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1.
Life Sci ; 255: 117818, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32445757

RESUMO

Activation of hepatic stellate cells (HSCs) is a central event in the pathogenesis of liver fibrosis and is characterized by the disappearance of lipid droplets. Although the exogenous supplementation of lipid droplet content can effectively reverse the activation of HSCs, the underlying molecular mechanisms are largely unknown. In our current study, we sought to investigate the role of lncRNA-H19 in the process of lipid droplets disappearance and to further examine the underlying molecular mechanisms. We found that the lncRNA-H19 level was increased in CCl4-induced fibrotic liver, which activated HSCs. Further research showed that hypoxia inducible factor-1α (HIF-1α) significantly increased lncRNA-H19 expression by binding to the lncRNA-H19 promoter at two hypoxia response element (HRE) sites located at 492-499 and 515-522 bp. Importantly, lncRNA-H19 knockdown markedly inhibited HSC activation and alleviated liver fibrosis, indicating that lncRNA-H19 may be a potential target for anti-fibrosis therapeutic approaches. Moreover, lncRNA-H19 knockdown could reverse the lipid droplet phenotype of activated HSCs, inhibiting the phosphorylated AMPKα-mediated lipid oxidation signaling pathway. The AMPK agonist AICAR promoted AMPKα phosphorylation and abrogated lipid droplets restoration in HSCs transfected with the lncRNA-H19 knockdown plasmid. Experimental molecular analysis showed that lncRNA-H19 triggered AMPKα to interact with LKB1 and resulted in AMPKα phosphorylation, which accelerating lipid droplets degradation and lipid oxidation. Taken together, our results highlighted the role of lncRNA-H19 in the metabolism of lipid droplets in HSCs, and revealed a new molecular target for alleviating liver fibrosis.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Estreladas do Fígado/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cirrose Hepática/patologia , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Gotículas Lipídicas/metabolismo , Cirrose Hepática/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Elementos de Resposta/genética
2.
Oxid Med Cell Longev ; 2020: 9741369, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31998447

RESUMO

Spinal cord injury (SCI) is a devastating disease that may lead to lifelong disability. Thus, seeking for valid drugs that are beneficial to promoting axonal regrowth and elongation after SCI has gained wide attention. Metformin, a glucose-lowering agent, has been demonstrated to play roles in various central nervous system (CNS) disorders. However, the potential protective effect of metformin on nerve regeneration after SCI is still unclear. In this study, we found that the administration of metformin improved functional recovery after SCI through reducing neuronal cell apoptosis and repairing neurites by stabilizing microtubules via PI3K/Akt signaling pathway. Inhibiting the PI3K/Akt pathway with LY294002 partly reversed the therapeutic effects of metformin on SCI in vitro and vivo. Furthermore, metformin treatment weakened the excessive activation of oxidative stress and improved the mitochondrial function by activating the nuclear factor erythroid-related factor 2 (Nrf2) transcription and binding to the antioxidant response element (ARE). Moreover, treatment with Nrf2 inhibitor ML385 partially abolished its antioxidant effect. We also found that the Nrf2 transcription was partially reduced by LY294002 in vitro. Taken together, these results revealed that the role of metformin in nerve regeneration after SCI was probably related to stabilization of microtubules and inhibition of the excessive activation of Akt-mediated Nrf2/ARE pathway-regulated oxidative stress and mitochondrial dysfunction. Overall, our present study suggests that metformin administration may provide a potential therapy for SCI.


Assuntos
Axônios/fisiologia , Metformina/farmacologia , Microtúbulos , Estresse Oxidativo/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Traumatismos da Medula Espinal , Animais , Cromonas/farmacologia , Microtúbulos/metabolismo , Microtúbulos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Morfolinas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Elementos de Resposta , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia
3.
Nat Commun ; 11(1): 575, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996678

RESUMO

mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.


Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Adipócitos Marrons/metabolismo , Lipogênese/fisiologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acetato-CoA Ligase/metabolismo , Animais , Proteínas de Transporte , Epigênese Genética , Ácido Graxo Sintases , Edição de Genes , Expressão Gênica , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Lipogênese/genética , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo , Fosforilação , Proteômica , Elementos de Resposta
4.
Proc Natl Acad Sci U S A ; 117(3): 1457-1467, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31900363

RESUMO

Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson's disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5' untranslated region (5' UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5' UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound's selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.


Assuntos
Proteínas Intrinsicamente Desordenadas/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Elementos de Resposta , alfa-Sinucleína/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , RNA Mensageiro/química , RNA Mensageiro/genética , alfa-Sinucleína/metabolismo
5.
Biochim Biophys Acta Gene Regul Mech ; 1863(2): 194488, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31926341

RESUMO

Polo-like kinase 4 (PLK4) is a member of the serine/threonine protein kinase family involved in cell-cycle regulation and cellular response to stresses. However, the alteration of PLK4 in response to endoplasmic reticulum (ER) stress has not been well described. In the present study, we focused on the regulation of PLK4 regulation in response to ER stress. PLK4 expression was dramatically reduced under ER stress induced by brefeldin A (BFA), tunicamycin (TM), or thapsigargin (TG) and down regulation of PLK4 expression was dependent on activating transcription factor 6 (ATF6) and CCAAT/enhancer-binding protein ß (C/EBPß). Luciferase activity analysis of the truncated PLK4 promoter indicated that region from -1343 to -1250 of the PLK4 promoter was sensitive to BFA or TG. Additionally, ChIP and ChIP Re-IP assays showed that ATF6 and C/EBPß were assembled on the same region of Plk4 promoter. Notably, we identified one C/EBPß responsive element at position -1284, to which ATF6 or C/EBPß binding was enhanced by BFA or TG under in vitro and in vivo conditions. Finally, overexpression of PLK4 inhibits apoptosis and promotes cell proliferation in response to ER stress. In summary, these results demonstrated that ER stress plays a crucial role in PLK4 expression. ATF6 may upregulate DNA-binding affinities after BFA treatment, via recruiting C/EBPß to the upstream promoter of PLK4. These findings may contribute to the understanding of the molecular mechanism of PLK4 regulation.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Neoplasias Ósseas/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica , Osteossarcoma/genética , Proteínas Serina-Treonina Quinases/genética , Apoptose , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Mutagênese , Osteossarcoma/enzimologia , Osteossarcoma/metabolismo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Elementos de Resposta , Transcrição Genética
6.
Nucleic Acids Res ; 48(2): 589-604, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31799619

RESUMO

IRF3, IRF5 and IRF9 are transcription factors, which play distinct roles in the regulation of antiviral and inflammatory responses. The determinants that mediate IRF-specific enhancer selection are not fully understood. To uncover regions occupied predominantly by IRF3, IRF5 or IRF9, we performed ChIP-seq experiments in activated murine dendritic cells. The identified regions were analysed with respect to the enrichment of DNA motifs, the interferon-stimulated response element (ISRE) and ISRE half-site variants, and chromatin accessibility. Using a machine learning method, we investigated the predictability of IRF-dominance. We found that IRF5-dominant regions differed fundamentally from the IRF3- and IRF9-dominant regions: ISREs were rare, while the NFKB motif and special ISRE half-sites, such as 5'-GAGA-3' and 5'-GACA-3', were enriched. IRF3- and IRF9-dominant regions were characterized by the enriched ISRE motif and lower frequency of accessible chromatin. Enrichment analysis and the machine learning method uncovered the features that favour IRF3 or IRF9 dominancy (e.g. a tripartite form of ISRE and motifs for NF-κB for IRF3, and the GAS motif and certain ISRE variants for IRF9). This study contributes to our understanding of how IRF members, which bind overlapping sets of DNA sequences, can initiate signal-dependent responses without activating superfluous or harmful programmes.


