Transcrição/genética , Estudos de Casos e Controles , Causalidade , Pré-Escolar , China/epidemiologia , Dupla Via de Saída do Ventrículo Direito/diagnóstico por imagem , Feminino , Cardiopatias Congênitas/diagnóstico por imagem , Cardiopatias Congênitas/epidemiologia , Cardiopatias Congênitas/genética , Heterozigoto , Hospitais Universitários , Humanos , Incidência , Lactente , Masculino , Mutação , Linhagem , Prognóstico , Estudos Retrospectivos , Medição de Risco
2.
Environ Pollut ; 252(Pt A): 216-226, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31151060

RESUMO

Microcystins (MCs) have been shown to be carcinogenic by animal and cellular experiments and found to be associated with the development of human hepatocellular carcinoma (HCC) through epidemiological studies. However, the molecular mechanism of microcystin-LR (MC-LR) induced HCC is still unclear. This study is determined to clarify the role and mechanism of LHX6 in MC-LR-induced hepatocarcinogenesis. Using the previously established MC-LR-induced malignant transformation model in L02 cells, we screened out LHX6, homeobox gene that was significantly changed. We found that LHX6 was significantly down-regulated in MC-LR treated L02 cells and the liver tissue of rats treated for 35 weeks with 10 µg/kg body weight of MC-LR. Expression of LHX6 in human tumor tissue was significantly down-regulated in high MC-LR-exposure group. LHX6 was hypermethylated in MC-LR treated L02 cells and up-regulated after treatment with 10 µM of 5-aza-2'-deoxycytidine. Furthermore, overexpression of LHX6 inhibited proliferation, invasion and migration of malignantly transformed L02 cells in vitro and in vivo, while knockdown of LHX6 resulted in an opposite phenotype. In addition, we found that up-regulation of P53 and Bax resulted in apoptosis, and that down-regulation of CTNNB1 and MMP7 led to migration of MC-LR treated L02 cells. Blockade of P53 and CTNNB1 by its inhibitor significantly diminished the effect of LHX6. These genes were working together during the process of MC-LR-induced hepatocarcinogenesis. Our study demonstrated for the first time that LHX6 gene expression is regulated by DNA methylation and can inhibit the proliferation, invasion and migration through Wnt/ß-catenin and P53 signaling pathways during the MC-LR-induced hepatocarcinogenesis. This result may suggest that LHX6 gene can be used as a potential target gene and a biomarker for liver cancer treatment.


Assuntos
Carcinoma Hepatocelular/induzido quimicamente , Transformação Celular Neoplásica/induzido quimicamente , Proteínas com Homeodomínio LIM/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Microcistinas/toxicidade , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Metilação de DNA/efeitos dos fármacos , Decitabina/farmacologia , Epigênese Genética , Humanos , Proteínas com Homeodomínio LIM/genética , Metaloproteinase 7 da Matriz/metabolismo , Proteínas do Tecido Nervoso/genética , Ratos , Transdução de Sinais , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismo
3.
BMC Med Genet ; 20(1): 71, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053111

RESUMO

BACKGROUND: Nail-patella syndrome (NPS) is an autosomal dominant developmental disorder most commonly characterized by dyplasia of nail or patella, the radial head or the humeral head hypoplasia, and, frequently ocular abnormalities and renal disease. It is caused by heterozygous loss-of-function mutations in the LMX1B gene, which encodes LIM homeodomain transcription factor and is essential for regulating the dorsal limb fate. METHODS: A five generation pedigree was recruited. Genomic DNA was extracted from the peripheral blood samples. Mutation detection was performed by Sanger sequencing the LMX1B gene. In silico functional annotation of the variant was performed using the in silico predictors SIFT, PolyPhen-2 and Mutation Taster. RESULTS: A novel heterozygous small deletion within exon 4 of LMX1B, c.712_714delTTC, was identified in a rare five-generation NPS pedigree. The mutation resulted in a deletion of the conserved amino acid phenylalanine at codon 238 (p.Phe238del), which located in the homeodomain of LMX1B may abolish DNA binding with the molecule. Conformational prediction showed that the variation could transform the helical structure comprising p.Phe234, p.Lys235, p.Ala236, and p.Ser237. CONCLUSION: We identified a novel NPS-causing LMX1B mutation and expanded the spectrum of mutations in the LMX1B gene. The c.712_714delTTC mutation may affect the quaternary structure of LMX1B, which is essential for the specification of dorsal limb fate at both zeugopodal and autopodal levels, leading to typical NPS.


