RESUMO
Anti-pigmentation peptides have been developed as alternative skin-lightening agents to replace conventional chemicals that have adverse effects on the skin. However, the maximum size of these peptides is often limited by their low skin and cell penetration. To address this issue, we used our intra-dermal delivery technology (IDDT) platform to identify peptides with hypo-pigmenting and high cell-penetrating activity. Using our cell-penetrating peptides (CPPs) from the IDDT platform, we identified RMNE1 and its derivative RMNE3, "DualPep-Shine", which showed levels of α-Melanocyte stimulating hormone (α-MSH)-induced melanin inhibition comparable to the conventional tyrosinase inhibitor, Kojic acid. In addition, DualPep-Shine was delivered into the nucleus and regulated the gene expression levels of melanogenic enzymes by inhibiting the promoter activity of microphthalmia-associated transcription factor-M (MITF-M). Using a 3D human skin model, we found that DualPep-Shine penetrated the lower region of the epidermis and reduced the melanin content in a dose-dependent manner. Furthermore, DualPep-Shine showed high safety with little immunogenicity, indicating its potential as a novel cosmeceutical ingredient and anti-pigmentation therapeutic agent.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Peptídeos Penetradores de Células , Melaninas , Melanócitos , Fator de Transcrição Associado à Microftalmia , Proteínas do Tecido Nervoso , Preparações Clareadoras de Pele , Pigmentação da Pele , Transcrição Gênica , Melaninas/antagonistas & inibidores , Pigmentação da Pele/efeitos dos fármacos , Fator de Transcrição Associado à Microftalmia/genética , Transcrição Gênica/efeitos dos fármacos , alfa-MSH/antagonistas & inibidores , alfa-MSH/metabolismo , Humanos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Preparações Clareadoras de Pele/química , Preparações Clareadoras de Pele/farmacologia , Melanoma Experimental , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Epiderme/efeitos dos fármacos , Epiderme/metabolismoRESUMO
Substance use disorders are associated with disruptions to both circadian rhythms and cellular metabolic state. At the molecular level, the circadian molecular clock and cellular metabolic state may be interconnected through interactions with the nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylase, sirtuin 1 (SIRT1). In the nucleus accumbens (NAc), a region important for reward, both SIRT1 and the circadian transcription factor neuronal PAS domain protein 2 (NPAS2) are highly enriched, and both are regulated by the metabolic cofactor NAD+ . Substances of abuse, like cocaine, greatly disrupt cellular metabolism and promote oxidative stress; however, their effects on NAD+ in the brain remain unclear. Interestingly, cocaine also induces NAc expression of both NPAS2 and SIRT1, and both have independently been shown to regulate cocaine reward in mice. However, whether NPAS2 and SIRT1 interact in the NAc and/or whether together they regulate reward is unknown. Here, we demonstrate diurnal expression of Npas2, Sirt1 and NAD+ in the NAc, which is altered by cocaine-induced upregulation. Additionally, co-immunoprecipitation reveals NPAS2 and SIRT1 interact in the NAc, and cross-analysis of NPAS2 and SIRT1 chromatin immunoprecipitation sequencing reveals several reward-relevant and metabolic-related pathways enriched among shared gene targets. Notably, NAc-specific Npas2 knock-down or a functional Npas2 mutation in mice attenuates SIRT1-mediated increases in cocaine preference. Together, our data reveal an interaction between NPAS2 and SIRT1 in the NAc, which may serve to integrate cocaine's effects on circadian and metabolic factors, leading to regulation of drug reward.