Assuntos
Elementos Facilitadores Genéticos/genética , Fator Regulador 3 de Interferon/genética , Fatores Reguladores de Interferon/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Animais , Linhagem Celular , Cromatina/genética , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Humanos , Aprendizado de Máquina , Camundongos , NF-kappa B/genética , Motivos de Nucleotídeos/genética , Análise de Componente Principal , Elementos de Resposta/genética , Fatores de Transcrição/genética
7.
Biochim Biophys Acta Gene Regul Mech ; 1863(1): 194475, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31870784

RESUMO

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-XL over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-XL expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- XL upregulation in AML cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Glucocorticoides/farmacologia , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Proteína bcl-X/genética , Apoptose , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Células U937 , Proteína bcl-X/metabolismo
8.
J Pharm Biomed Anal ; 177: 112855, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31561061

RESUMO

FSH plays a key role in the function of the reproductive system of human beings and is widely used both diagnostically and therapeutically in reproductive medicine. With the growing incidence of infertility, the demand for FSH pharmaceutical products is increasing. For this reason, the quality control process for FSH products is becoming more stringent. An accurate determination of bioactivity is crucial for the safety and efficacy of recombinant human follicle stimulating hormone (rhFSH). Up to now, in-vivo bioassay based on FSH-induced increases in rat ovarian weight has been the only method widely accepted by different pharmacopoeias. However this method has such drawbacks as the complex procedures, long assay period and high variability. Here, we established a reporter gene assay (RGA) based on the CHO-K1-FSHR-CRE-Luc cell line that stably expresses human follicle stimulating hormone receptor (hFSHR), as well as a luciferase reporter under the control of cyclic adenosine monophosphate (cAMP) response elements (CRES). Our study showed that our new assay not only has good dose-dependent responsiveness to rhFSH, but it also performs excellently in terms of specificity, precision, linearity, and simplicity compared with in-vivo rat bioassays. These results implied that this robust reporter gene assay may be a viable supplement to the animal in-vivo bioassay and may be employed in potency determination of rhFSH pharmaceutical products.


Assuntos
Bioensaio/métodos , Hormônio Foliculoestimulante Humano/farmacologia , Genes Reporter/genética , Receptores do FSH/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , AMP Cíclico/genética , Estudos de Viabilidade , Luciferases/química , Luciferases/genética , Receptores do FSH/genética , Proteínas Recombinantes/farmacologia , Elementos de Resposta/genética
9.
PLoS One ; 14(12): e0224850, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31825959

RESUMO

N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.


Assuntos
Adenosina/análogos & derivados , RNA Viral/química , RNA Viral/metabolismo , Elementos de Resposta , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Adenosina/química , Pareamento de Bases , Sequência de Bases , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética
10.
Cell Mol Biol Lett ; 24: 65, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827541

RESUMO

Objective: Hypoestrogenism in women is strongly associated with menopause and it can lead to lipid disorder, which predisposes people to premature cardiovascular disease. However, the mechanism of lipid disorder remains unclear. Sterol regulatory element-binding protein 2 (SREBP2) is the key transcription factor regulating cholesterol metabolism. We hypothesize that estrogen regulates SREBP2 transcription through an estrogen response element (ERE) in the SREBP2 promoter region. Methods: Human hepatoblastoma cells (HepG2) were treated with dose-dependent concentrations of estradiol (E2) for 24 h. Then, SREBP2 expression was determined via real-time PCR and immunofluorescence. The expressions of the SREBP2 downstream target genes HMGCR and LDLR were determined via real-time PCR. Lipid secretion in the culture media of HepG2 cells was measured using ELISA. Through bioinformatics analysis, we identified high-scoring ERE-like sequences in the SREBP2 gene promoter. Chromatin immunoprecipitation analysis was used to confirm the ERE. DNA fragments of the putative or mutated ERE-like sequence were synthesized and ligated into pGL3-basic plasmid to construct the SREBP2 promoter luciferase reporter systems. SREBP2-Luciferase (SREBP2-Luc), SREBP2-Mutation (SREBP2-Mut) and the blank control were transfected into hepatic cell lines. Luciferase activities were measured using the dual-luciferase reporter assay system. Chromatin immunoprecipitation analysis and the luciferase reporter assay were repeated in human hepatoma cells (HuH-7). Results: We found that E2 dose-dependently increased the expression of SREBP2 in HepG2 cells and that the increased levels were blocked when treated with an estrogen receptor-alpha antagonist. Additionally, E2 increased both HMGCR and LDLR expression and lipid secretion in HepG2 cells. Notably, we identified a functional ERE in the SREBP2 gene promoter, to which E2 could specifically bind and induce transcription. Conclusions: An ERE was identified in the SREBP2 gene promoter. It mediates the regulation of SREBP2 expression by estrogen in hepatocytes. This study provides a mechanism to link cardiovascular disease with estrogen.