Assuntos
Deleção de Genes , Proteínas com Homeodomínio LIM/genética , Síndrome da Unha-Patela/genética , Fatores de Transcrição/genética , China , Códon , Éxons , Feminino , Heterozigoto , Humanos , Mutação com Perda de Função , Masculino , Linhagem
4.
Cell Prolif ; 52(4): e12612, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31012189

RESUMO

OBJECTIVE: POU5F1 (OCT4) is implicated in cancer stem cell self-renewal. Currently, some studies have shown that OCT4 has a dual function in suppressing or promoting cancer progression. However, the precise molecular mechanism of OCT4 in breast cancer progression remains unclear. MATERIALS AND METHODS: RT-PCR and Western blot were utilized to investigate OCT4 expression in breast cancer tissues and cells. Cell proliferation assays and mouse models were applied to determine the effects of OCT4 on breast cancer cell proliferation. DNMT1 inhibitors, ChIP, CoIP, IHC and ERα inhibitors were used to explore the molecular mechanism of OCT4 in breast cancer. RESULTS: OCT4 was down-regulated in breast cancer tissues, and the overexpression of OCT4 promoted MDA-MB-231 cell proliferation and inhibited the proliferation of MCF-7 cells in vitro and in vivo, respectively. Two DNMT1 inhibitors (5-aza-dC and zebularine) suppressed OCT4-induced MDA-MB-231 cell proliferation through Ras/Raf1/ERK inactivation by targeting ISL1, which is the downstream of DNMT1. In contrast, OCT4 interacted with ERα, decreased DNMT1 expression and inactivated the Ras/Raf1/ERK signalling pathway in MCF-7 cells. Moreover, ERα inhibitor (AZD9496) reversed the suppression of OCT4-induced proliferation in MCF-7 cells via the activation of ERK signalling pathway. CONCLUSIONS: OCT4 is dependent on ERα to suppress the proliferation of breast cancer cells through DNMT1/ISL1/ERK axis.


Assuntos
Neoplasias da Mama/genética , Proliferação de Células/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Receptor alfa de Estrogênio/genética , Proteínas com Homeodomínio LIM/genética , Sistema de Sinalização das MAP Quinases/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/genética
5.
Mol Med Rep ; 19(5): 3584-3592, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864738

RESUMO

Biological pacemakers that combine cell­based and gene­based therapies are a promising treatment for sick sinus syndrome or severe atrioventricular block. The current study aimed to induce differentiation of adipose tissue­derived stem cells (ADSCs) into cardiac pacemaker cells through co­expression of the transcription factors insulin gene enhancer binding protein 1 (ISL­1) and T­box18 (Tbx18). ADSCs were transfected with green fluorescent protein, ISL­1, Tbx18 or ISL­1+Tbx18 fluorescent protein lentiviral vectors, and subsequently co­cultured with neonatal rat ventricular cardiomyocytes in vitro for 7 days. The potential for regulating the differentiation of ADSCs into pacemaker­like cells was evaluated by cell morphology, beating rate, reverse transcription­quantitative polymerase chain reaction, western blotting, immunofluorescence and electrophysiological activity. ADSCs were successfully transformed into spontaneously beating cells that exhibited a behavior similar to that of co­cultured pacemaker cells. This effect was significantly increased in the combined ISL­1 and Tbx18 group. These results provide a potential strategy for enriching the cardiac pacemaker cell population from ADSCs.