Assuntos
Cocaína , Núcleo Accumbens , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Ritmo Circadiano/fisiologia , Cocaína/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Recompensa , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Antimicrobial peptides (AMPs) are short, positively charged host defense peptides, found in various life forms from microorganisms to humans. AMPs are gaining more attention as substitutes for antibiotics in order to combat the risk posed by multi-drug- resistant pathogens. The nematode Caenorhabditis elegans relies solely on its innate immune defense to cope with its challenging life-style. Bacterial infection in C. elegans leads to induction of antimicrobial proteins, defensins, nemapores, cecropins, and neuropeptide-like proteins, which act to limit bacterial proliferation. This study reports how the C. elegans recombinant antibacterial factor (ABF-1) rapidly inhibited bacterial growth (Salmonella Typhi, Klebsiella pneumonia, Shigella sonnei and Vibrio alginolyticus). The ABF-1 exposure on S. Typhi, showed differential regulation in cell-cycle, DNA repair mechanism, membrane stability, and stress related proteins. The exogenous supply of ABF-1 protein has extended C. elegans survival by reducing the bacterial colony forming units on the nematode intestine. Together, these findings indicate the valuable and potential therapeutic applications of ABF-1 protein as antimicrobial agents against intracellular pathogens.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Proteínas de Caenorhabditis elegans/farmacologia , Caenorhabditis elegans , Proteínas Recombinantes/farmacologia , Salmonella typhi/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Humanos , Intestinos/microbiologia , Testes de Sensibilidade Microbiana , Proteínas Recombinantes/genética , Análise de SobrevidaRESUMO
BACKGROUND: In the developing mouse embryo, the bHLH transcription factor Neurog2 is transiently expressed by retinal progenitor cells and required for the initial wave of neurogenesis. Remarkably, another bHLH factor, Ascl1, normally not present in the embryonic Neurog2 retinal lineage, can rescue the temporal phenotypes of Neurog2 mutants. RESULTS: Here we show that Neurog2 simultaneously promotes terminal cell cycle exit and retinal ganglion cell differentiation, using mitotic window labeling and integrating these results with retinal marker quantifications. We also analyzed the transcriptomes of E12.5 GFP-expressing cells from Neurog2GFP/+ , Neurog2GFP/GFP , and Neurog2Ascl1KI/GFP eyes, and validated the most significantly affected genes using qPCR assays. CONCLUSIONS: Our data support the hypothesis that Neurog2 acts at the top of a retinal bHLH transcription factor hierarchy. The combined expression levels of these downstream factors are sufficiently induced by ectopic Ascl1 to restore RGC genesis, highlighting the robustness of this gene network during retinal ganglion cell neurogenesis. Developmental Dynamics 247:965-975, 2018. © 2018 Wiley Periodicals, Inc.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Ciclo Celular , Proteínas do Tecido Nervoso/fisiologia , Neurogênese , Células Ganglionares da Retina/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos , Camundongos , Transcriptoma/efeitos dos fármacosRESUMO
Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Proteínas de Homeodomínio/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Fatores de Transcrição Maf Maior/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Transativadores/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Reprogramação Celular/efeitos dos fármacos , Cinamatos/farmacologia , Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Niacinamida/farmacologia , Transativadores/genética , Transativadores/metabolismo , Tretinoína/farmacologia , Alcaloides de Veratrum/farmacologiaRESUMO
BACKGROUND: Neurogenin2 (Ngn2) is a proneural gene that directs neuronal differentiation of progenitor cells during development. Here, we investigated whether Ngn2 can reprogram MSCs to adopt a neural precursor fate and enhance the therapeutic effects of MSCs after experimental stroke. METHODS: In vitro, MSCs were transfected with lenti-GFP or lenti-Ngn2. Following neuronal induction, cells were identified by immunocytochemistry, Western blot and electrophysiological analyses. In a stroke model induced by transient right middle cerebral artery occlusion (MCAO), PBS, GFP-MSCs or Ngn2-MSCs were injected 1 day after MCAO. Behavioral tests, neurological and immunohistochemical assessments were performed. RESULTS: In vitro, Ngn2-MSCs expressed neural stem cells markers (Pax6 and nestin) and lost the potential to differentiate into mesodermal cell types. Following neural induction, Ngn2-MSCs expressed higher levels of neuron-specific proteins MAP2, Tuj1 and NeuN, and also expressed voltage-gated Na+ channel, which was absent in GFP-MSCs. In vivo, after transplantation, Ngn2-MSCs significantly reduced apoptotic cells, decreased infarct volume, and increased the expression of VEGF and BDNF. Finally, Ngn2-MSCs treated animals showed the highest functional recovery among the three groups. CONCLUSIONS: Ngn2 was sufficient to convert MSCs into a neural precursor fate and transplantation of Ngn2-MSCs was advantageous for the treatment of stroke rats.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Células-Tronco Neurais/metabolismo , Acidente Vascular Cerebral/terapia , Aloenxertos , Animais , Antígenos de Diferenciação/biossíntese , Masculino , Células-Tronco Mesenquimais/patologia , Células-Tronco Neurais/patologia , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