Assuntos
Estradiol/farmacologia , Gotículas Lipídicas/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Genes Reporter , Células Hep G2 , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Luciferases/genética , Luciferases/metabolismo , Mutação , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais
11.
Genes (Basel) ; 10(12)2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817743

RESUMO

The phosphate starvation response (PHR) protein family exhibits the MYB and coiled-coil domains. In plants, within the either 5' untranslated regions (UTRs) or promoter regions of phosphate starvation-induced (PSI) genes are characteristic cis-regulatory elements, namely PHR1 binding sequence (P1BS). The most widely studied PHR protein family members, such as AtPHR1 in Arabidopsis thaliana (L.) and OsPHR2 in Oryza sativa (L.), may activate the gene expression of a broad range of PSI genes by binding to such elements in a phosphate (Pi) dependent manner. In Pi signaling, PHR transcription factors (TFs) can be selectively activated or deactivated by other proteins to execute the final step of signal transduction. Several new proteins have been associated with the AtPHR1/OsPHR2 signaling cascade in the last few years. While the PHR TF transcriptional role has been studied intensively, here we highlight the recent findings of upstream molecular components and other signaling pathways that may interfere with the PHR final mode of action in plants. Detailed information about transcriptional regulation of the AtPHR1 gene itself and its upstream molecular events has been reviewed.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Oryza , Elementos de Resposta , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Oryza/genética , Oryza/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Medicine (Baltimore) ; 98(51): e18442, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31861015

RESUMO

Genetic variation and genotype of Hepatitis B virus (HBV) are related to the efficiency of interferon alpha (IFN-α)-based antiviral therapy. However, the correlation of variation in interferon-stimulated response element (ISRE) and HBV genotype response to IFN-α therapy remains elusive.Differences of ISRE between genotype B and C HBV were explored using the HBV sequences retrieved from GenBank, and further investigated by ISRE region cloning and sequencing from 60 clinical samples post-IFN-α therapy. Additionally, ISRE mutants were constructed and their relation to responsiveness of IFN-α was evaluated by real-time PCR and Southern blot analysis.ISRE pattern between genotype B and C were found based on both clinical sample sequencing and full-length sequence alignment. The primary difference is the fourth base within the ISRE region, with T and C for genotype B and C, respectively. HBV with genotype C-type ISRE had a higher replicative capability as compared to HBV with genotype B-type ISRE after IFN-α treatment in huh7 cells. CONCLUSION:: Preference of ISRE between genotype B and C HBV are distinct. Single nucleotide difference (C to T) within the HBV ISRE region may link to the efficacy of IFN-α therapy to genotype B and C HBV. Therefore, this study provides a clue for the determination of IFN-α therapy response to HBV treatment.


Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Interferon-alfa/farmacologia , Elementos de Resposta , Adulto , Feminino , Genótipo , Vírus da Hepatite B/genética , Humanos , Interferon-alfa/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação , Adulto Jovem
13.
Int J Mol Sci ; 21(1)2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878115

RESUMO

p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.