Assuntos
Diferenciação Celular/genética , Sistema de Condução Cardíaco/citologia , Sistema de Condução Cardíaco/metabolismo , Fatores de Transcrição/genética , Tecido Adiposo/citologia , Animais , Biomarcadores , Reprogramação Celular , Fenômenos Eletrofisiológicos , Expressão Gênica , Humanos , Proteínas com Homeodomínio LIM/genética , Masculino , Ratos , Células-Tronco/citologia , Proteínas com Domínio T/genética , Transdução Genética
6.
J Exp Clin Cancer Res ; 38(1): 97, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30791932

RESUMO

BACKGROUND: Epithelial-mesenchymal transition (EMT)-associated proteins play key roles in cancer progression and metastasis with the involvement of microRNAs (miRNAs). This study aims to assess the role of miR-506 working in tandem with LIM Homeobox 2 (LHX2) in EMT and metastasis through the Wnt/ß-catenin signaling pathway in nasopharyngeal carcinoma (NPC). METHODS: Differentially expressed genes associated with NPC were screened using microarray analyses, from which LHX2 was identified. Next, the potential relationship between miR-506 and LHX2 was analyzed. In order to explore the effect of miR-506 or LHX2 on NPC cell proliferation, migration, invasion and apoptosis, serials of mimics, inhibitors or siRNA against LHX2 were transfected into NPC cells. Then, the expression patterns of LHX2, Wnt1, ß-catenin, E-cadherin, Vimentin, TCF4 and Twist were determined to assess the influence of miR-506 or LHX2 on EMT as well as the relationship between the Wnt/ß-catenin signaling pathway and TCF4. The tumorigenicity and lymph node metastasis (LNM) in xenograft tumors of nude mice were observed. RESULTS: The has-miR-506-3p was identified as the down-regulated gene in NPC based on the microarray data while LHX2 was negatively regulated by miR-506. Over-expression of miR-506 or silencing of LHK2 inhibited NPC cell proliferation, migration, invasion, tumorigenicity and LNM but promoted apoptosis indicated by decreased Wnt1, ß-catenin, Vimentin, TCF4 and Twist expressions along with increased E-cadherin expressions. CONCLUSIONS: miR-506 inhibits tumor growth and metastasis in NPC via inhibition of Wnt/ß-catenin signaling by down-regulating LHX2, accompanied by decreased TCF4. Taken together, miR-506 targeted-inhibition LHX2 presents a promising therapeutic strategy for the treatment of NPC. TRIAL REGISTRATION: ChiCTR1800018889 . Registered 15 October 2018.


Assuntos
Proliferação de Células/genética , Proteínas com Homeodomínio LIM/genética , MicroRNAs/genética , Carcinoma Nasofaríngeo/genética , Metástase Neoplásica/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Adulto , Idoso , Animais , Apoptose/genética , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , RNA Interferente Pequeno/genética , Fator de Transcrição 4/genética , Vimentina/genética
7.
Dev Cell ; 48(4): 475-490.e7, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30713072

RESUMO

The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5+ population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1+TNNT2+ intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations.


Assuntos
Diferenciação Celular/fisiologia , Miócitos Cardíacos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Análise de Célula Única , Animais , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Ventrículos do Coração/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Proteínas com Homeodomínio LIM/genética , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes , Miocárdio/metabolismo , Organogênese , Análise de Célula Única/métodos
8.
Nat Commun ; 10(1): 376, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670697

RESUMO

Many genetic loci affect circulating lipid levels, but it remains unknown whether lifestyle factors, such as physical activity, modify these genetic effects. To identify lipid loci interacting with physical activity, we performed genome-wide analyses of circulating HDL cholesterol, LDL cholesterol, and triglyceride levels in up to 120,979 individuals of European, African, Asian, Hispanic, and Brazilian ancestry, with follow-up of suggestive associations in an additional 131,012 individuals. We find four loci, in/near CLASP1, LHX1, SNTA1, and CNTNAP2, that are associated with circulating lipid levels through interaction with physical activity; higher levels of physical activity enhance the HDL cholesterol-increasing effects of the CLASP1, LHX1, and SNTA1 loci and attenuate the LDL cholesterol-increasing effect of the CNTNAP2 locus. The CLASP1, LHX1, and SNTA1 regions harbor genes linked to muscle function and lipid metabolism. Our results elucidate the role of physical activity interactions in the genetic contribution to blood lipid levels.