BACKGROUND: Cardiovascular patients suffer from reduced blood flow leading to ischaemia and impaired tissue metabolism. Unfortunately, an increasing group of elderly patients cannot be treated with current revascularization methods. Thus, new treatment strategies are urgently needed. Hypoxia-inducible factors (HIFs) upregulate the expression of angiogenic mediators together with genes involved in energy metabolism and recovery of ischaemic tissues. Especially, HIF-2α is a novel factor, and only limited information is available about its therapeutic potential. METHODS: Gene transfers with adenoviral HIF-1α and HIF-2α were performed into the mouse heart and rabbit ischaemic hindlimbs. Angiogenesis was evaluated by histology. Left ventricle function was analysed with echocardiography. Perfusion in rabbit skeletal muscles and energy recovery after electrical stimulation-induced exercise were measured with ultrasound and (31)P-magnetic resonance spectroscopy ((31)P-MRS), respectively. RESULTS: HIF-1α and HIF-2α gene transfers increased capillary size up to fivefold in myocardium and ischaemic skeletal muscles. Perfusion in skeletal muscles was increased by fourfold without oedema. Especially, AdHIF-1α enhanced the recovery of ischaemic muscles from electrical stimulation-induced energy depletion. Special characteristic of HIF-2α gene transfer was a strong capillary growth in muscle connective tissue and that HIF-2α gene transfer maintained left ventricle function. CONCLUSIONS: We conclude that both AdHIF-1α and AdHIF-2α gene transfers induced beneficial angiogenesis in vivo. Transient moderate increases in angiogenesis improved energy recovery after exercise in ischaemic muscles. This study shows for the first time that a moderate increase in angiogenesis is enough to improve tissue energy metabolism, which is potentially a very useful feature for cardiovascular gene therapy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/farmacologia , Músculo Esquelético/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Capilares/fisiologia , Vasos Coronários/fisiologia , Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Isquemia/terapia , Camundongos Endogâmicos C57BL , Músculo Esquelético/irrigação sanguínea , Miocárdio/metabolismo , CoelhosRESUMO
Glioblastoma multiform (GBM) are devastating brain tumors containing a fraction of multipotent stem-like cells which are highly tumorigenic. These cells are resistant to treatments and are likely to be responsible for tumor recurrence. One approach to eliminate GBM stem-like cells would be to force their terminal differentiation. During development, neurons formation is controlled by neurogenic transcription factors such as Ngn1/2 and NeuroD1. We found that in comparison with oligodendrogenic genes, the expression of these neurogenic genes is low or absent in GBM tumors and derived cultures. We thus explored the effect of overexpressing these neurogenic genes in three CD133(+) Sox2(+) GBM stem-like cell cultures and the U87 glioma line. Introduction of Ngn2 in CD133(+) cultures induced massive cell death, proliferation arrest and a drastic reduction of neurosphere formation. Similar effects were observed with NeuroD1. Importantly, Ngn2 effects were accompanied by the downregulation of Olig2, Myc, Shh and upregulation of Dcx and NeuroD1 expression. The few surviving cells adopted a typical neuronal morphology and some of them generated action potentials. These cells appeared to be produced at the expense of GFAP(+) cells which were radically reduced after differentiation with Ngn2. In vivo, Ngn2-expressing cells were unable to form orthotopic tumors. In the U87 glioma line, Ngn2 could not induce neuronal differentiation although proliferation in vitro and tumoral growth in vivo were strongly reduced. By inducing cell death, cell cycle arrest or differentiation, this work supports further exploration of neurogenic proteins to oppose GBM stem-like and non-stem-like cell growth.