Assuntos
Quadruplex G , Isoformas de Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Elementos de Resposta/genética , Proteína Supressora de Tumor p53/genética
14.
Technol Cancer Res Treat ; 18: 1533033819875166, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31769345

RESUMO

OBJECTIVE: To construct plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3 and elaborate the effects of overexpressed TK/VP3 on nasopharyngeal carcinoma cells. METHODS: Four plasmids were constructed, including pcDNA3.1-CMV-TK/VP3, pcDNA3.1-Hre2.TK/VP3, pcDNA3.1-Grp78.TK/VP3, and pcDNA3.1-Hre2.Grp78.TK/VP3. The human nasopharyngeal carcinoma cell line HNE1 cells were transfected with the 4 plasmids, respectively. Cell viabilities were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was conducted using flow cytometry analysis. The expression of TK, VP3, Grp78, and hypoxia-inducible factor 1α and apoptosis-related proteins was determined by real-time quantitative polymerase chain reaction and Western blotting. RESULTS: The recombinant plasmids that could steadily overexpress TK and VP3 were successfully constructed. Expression of TK and VP3 in cells transfected with pcDNA3.1-Hre2.TK/VP3 and pcDNA3.1-Grp78.TK/VP3 was significantly higher than pcDNA3.1-CMV-TK/VP3, and expression in cells transfected with pcDNA3.1-Hre2.Grp78.TK/VP3 was the highest. Under glucose deprivation or hypoxia condition, Grp78 or hypoxia-inducible factor 1α was overexpressed so that expression of TK and VP3 was significantly upregulated, which could further inhibit cell proliferation and enhance cell apoptosis. CONCLUSION: We successfully constructed 4 plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3, which could significantly inhibit the proliferation as well as enhance the apoptosis of nasopharyngeal carcinoma cells under glucose deprivation or hypoxia condition.


Assuntos
Proteínas de Choque Térmico/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta , Transativadores/metabolismo , Apoptose/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transativadores/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
Int J Mol Sci ; 20(22)2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31739570

RESUMO

The WRKY transcription factor superfamily is known to participate in plant growth and stress response. However, the role of this family in wheat (Triticum aestivum L.) is largely unknown. Here, a salt-induced gene TaWRKY13 was identified in an RNA-Seq data set from salt-treated wheat. The results of RT-qPCR analysis showed that TaWRKY13 was significantly induced in NaCl-treated wheat and reached an expression level of about 22-fold of the untreated wheat. Then, a further functional identification was performed in both Arabidopsis thaliana and Oryza sativa L. Subcellular localization analysis indicated that TaWRKY13 is a nuclear-localized protein. Moreover, various stress-related regulatory elements were predicted in the promoter. Expression pattern analysis revealed that TaWRKY13 can also be induced by polyethylene glycol (PEG), exogenous abscisic acid (ABA), and cold stress. After NaCl treatment, overexpressed Arabidopsis lines of TaWRKY13 have a longer root and a larger root surface area than the control (Columbia-0). Furthermore, TaWRKY13 overexpression rice lines exhibited salt tolerance compared with the control, as evidenced by increased proline (Pro) and decreased malondialdehyde (MDA) contents under salt treatment. The roots of overexpression lines were also more developed. These results demonstrate that TaWRKY13 plays a positive role in salt stress.