Assuntos
Exercício , Loci Gênicos/genética , Lipídeos/sangue , Lipídeos/genética , Adolescente , Adulto , Grupo com Ancestrais do Continente Africano/genética , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático/genética , Brasil , Proteínas de Ligação ao Cálcio/genética , Colesterol/sangue , HDL-Colesterol/sangue , HDL-Colesterol/genética , LDL-Colesterol/sangue , LDL-Colesterol/genética , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Hispano-Americanos/genética , Humanos , Proteínas com Homeodomínio LIM/genética , Metabolismo dos Lipídeos/genética , Masculino , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Triglicerídeos/sangue , Triglicerídeos/genética , Adulto Jovem
9.
Forensic Sci Int Genet ; 38: 1-8, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30300865

RESUMO

Many studies have reported age-associated DNA methylation changes and age-predictive models in various tissues and body fluids. Although age-associated DNA methylation changes can be tissue-specific, a multi-tissue age predictor that is applicable to various tissues and body fluids with considerable prediction accuracy might be valuable. In this study, DNA methylation at 5 CpG sites from the ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59 genes were investigated in 448 samples from blood, saliva, and buccal swabs. A multiplex methylation SNaPshot assay was developed to measure DNA methylation simultaneously at the 5 CpG sites. Among the 5 CpG sites, 3 CpG sites in the ELOVL2, KLF14 and TRIM59 genes demonstrated strong correlation between DNA methylation and age in all 3 sample types. Age prediction models built separately for each sample type using the DNA methylation values at the 5 CpG sites showed high prediction accuracy with a Mean Absolute Deviation from the chronological age (MAD) of 3.478 years in blood, 3.552 years in saliva and 4.293 years in buccal swab samples. A tissue-combined model constructed with 300 training samples including 100 samples from each blood, saliva and buccal swab samples demonstrated a very strong correlation between predicted and chronological ages (r = 0.937) and a high prediction accuracy with a MAD of 3.844 years in the 148 independent test set samples of 50 blood, 50 saliva and 48 buccal swab samples. Although more validation might be needed, the tissue-combined model's prediction accuracies in each sample type were very much similar to those obtained from each tissue-specific model. The multiplex methylation SNaPshot assay and the age prediction models in our study would be useful in forensic analysis, which frequently involves DNA from blood, saliva, and buccal swab samples.


Assuntos
Envelhecimento/genética , Análise Química do Sangue , Metilação de DNA , Mucosa Bucal/química , Saliva/química , Acetiltransferases/genética , Adolescente , Adulto , Idoso , Ilhas de CpG/genética , Genética Forense , Marcadores Genéticos , Técnicas de Genotipagem/instrumentação , Humanos , Proteínas com Homeodomínio LIM/genética , Proteínas de Membrana/genética , Metaloproteínas/genética , Pessoa de Meia-Idade , Proteínas Musculares/genética , Análise de Sequência de DNA , Fatores de Transcrição Sp/genética , Fatores de Transcrição/genética , Adulto Jovem
10.
BMC Nephrol ; 19(1): 382, 2018 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-30594156

RESUMO

BACKGROUND: Congenital nephrotic syndrome (CNS) is characterised by increased proteinuria, hypoproteinemia, and edema beginning in the first 3 months of life. Recently, molecular genetic studies have identified several genes involved in the pathogenesis of CNS. A systematic investigation of the genes for CNS in China has never been performed; therefore, we conducted a mutational analysis in 12 children with CNS,with the children coming from 10 provinces and autonomous regions in China. METHODS: Twelve children with CNS were enrolled from 2009 to 2016. A mutational analysis was performed in six children by Sanger sequencing in eight genes (NPHS1, NPHS2, PLCE1, WT1, LAMB2, LMXIB, COQ6 and COQ2) before 2014, and whole-exome sequencing was used from 2014 to 2016 in another six children. Significant variants that were detected by next generation sequencing were confirmed by conventional Sanger sequencing in the patients' families. RESULTS: Of the 12 children, eight patients had a compound heterozygous NPHS1 mutation, one patient had a de novo mutation in the WT1 gene, and another patient with extrarenal symptoms had a homozygous mutation in the COQ6 gene. No mutations were detected in genes NPHS2, PLCE1, LAMB2, LMXIB, and COQ2 in the 12 patients. CONCLUSIONS: This study demonstrates that the majority of CNS cases (67%, 8/12 patients) are caused by genetic defects, and the NPHS1 mutation is the most common cause of CNS in Chinese patients. A mutational analysis of NPHS1 should be recommended in Chinese patients with CNS in all exons of NPHS1 and in the intron-exon boundaries.