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Neoplasias Encefálicas/patologia , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Fatores de Transcrição/farmacologia , Antígeno AC133 , Antígenos CD/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Morte Celular , Citometria de Fluxo , Proteína Glial Fibrilar Ácida/metabolismo , Glicoproteínas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Fator de Transcrição 2 de Oligodendrócitos , Proteína Oncogênica p55(v-myc)/metabolismo , Peptídeos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Long noncoding RNAs (lncRNAs) are emerging regulators of vascular diseases, yet their role in diabetic vascular calcification/aging remains poorly understood. In this study, we identified a down-expressed lncRNA SNHG1 in high glucose (HG)-induced vascular smooth muscle cells (HA-VSMCs), which induced excessive autophagy and promoted HA-VSMCs calcification/senescence. Overexpression of SNHG1 alleviated HG-induced HA-VSMCs calcification/senescence. The molecular mechanisms of SNHG1 in HA-VSMCs calcification/senescence were explored by RNA pull-down, RNA immunoprecipitation, RNA stability assay, luciferase reporter assay, immunoprecipitation and Western blot assays. In one mechanism, SNHG1 directly interacted with Bhlhe40 mRNA 3'-untranslated region and increased Bhlhe40 mRNA stability and expression. In another mechanism, SNHG1 enhanced Bhlhe40 protein SUMOylation by serving as a scaffold to facilitate the binding of SUMO E3 ligase PIAS3 and Bhlhe40 protein, resulting in increased nuclear translocation of Bhlhe40 protein. Moreover, Bhlhe40 suppressed the expression of Atg10, which is involved in the process of autophagosome formation. Collectively, the protective effect of SNHG1 on HG-induced HA-VSMCs calcification/senescence is accomplished by stabilizing Bhlhe40 mRNA and promoting the nuclear translocation of Bhlhe40 protein. Our study could provide a novel approach for diabetic vascular calcification/aging.
Assuntos
Proteínas Relacionadas à Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , MicroRNAs , RNA Longo não Codificante , Calcificação Vascular , Humanos , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Glucose/metabolismo , Proteínas de Homeodomínio , MicroRNAs/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/farmacologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Inibidoras de STAT Ativados/farmacologia , RNA Longo não Codificante/metabolismoRESUMO
Olig2 protein, a member of the basic helix-loop-helix transcription factor family, was introduced into the mouse embryonic carcinoma cell line P19 for induction of motor neuron differentiation. We show that Olig2 protein has the ability to permeate the cell membrane without the addition of a protein transduction domain (PTD), similar to other basic helix-loop-helix transcription factors such as MyoD and NeuroD2. Motor neuron differentiation was evaluated for the elongation of neurites and the expression of choline acetyltransferase (ChAT) mRNA, a differentiation marker of motor neurons. By addition of Olig2 protein, motor neuron differentiation was induced in P19 cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Linhagem Celular Tumoral , Colina O-Acetiltransferase/biossíntese , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Neuritos/metabolismo , Neuritos/fisiologia , Neurogênese/efeitos dos fármacos , Fator de Transcrição 2 de Oligodendrócitos , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/biossínteseRESUMO
Saponins from bitter melon (BMS) exert potential bioactivities and pharmacological activities, including anti-oxidation and lifespan extension. However, the exact mechanisms of BMS in response to oxidative stress remain unknown. Results demonstrated that bitter melon saponins could strengthen locomotive activities (body bend and head thrashing) accompanied by delaying the muscle fiber damage with age in Caenorhabditis elegans. In addition, BMS inhibited the ROS accumulation, improved the activities of antioxidant enzymes like SOD (by 57.90% and 94.34% for 100 µg/ml and 200 µg/ml BMS, respectively) and CAT (by 51.45% and 56.91% for 100 µg/ml and 200 µg/ml BMS, respectively), and extend the lifespan of N2 and CL2006 worms under paraquat-induced oxidative stress. Mechanism study suggested that BMS modulated the mRNA expressions of oxidation-related regulators, like the upregulation of cat-1, hsf-1, sir-2.1, and hlh-30. Furthermore, gene-deficient mutants verified that IIS (insulin/insulin-like growth factor-1 signaling) pathway linked with sir-2.1 and hlh-30 factors were involved in the BMS's lifespan-extension effects under oxidative stress. In general, this study supplemented the explanation of BMS in promoting oxidation-resistance and lifespan-extension activities, which could be served as a potential candidate for anti-aging. PRACTICAL APPLICATIONS: Our previous studies have suggested that saponins from bitter melon exhibited fat-lowering activity in C. elegans. However, little was known about the mechanism underlying the anti-oxidation effects of BMS in C. elegans. Current results indicated that the IIS pathway linked with sir-2.1 and hlh-30 transcriptional factors jointly to increase the lifespan in BMS' responses to oxidative stress. Our findings are beneficial to understand the main nutritional ingredients in bitter melon, which are ideal and expected in functional foods for aging.