Assuntos
Tolerância ao Sal/genética , Fatores de Transcrição/genética , Triticum/genética , Triticum/metabolismo , Mapeamento Cromossômico , Cromossomos de Plantas , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Genômica/métodos , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Estresse Fisiológico/genética
16.
Int J Mol Sci ; 20(23)2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31775269

RESUMO

Plants have a series of response mechanisms to adapt when they are subjected to external stress. Calcium-dependent protein kinases (CDPKs) in plants function against a variety of abiotic stresses. We screened 17 CDPKs from drought- and salt-induced soybean transcriptome sequences. The phylogenetic tree divided CDPKs of rice, Arabidopsis and soybean into five groups (I-V). Cis-acting element analysis showed that the 17 CDPKs contained some elements associated with drought and salt stresses. Quantitative real-time PCR (qRT-PCR) analysis indicated that the 17 CDPKs were responsive after different degrees of induction under drought and salt stresses. GmCDPK3 was selected as a further research target due to its high relative expression. The subcellular localization experiment showed that GmCDPK3 was located on the membrane of Arabidopsis mesophyll protoplasts. Overexpression of GmCDPK3 improved drought and salt resistance in Arabidopsis. In the soybean hairy roots experiment, the leaves of GmCDPK3 hairy roots with RNA interference (GmCDPK3-RNAi) soybean lines were more wilted than those of GmCDPK3 overexpression (GmCDPK3-OE) soybean lines after drought and salt stresses. The trypan blue staining experiment further confirmed that cell membrane damage of GmCDPK3-RNAi soybean leaves was more severe than in GmCDPK3-OE soybean lines. In addition, proline (Pro) and chlorophyll contents were increased and malondialdehyde (MDA) content was decreased in GmCDPK3-OE soybean lines. On the contrary, GmCDPK3-RNAi soybean lines had decreased Pro and chlorophyll content and increased MDA. The results indicate that GmCDPK3 is essential in resisting drought and salt stresses.


Assuntos
Secas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Estresse Salino/genética , Cloreto de Sódio/efeitos adversos , Soja/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Oryza/efeitos dos fármacos , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Soja/efeitos dos fármacos , Soja/crescimento & desenvolvimento , Soja/metabolismo
17.
Int J Mol Sci ; 20(23)2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31775380

RESUMO

Human cytochrome P450 1B1 (CYP1B1)-mediated biotransformation of endobiotics and xenobiotics plays an important role in the progression of human breast cancer. In this study, we investigated the effects of WY-14643, a peroxisome proliferator-activated receptor α (PPARα) agonist, on CYP1B1 expression and the related mechanism in MCF7 breast cancer cells. We performed quantitative reverse transcription-polymerase chain reaction, transient transfection, and chromatin immunoprecipitation to evaluate the effects of PPARα on peroxisome proliferator response element (PPRE)-mediated transcription. WY-14643 increased the protein and mRNA levels of CYP1B1, as well as promoter activity, in MCF-7 cells. Moreover, WY-14643 plus GW6471, a PPARα antagonist, significantly inhibited the WY-14643-mediated increase in CYP1B1 expression. PPARα knockdown by a small interfering RNA markedly suppressed the induction of CYP1B1 expression by WY-14643, suggesting that WY-14643 induces CYP1B1 expression via a PPARα-dependent mechanism. Bioinformatics analysis identified putative PPREs (-833/-813) within the promoter region of the CYP1B1 gene. Inactivation of these putative PPREs by deletion mutagenesis suppressed the WY-14643-mediated induction of CYP1B1 promoter activation. Furthermore, WY-14643 induced PPARα to assume a form capable of binding specifically to the PPRE-binding site in the CYP1B1 promoter. Our findings suggest that WY-14643 induces the expression of CYP1B1 through activation of PPARα.


Assuntos
Neoplasias da Mama/metabolismo , Citocromo P-450 CYP1B1/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , PPAR alfa/metabolismo , Proliferadores de Peroxissomos/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células , Citocromo P-450 CYP1B1/metabolismo , Feminino , Humanos , PPAR alfa/genética , Regiões Promotoras Genéticas , Elementos de Resposta , Células Tumorais Cultivadas
18.
Nat Genet ; 51(10): 1494-1505, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570894

RESUMO

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells.