Assuntos
Proteínas de Membrana/genética , Síndrome Nefrótica/genética , Alquil e Aril Transferases/genética , Grupo com Ancestrais do Continente Asiático/genética , China , Análise Mutacional de DNA , Feminino , Heterozigoto , Homozigoto , Humanos , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Homeodomínio LIM/genética , Laminina/genética , Masculino , Síndrome Nefrótica/congênito , Fosfoinositídeo Fosfolipase C/genética , Fatores de Transcrição/genética , Ubiquinona/genética , Proteínas WT1/genética , Sequenciamento Completo do Exoma
11.
Neural Dev ; 13(1): 21, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30217225

RESUMO

BACKGROUND: Homeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation. METHODS: Differential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures. RESULTS: We identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures. CONCLUSIONS: Tcf12 expression in the SVZ of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Corpos Geniculados/citologia , Corpos Geniculados/embriologia , Fator Nuclear 1 de Tireoide/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Embrião de Mamíferos , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/fisiologia , Genômica , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Interneurônios/fisiologia , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/genética , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Técnicas de Cultura de Órgãos , Elementos Reguladores de Transcrição/fisiologia , Fator Nuclear 1 de Tireoide/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Gynecol Oncol ; 150(3): 545-551, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29960712

RESUMO

OBJECTIVES: The methylation profile of genes in precursor lesions in cervical cancer was characterized to improve screening techniques for high-grade intraepithelial neoplasia. METHODS: A total of 447 cervical cytology samples obtained from women who underwent colposcopy were examined. The cases were distributed as follows: (1) cervices without cervical intraepithelial neoplasia (CIN; n = 152); (2) cervices with a CIN grade of 1 (CIN 1; n = 147); and (3) cervices with a CIN grade of 2 or 3 (CIN 2/3; n = 148). The methylation pattern for a panel of 15 genes was analysed by quantitative methylation-specific PCR (qMSP) and compared between the groups (≤CIN 1 vs. CIN 2+). RESULTS: In the validation set, seven genes presented significantly different methylation profiles according to diagnosis, namely, DAPK1 (p = 0.001), EPB41L3 (p = 0.001), HIC1 (p = 0.028), hsa-miR-124-2 (p = 0.001), LMX1A (p = 0.001), SOX1 (p = 0.001), and TERT (p = 0.001). Six genes showed a significant increase in the frequency of methylation in the presence of hr-HPV, namely, DAPK1 (p = 0.001), EPB41L3 (p = 0.001), hsa-miR-124-2 (p = 0.001), LMX1A (p = 0.001), SOX1 (p = 0.001), and TERT (p = 0.001). The methylation of the hsa-miR-124 gene showed sensitivity and specificity (86.7% and 61.3%, respectively) similar to that of the HPV test (91.3% and 50.0%, respectively). The independent factors associated with the diagnosis of CIN 2+ and the methylation of the hsa-miR-124-2 (OR = 5.1), SOX1 (OR = 2.8), TERT (OR = 2.2), and LMX1A (OR = 2.0) genes were a positive test for hr-HPV (odds ratio [OR] = 5.5). CONCLUSIONS: Hypermethylation of the hsa-miR-124-2, SOX1, TERT, and LMX1A genes may be a promising biomarker for precursor lesions in cervical cancer regardless of the hr-HPV status.