Assuntos
Proteínas de Caenorhabditis elegans , Momordica charantia , Saponinas , Sirtuínas , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Saponinas/farmacologia , Estresse Oxidativo , Envelhecimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sirtuínas/metabolismo , Sirtuínas/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologiaRESUMO
OBJECTIVE: To observe the mutation and expression of TCF3 gene in Burkitt's lymphoma (BL), and explore its effect on the proliferation of BL cells and clinical efficacy and prognosis. METHODS: The mutation and expression of TCF3 in tumor tissues from BL patients were observed by the second-generation sequencing and real-time quantitative PCR. The proliferation and apoptosis of lymphoma cells after TCF3 knocked down were observed by siRNA interference technique and CCK-8 method. Survival analysis was used to observe the relationship between TCF3 mutation and the treatment efficacy and prognosis of BL patients. RESULTS: There were high frequency mutation rate (mutation rate was 23.7%) and high expression of TCF3 in BL patients. After TCF3 knocked down, cell proliferation was inhibited and apoptosis was promoted. In TCF3-siRNA group and control group, the cell proliferation rate at 48 h was (50.2±5.9)% and (96.6±11.4)%, and apoptosis rate was 30.1% and 1.5%, respectively, which showed significantly different between the two groups (P<0.001, P=0.005). The complete remission rate of patients with TCF3 mutation was low. The complete remission rate of mutant group and wild-type group was 44.4% and 82.8%, respectively (P=0.023). The 2-year progression-free survival rate and overall survival rate of the patients with TCF3 mutation was 55.6% and 61.0%, respectively, which was lower than 83.2% and 85.2% of the patients without mutation, but the differences were not statistically significant. CONCLUSION: There are mutation and abnormal expression of TCF3 in patients with BL. Patients with TCF3 mutations have low remission rate and poor prognosis.
Assuntos
Linfoma de Burkitt , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/uso terapêutico , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/genética , Humanos , Prognóstico , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêuticoRESUMO
The effect of long intergenic noncoding RNA 01315 (LINC01315) on colorectal cancer has widely been proved. Nevertheless, how LINC01315 functions in the stemness of colorectal cancer and whether LINC01315 exists in colorectal cancer stem-like cell-derived exosomes remain dim, which are thus investigated in this research. CD133+/CD44+ colorectal cancer stem cells were sorted and verified through flow cytometry. Exosomes derived from CD133+/CD44+ colorectal cancer stem cells were collected. The viability, proliferation, stemness and migration of CD133+/CD44+, CD133-/CD44-, and colorectal cancer cells after transfection or the co-culture with exosomes were detected by MTT, colony formation, spheroid, and wound healing assays, respectively. Expressions of LINC01315, BCL-2, Bax, cleaved caspase-3, MMP-9, E-cadherin, and vimentin in cells or exosomes were analyzed using western blot or qRT-PCR. Genes interacted with LINC01315 in colorectal cancer were predicted by bioinformatics analysis. The results showed that LINC01315 was high-expressed in CD133+/CD44+ colorectal cancer stem cells and exosomes. Compared with colorectal cancer cells, the viability, proliferation, stemness, and migration of CD133+/CD44+ cancer cells were stronger, while these of CD133-/CD44- cancer cells were weaker. Besides, LINC01315 silencing decreased the viability, proliferation, stemness, and migration of CD133+/CD44+ cancer cells, while sh-LINC01315 inhibited the promotive effects of CD133+/CD44+ cancer cell-derived exosomes on the viability, proliferation, stemness, and migration of colorectal cancer cells. LINC01315 was also found to be correlated with DPEP1, KRT23, ASCL2, AXIN2, and DUSP4 in colorectal cancer. In conclusion, colorectal cancer stem cell-derived exosomal LINC01315 promotes the proliferation, migration, and stemness of colorectal cancer cells.
Assuntos
Neoplasias Colorretais , RNA Longo não Codificante , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/metabolismoRESUMO
OBJECTIVE: Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins. METHODS: Quantitative reverse transcription-polymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity. RESULTS: ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1α and HIF-2α resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1ß-null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1α and HIF-2α in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxia-responsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state. CONCLUSION: Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions.