Assuntos
Linfócitos B/imunologia , Cromatina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Elementos de Resposta/genética , Linfócitos T/imunologia , Desequilíbrio Alélico , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/imunologia , Epigênese Genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Polissacarídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transcriptoma
19.
PLoS One ; 14(10): e0223521, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31603924

RESUMO

The adaptation of plants to land required multiple morphological innovations. Among these include a variety of lateral organs that are initiated from apical meristems, in which the mantainance of undifferentiated stem cells is regulated by the homeodomain WUSCHEL-RELATED (WOX) transcription factors. Expansion of the WOX gene family has been associated with whole genome duplication (WGD) events and postulated to have been pivotal to the evolution of morphological complexity in land plants. Previous studies have classified the WOX gene family into three superclades (e.g., the ancient clade, the intermediate clade, and the modern clade). In order to improve our understanding of the evolution of the WOX gene family, we surveyed the WOX gene sequences from 38 genomes and 440 transcriptomes spanning the Viridiplantae and Rhodophyta. The WOX phylogeny inferred from 1039 WOX proteins drawn from 267 species with improved support along the backbone of the phylogeny suggests that the plant-specific WOX family contains three ancient superclades, which we term Type 1 (T1WOX, the WOX10/13/14 clade), Type 2 (T2WOX, the WOX8/9 and WOX11/12 clades), and Type 3 (T3WOX, the WUS, WOX1/6, WOX2, WOX3, WOX4 and WOX5/7 clades). Divergence of the T1WOX and T2WOX superclades may predate the diversification of vascular plants. Synteny analysis suggests contribution of WGD to expansion of the WOX family. Promoter analysis finds that the capacity of the WOX genes to be regulated by the auxin and cytokinin signaling pathways may be deeply conserved in the Viridiplantae. This study improves our phylogenetic context for elucidating functional evolution of the WOX gene family, which has likely contributed to the morphological complexity of land plants.


Assuntos
Genes Homeobox , Filogenia , Plantas/classificação , Plantas/genética , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Arabidopsis/genética , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Funções Verossimilhança , Família Multigênica , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética
20.
Cell Struct Funct ; 44(2): 137-151, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31534067

RESUMO

The Golgi apparatus is an organelle where membrane or secretory proteins receive post-translational modifications such as glycosylation and sulfation, after which the proteins are selectively transported to their final destinations through vesicular transport. When the synthesis of secretory or membrane proteins is increased and overwhelms the capacity of the Golgi (Golgi stress), eukaryotic cells activate a homeostatic mechanism called the Golgi stress response to augment the capacity of the Golgi. Four response pathways of the Golgi stress response have been identified, namely the TFE3, CREB3, HSP47, and proteoglycan pathways, which regulate the general function of the Golgi, apoptosis, cell survival, and proteoglycan glycosylation, respectively. Here, we identified a novel response pathway that augments the expression of glycosylation enzymes for mucins in response to insufficiency in mucin-type glycosylation in the Golgi (mucin-type Golgi stress), and we found that expression of glycosylation enzymes for mucins such as GALNT5, GALNT8, and GALNT18 was increased upon mucin-type-Golgi stress. We named this pathway the mucin pathway. Unexpectedly, mucin-type Golgi stress induced the expression and activation of TFE3, a key transcription factor regulating the TFE3 pathway, suggesting that the activated mucin pathway sends a crosstalk signal to the TFE3 pathway. We identified an enhancer element regulating transcriptional induction of TFE3 upon mucin-type Golgi stress, and named it the mucin-type Golgi stress response element, of which consensus was ACTTCC(N9)TCCCCA. These results suggested that crosstalk from the mucin pathway to the TFE3 pathway has an important role in the regulation of the mammalian Golgi stress response.Key words: Golgi stress, mucin, TFE3, organelle autoregulation, organelle zone.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Complexo de Golgi/metabolismo , Mucinas/metabolismo , Elementos de Resposta/genética , Complexo de Golgi/genética , Células HT29 , Células HeLa , Humanos , Mucinas/genética , Mutação Puntual
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