Assuntos
Neoplasia Intraepitelial Cervical/diagnóstico , Neoplasia Intraepitelial Cervical/genética , Metilação de DNA , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Adulto , Biomarcadores Tumorais/genética , Neoplasia Intraepitelial Cervical/patologia , Neoplasia Intraepitelial Cervical/virologia , Detecção Precoce de Câncer , Feminino , Humanos , Proteínas com Homeodomínio LIM/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Papillomaviridae , Infecções por Papillomavirus/complicações , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Sensibilidade e Especificidade , Telomerase/genética , Fatores de Transcrição/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
13.
Development ; 145(14)2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-29945864

RESUMO

Epigenetic regulation of gene expression has a crucial role allowing for the self-renewal and differentiation of stem and progenitor populations during organogenesis. The mammalian kidney maintains a population of self-renewing stem cells that differentiate to give rise to thousands of nephrons, which are the functional units that carry out filtration to maintain physiological homeostasis. The polycomb repressive complex 2 (PRC2) epigenetically represses gene expression during development by placing the H3K27me3 mark on histone H3 at promoter and enhancer sites, resulting in gene silencing. To understand the role of PRC2 in nephron differentiation, we conditionally inactivated the Eed gene, which encodes a nonredundant component of the PRC2 complex, in nephron progenitor cells. Resultant kidneys were smaller and showed premature loss of progenitor cells. The progenitors in Eed mutant mice that were induced to differentiate did not develop into properly formed nephrons. Lhx1, normally expressed in the renal vesicle, was overexpressed in kidneys of Eed mutant mice. Thus, PRC2 has a crucial role in suppressing the expression of genes that maintain the progenitor state, allowing nephron differentiation to proceed.


Assuntos
Diferenciação Celular/fisiologia , Epigênese Genética/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Néfrons/embriologia , Complexo Repressor Polycomb 2/biossíntese , Células-Tronco/metabolismo , Animais , Proteínas com Homeodomínio LIM/biossíntese , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Transgênicos , Mutação , Néfrons/citologia , Complexo Repressor Polycomb 2/genética , Células-Tronco/citologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
14.
Development ; 145(10)2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29712641

RESUMO

Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a, as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles.


Assuntos
Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Néfrons/embriologia , Organogênese/genética , Células-Tronco/citologia , Transcrição Genética/genética , Animais , Linhagem Celular , Proliferação de Células/genética , Células HEK293 , Fator 1-alfa Nuclear de Hepatócito/biossíntese , Fator 4 Nuclear de Hepatócito/biossíntese , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Nefropatias/genética , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Knockout , Néfrons/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética
15.
Gene ; 669: 99-106, 2018 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-29800735

RESUMO

PURPOSE: Increasing evidence demonstrates that the four and a half LIM domain (FHL) gene and its protein products have different functions in the progression of various malignancies. However, the role of FHL protein 2 (FHL2) in cervical cancer (CC) has not been fully elucidated. In this study, we investigated the prognostic value of FHL2 expression in human CC tissues and the potential molecular mechanisms through which FHL2 modulates CC cell proliferation and apoptosis. MATERIALS AND METHODS: We measured FHL2 expression in CC cell lines and tissues by quantitative real-time polymerase chain reaction and Western blot assays. The effects of FHL2 knockdown on cell proliferation and apoptosis in two CC cell lines were examined using RNA interference, cell counting kit-8, Western blot and flow cytometry assays. Furthermore, we assessed phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) expression in two CC cell lines to determine whether the AKT/mTOR pathway is involved in the effects of FHL2 silencing on cell proliferation and apoptosis. Nude mice tumorigenicity experiments were also performed to evaluate the effects of FHL2 on HeLa cell growth in vivo. RESULTS: We found that FHL2 was significantly upregulated in CC cell lines and tissues. According to survival curves, high FHL2 expression levels in patients were correlated with poor prognosis. Moreover, by decreasing p-AKT and p-mTOR protein levels, silencing FHL2 significantly inhibited cell proliferation and induced apoptosis. FHL2 knockdown also induced apoptosis by increasing the Bax-to-Bcl2 ratio. By contrast, FHL2 overexpression significantly promoted cell proliferation. Finally, decreased tumour growth in an in vivo animal model also demonstrated the tumour-suppressing effects of FHL2 knockdown. CONCLUSION: Our findings indicate that FHL2 is an important prognostic factor in CC and that it plays a crucial oncoprotein role by promoting cell proliferation and inhibiting apoptosis in CC, possibly by targeting the AKT/mTOR pathway.