Assuntos
Anquirinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Calcinose/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Disco Intervertebral/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anquirinas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Western Blotting , Calcinose/induzido quimicamente , Calcinose/patologia , Hipóxia Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/patologia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , RatosRESUMO
OBJECTIVE: In various cancers, migration and invasion inhibitory protein (MIIP) is expressed at low level and is involved in cancer pathogenesis. Herein, we sought to explore the function of MIIP in clear cell renal cell carcinoma (ccRCC). METHODS: CCK-8, colony formation, cell cycle, and endothelial cell tube formation assays were performed to evaluate the roles of MIIP in ccRCC proliferation and angiogenesis. To explore the underlying mechanism, we conducted RNA-sequencing, GSEA, qRT-PCR, Western blot, ELISA, cell transfection, coimmunoprecipitation, and ubiquitination assays in ccRCC cell lines. Furthermore, xenograft tumor growth in nude mice, and Ki-67 and CD31 staining in xenograft tissues were examined. Finally, the association of MIIP expression with clinical pathology and the expression status of HIF-2α and cysteine-rich 61 (CYR61) were further analyzed in human RCC tissues through Western blot and immunohistochemistry. RESULTS: Both in vitro and in vivo functional experiments indicated that forced expression of MIIP inhibited ccRCC proliferation and angiogenesis, whereas silencing MIIP either in normal HK-2 cells or in ccRCC cells had the opposite effect (P < 0.05). Mechanistically, CYR61 was identified as a gene significantly downregulated by MIIP overexpression, and was required for the suppressive role of MIIP in ccRCC. MIIP was found to promote HSP90 acetylation and thus impair its chaperone function toward HIF-2α. Consequently, RACK1 binds HIF-2α and causes its ubiquitination and proteasomal degradation, thus decreasing the transcription of its target, CYR61. Finally, analyses of clinical samples demonstrated that MIIP is significantly downregulated in cancer vs. normal tissues in RCC cases, and its expression is negatively associated with histological grade, metastasis, the prognosis of patients with RCC, and the expression of HIF-2α and CYR61 (P < 0.05). CONCLUSIONS: MIIP is a novel tumor suppressor in ccRCC via negative regulation of HIF-2α-CYR61 axis.
Assuntos
Carcinoma de Células Renais , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Renais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Proliferação de Células , Cisteína/genética , Cisteína/metabolismo , Cisteína/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Processos NeoplásicosRESUMO
Nurr1 is a transcription factor specific for the development and maintenance of the midbrain dopamine (DA) neurons. Exogenous Nurr1 in neural precursor (NP) cells induces the differentiation of DA neurons in vitro that are capable of reversing motor dysfunctions in a rodent model for Parkinson disease. The promise of this therapeutic approach, however, is unclear due to poor cell survival and phenotype loss of DA cells after transplantation. We herein demonstrate that Nurr1 proteins undergo ubiquitin-proteasome-system-mediated degradation in differentiating NP cells. The degradation process is activated by a direct Akt-mediated phosphorylation of Nurr1 proteins and can be prevented by abolishing the Akt-target sequence in Nurr1 (Nurr1(Akt)). Overexpression of Nurr1(Akt) in NP cells yielded DA neurons in which Nurr1 protein levels were maintained for prolonged periods. The sustained Nurr1 expression endowed the Nurr1(Akt)-induced DA neurons with resistance to toxic stimuli, enhanced survival, and sustained DA phenotypes in vitro and in vivo after transplantation.