Assuntos
Proteínas com Homeodomínio LIM/metabolismo , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Animais , Apoptose , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Proteínas com Homeodomínio LIM/antagonistas & inibidores , Proteínas com Homeodomínio LIM/genética , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Regulação para Cima , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/mortalidade
16.
Proc Natl Acad Sci U S A ; 115(18): 4643-4648, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29666277

RESUMO

Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons. The protein-protein interactions within these complexes are mediated by ordered LIM domains in the LIM-HD proteins and intrinsically disordered LIM interaction domains (LIDs) in LDB1 and ISL1; however, little is known about how the strength or rates of binding contribute to complex assemblies. We have measured the interactions of LIM:LID complexes using FRET-based protein-protein interaction studies and EMSAs and used these data to model population distributions of complexes. The protein-protein interactions within the ternary complexes are much weaker than those in the binary complex, yet surprisingly slow LDB1:ISL1 dissociation kinetics and a substantial increase in DNA binding affinity promote formation of the ternary complex over the binary complex in motor neurons. We have used mutational and protein engineering approaches to show that allostery and modular binding by tandem LIM domains contribute to the LDB1LID binding kinetics. The data indicate that a single intrinsically disordered region can achieve highly disparate binding kinetics, which may provide a mechanism to regulate the timing of transcriptional complex assembly.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteínas Intrinsicamente Desordenadas/química , Proteínas com Domínio LIM/química , Proteínas com Homeodomínio LIM/química , Complexos Multiproteicos/química , Fatores de Transcrição/química , Iniciação da Transcrição Genética , Animais , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Development ; 145(9)2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29650591

RESUMO

Precise control of the relative ratio of retinal neurons and glia generated during development is essential for visual function. We show that Lhx2, which encodes a LIM-homeodomain transcription factor essential for specification and differentiation of retinal Müller glia, also plays a crucial role in the development of retinal neurons. Overexpression of Lhx2 with its transcriptional co-activator Ldb1 triggers cell cycle exit and inhibits both Notch signaling and retinal gliogenesis. Lhx2/Ldb1 overexpression also induces the formation of wide-field amacrine cells (wfACs). In contrast, Rnf12, which encodes a negative regulator of LDB1, is necessary for the initiation of retinal gliogenesis. We also show that Lhx2-dependent neurogenesis and wfAC formation requires Ascl1 and Neurog2, and that Lhx2 is necessary for their expression, although overexpression of Lhx2/Ldb1 does not elevate expression of these proneural bHLH factors. Finally, we demonstrate that the relative level of the LHX2-LDB1 complex in the retina decreases in tandem with the onset of gliogenesis. These findings show that control of Lhx2 function by Ldb1 and Rnf12 underpins the coordinated differentiation of neurons and Müller glia in postnatal retina.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Ependimogliais/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Neurogênese/fisiologia , Neurônios Retinianos/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Células Ependimogliais/citologia , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios Retinianos/citologia , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
18.
Development ; 145(8)2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29615466