Assuntos
Dopamina/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Western Blotting , Butadienos/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Imunoprecipitação , Mesencéfalo/citologia , Morfolinas/farmacologia , Nitrilas/farmacologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Estabilidade Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Caudal-related homeobox transcription factors are involved in the definition of the anteroposterior axis and intestinal development. Recent reports indicate that dysregulation of CDX1 and CDX2, the human homologues of Drosophila caudal, are associated with several types of cancer. Very little is known, however, about the regulatory mechanisms that direct the caudal-related homeobox gene expression. In this study, we have identified the binding sites for bHLH-PAS proteins, referred to as CNS midline element (CME), in the 5'-flanking region of the Drosophila caudal gene. Analyses using transgenic flies carrying a caudal-lacZ fusion gene bearing a wild-type or mutant CME indicate that the CME sites are required for caudal gene expression in vivo. We also determined that the caudal promoter activity can be regulated by Trachealess (Trh)/Tango (Tgo) bHLH-PAS proteins, via the CME sites. Our results suggest that the Drosophila caudal gene is a target of the Trh/Tgo bHLH-PAS proteins.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Proteínas de Drosophila/farmacologia , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Proteínas de Drosophila/genética , Embrião não Mamífero , TransgenesRESUMO
Members of the Caenorhabditis elegans REF-1 family of bHLH proteins are atypical in that each protein contains two bHLH domains. In this study we describe a functional and molecular characterization of the REF-1 family members, hlh-29/hlh-28. 5'-RACE results confirm the presence of two bHLH domain coding regions in a single transcript and quantitative PCR (qPCR) shows that hlh-29/hlh-28 mRNA is detected in wild-type animals throughout development. A promoter fusion of hlh-29 to the green fluorescent protein shows post-embryonic reporter activity in cells of the vulva, the somatic gonad, the intestine and in neuronal cells of the head and tail. Loss of hlh-29/hlh-28 function via RNA interference (RNAi) results in multiple phenotypes including late embryonic lethality, yolk protein accumulation, everted vulva, bordering behavior, and alter chemosensory responses.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/farmacologia , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/embriologia , Gônadas/metabolismo , Dados de Sequência Molecular , Neurônios/metabolismo , Interferência de RNA , Distribuição Tecidual , Vulva/embriologia , Vulva/metabolismoRESUMO
Runt-related transcription factor 2 (Runx2) is a key transcription factor for osteogenic gene expression and osteoblast differentiation. In order to maintain bone homeostasis, the transcriptional activity of Runx2 is tightly modulated by many intra- and extra-cellular factors. Here, we reveal the mechanism by which Hairy Enhancer of Split1 (Hes1) regulates the transcriptional activity of Runx2, and elucidate the potential role of Hes1 during osteoblast differentiation. Coexpression of Hes1 with Runx2 promoted an increase in Runx2 protein levels by increasing the half-life of Runx2; Hes1 thereby augmented the formation of a Runx2-DNA complex at Runx2 target sites. During osteoblast differentiation, the retroviral overexpression of Hes1 accelerated osteogenesis and stimulated the expression of osteogenic marker genes, including osteopontin and type 1 collagen. Taken together, these results suggest that Hes1 augments the protein level and transcriptional activity of Runx2, resulting in the stimulation of osteoblast differentiation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/farmacologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Células 3T3 , Animais , Sítios de Ligação , Diferenciação Celular/fisiologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/química , DNA/química , DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Meia-Vida , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/fisiologia , Fatores de Transcrição HES-1 , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: To determine the role of hypoxia-inducible factor-2alpha (HIF2alpha) on the sensitivity of renal cell carcinoma (RCC) cell lines to ionizing radiation and to determine if the mTOR antagonist, rapamycin, could decrease HIF2alpha protein levels. MATERIALS AND METHODS: Cell lines expressing stable short-hairpin RNAs (shRNAs) encoding HIF2alpha shRNAs or an empty vector were transfected with a hypoxia responsive element (HRE)-driven firefly luciferase reporter gene. Two separate paired cell lines were assayed for their response to increasing doses of ionizing radiation. Proliferation and cell cycle kinetics were compared for cell lines expressing HIF2alpha shRNAs and empty vectors. The effect of an mTOR antagonist, rapamycin on HIF1alpha and HIF2alpha proteins levels was also assessed. RESULTS: We confirmed that the 786-O RCC lines with stably integrated shRNAs against HIF2alpha had decreased activation of a plasmid with a HRE-driven firefly luciferase reporter gene. Lines from two separate cell clones with decreased HIF2alpha levels showed a significant increase in radiation sensitivity and an increase in G2 cell cycle arrest. Rapamycin, while effective in decreasing HIF1alpha protein levels, did not affect HIF2alpha levels in either of the RCC cell lines. CONCLUSIONS: These results show that decreasing levels of HIF2alpha leads to an increased sensitivity to ionizing radiation. This finding may explain in part, the known resistance of RCC to radiation therapy. Although mTOR antagonists are approved for the treatment of RCC, these agents do not decrease HIF2alpha levels and therefore might not be effective in enhancing the radio-sensitivity of these tumours.