RESUMO

The Drosophila ovary serves as a model for pioneering studies of stem cell niches, with defined cell types and signaling pathways supporting both germline and somatic stem cells. The establishment of the niche units begins during larval stages with the formation of terminal filament-cap structures; however, the genetics underlying their development remains largely unknown. Here, we show that the transcription factor Lmx1a is required for ovary morphogenesis. We found that Lmx1a is expressed in early ovarian somatic lineages and becomes progressively restricted to terminal filaments and cap cells. We show that Lmx1a is required for the formation of terminal filaments, during the larval-pupal transition. Finally, our data demonstrate that Lmx1a functions genetically downstream of Bric-à-Brac, and is crucial for the expression of key components of several conserved pathways essential to ovarian stem cell niche development. Importantly, expression of chicken Lmx1b is sufficient to rescue the null Lmx1a phenotype, indicating functional conservation across the animal kingdom. These results significantly expand our understanding of the mechanisms controlling stem cell niche development in the fly ovary.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas com Homeodomínio LIM/metabolismo , Ovário/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Linhagem da Célula , Galinhas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Genes de Insetos , Proteínas com Homeodomínio LIM/genética , Mutação , Ovário/citologia , Ovário/metabolismo , Transdução de Sinais , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/fisiologia , Fatores de Transcrição/genética
19.
Nat Commun ; 9(1): 1422, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29651049

RESUMO

We report that half striatal cholinergic interneurons are dual transmitter cholinergic and GABAergic interneurons (CGINs) expressing ChAT, GAD65, Lhx7, and Lhx6 mRNAs, labeled with GAD and VGAT, generating monosynaptic dual cholinergic/GABAergic currents and an inhibitory pause response. Dopamine deprivation increases CGINs ongoing activity and abolishes GABAergic inhibition including the cortico-striatal pause because of high [Cl-]i levels. Dopamine deprivation also dramatically increases CGINs dendritic arbors and monosynaptic interconnections probability, suggesting the formation of a dense CGINs network. The NKCC1 chloride importer antagonist bumetanide, which reduces [Cl-]i levels, restores GABAergic inhibition, the cortico-striatal pause-rebound response, and attenuates motor effects of dopamine deprivation. Therefore, most of the striatal cholinergic excitatory drive is balanced by a concomitant powerful GABAergic inhibition that is impaired by dopamine deprivation. The attenuation by bumetanide of cardinal features of Parkinson's disease paves the way to a novel therapeutic strategy based on a restoration of low [Cl-]i levels and GABAergic inhibition.


Assuntos
Neurônios Colinérgicos/metabolismo , Corpo Estriado/metabolismo , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Interneurônios/metabolismo , Doença de Parkinson Secundária/metabolismo , Ácido gama-Aminobutírico/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bumetanida/farmacologia , Cloretos/metabolismo , Colinérgicos/metabolismo , Colinérgicos/farmacologia , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/patologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Dopamina/deficiência , Regulação da Expressão Gênica , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Humanos , Interneurônios/efeitos dos fármacos , Interneurônios/patologia , Transporte de Íons , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Doença de Parkinson Secundária/genética , Doença de Parkinson Secundária/patologia , Técnicas de Patch-Clamp , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Ácido gama-Aminobutírico/farmacologia
20.
Genesis ; 56(4): e23098, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29508544

RESUMO

LHX3, a LIM-homeodomain transcription factor, is broadly expressed in the developing pituitary, spinal cord, medulla, retina and inner ear, and plays essential roles during embryonic development. Mice with homozygous Lhx3 null mutation exhibit failure in the formation of pituitary gland and die perinatally. To facilitate the functional study of Lhx3 in mice, we engineered and characterized two novel Lhx3 mouse strains: Lhx3GFP reporter knock-in and Lhx3loxP conditional knockout mice. Coimmunolabeling of LHX3 and GFP shows that the expression pattern of the knock-in GFP reporter recapitulates that of endogenous LHX3 in cochlea, vestibule, retina, and spinal cord. By crossing Lhx3loxP mice with the ubiquitous CMV-Cre mice, we have demonstrated a high efficiency of Cre recombinase-mediated removal of exons 3 to 5 of Lhx3, which encode the second LIM-domain and the HD domain of LHX3, resulting global knockout of Lhx3. Thus, Lhx3GFP and Lhx3loxP mice serve as valuable genetic tools to dissect the tissue-specific roles of Lhx3 at late-gestation and postnatal stages in mice.


Assuntos
Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Éxons , Regulação da Expressão Gênica no Desenvolvimento/genética , Engenharia Genética/métodos , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Mutação , Hipófise/metabolismo , Hipófise/fisiologia
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