RESUMEN
MicroRNA-22 (miR-22) is emerging as a critical regulator in organ development and various cancers. However, its role in normal hematopoiesis and leukaemogenesis remains unclear. Here, we detected its increased expression during monocyte/macrophage differentiation of HL-60, THP1 cells and CD34+ hematopoietic stem/progenitor cells, and confirmed that PU.1, a key transcriptional factor for monocyte/macrophage differentiation, is responsible for transcriptional activation of miR-22 during the differentiation. By gain- and loss-of-function experiments, we demonstrated that miR-22 promoted monocyte/macrophage differentiation, and MECOM (EVI1) mRNA is a direct target of miR-22 and MECOM (EVI1) functions as a negative regulator in the differentiation. The miR-22-mediated MECOM degradation increased c-Jun but decreased GATA2 expression, which results in increased interaction between c-Jun and PU.1 via increasing c-Jun levels and relief of MECOM- and GATA2-mediated interference in the interaction, and thus promoting monocyte/macrophage differentiation. We also observed significantly down-regulation of PU.1 and miR-22 as well as significantly up-regulation of MECOM in acute myeloid leukemia (AML) patients. Reintroduction of miR-22 relieved the differentiation blockage and inhibited the growth of bone marrow blasts of AML patients. Our results revealed new function and mechanism of miR-22 in normal hematopoiesis and AML development and demonstrated its potential value in AML diagnosis and therapy.
Asunto(s)
Proteínas de Unión al ADN/genética , Factor de Transcripción GATA2/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proto-Oncogenes/genética , Transactivadores/biosíntesis , Factores de Transcripción/genética , Diferenciación Celular/genética , Regulación Neoplásica de la Expresión Génica , Células HL-60 , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/patología , Proteína del Locus del Complejo MDS1 y EV11 , Macrófagos/metabolismo , MicroARNs/biosíntesis , Monocitos/metabolismo , Proteínas Proto-Oncogénicas/genética , Transactivadores/genéticaRESUMEN
Emerging evidence has shown that microRNAs have key roles in regulating various normal physiological processes, whereas their deregulated expression is correlated with various diseases. The miR-146 family includes miR-146a and miR-146b, with a distinct expression spectrum in different hematopoietic cells. Recent work indicated that miR-146a has a close relationship with inflammation and autoimmune diseases. miR-146-deficient mice have developed some abnormal hematopoietic phenotypes, suggesting the potential functions of miR-146 in hematopoietic development. In this study, we found that miR-146b was consistently up-regulated in both K562 and CD34(+) hematopoietic stem/progenitor cells (HSPCs) undergoing either erythroid or megakaryocytic differentiation. Remarkably, erythroid and megakaryocytic maturation of K562 cells was induced by excess miR-146b but inhibited by decreased miR-146b levels. More importantly, an mRNA encoding receptor tyrosine kinase, namely platelet-derived growth factor receptor α (PDGFRA), was identified and validated as a direct target of miR-146b in hematopoietic cells. Gain-of-function and loss-of-function assays showed that PDGFRA functioned as a negative regulator in erythroid and megakaryocytic differentiation. miR-146b could ultimately affect the expression of the GATA-1 gene, which is regulated by HEY1 (Hairy/enhancer-of-split related with YRPW motif protein 1), a transcriptional repressor, via inhibition of the PDGFRA/JNK/JUN/HEY1 pathway. Lentivirus-mediated gene transfer also demonstrated that the overexpression of miR-146b promoted erythropoiesis and megakaryocytopoiesis of HSPCs via its regulation on the PDGFRA gene and effects on GATA-1 expression. Moreover, we confirmed that the binding of GATA-1 to the miR-146b promoter and induction of miR-146b during hematopoietic maturation were dependent on GATA-1. Therefore, miR-146b, PDGFRA, and GATA-1 formed a regulatory circuit to promote erythroid and megakaryocytic differentiation.
Asunto(s)
Células Eritroides/metabolismo , Eritropoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Megacariocitos/metabolismo , MicroARNs/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Trombopoyesis/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Células Eritroides/citología , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Células K562 , Megacariocitos/citología , Ratones , MicroARNs/genética , Regiones Promotoras Genéticas/fisiología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
We previously characterized the zinc finger protein gene HZF1 [also known as ZNF16 (zinc finger protein 16)] and demonstrated its important roles in erythroid and megakaryocytic differentiation of K562 cells. In the present study, we investigated its effect on erythroid and megakaryocytic differentiation of HSPCs (haemopoietic stem/progenitor cells). We observed up-regulation of ZNF16 during erythroid and megakaryocytic differentiation of the CD34+ HSPCs, and demonstrated that ZNF16 promotes erythroid and megakaryocytic differentiation by gain-of-function and loss-of-function experiments. Using a luciferase reporter and ChIP assays ZNF16 was demonstrated to bind to the c-KIT gene promoter and inhibit its expression in K562 cells. Enforced expression and knockdown of ZNF16 down-regulated and up-regulated the expression of the c-KIT gene in K562 cells and HSPCs respectively. Significantly decreased levels of the c-Kit protein were observed following erythroid and megakaryocytic differentiation of K562 and CD34+ cells. The knockdown of c-KIT partially rescued the differentiation inhibition caused by ZNF16 knockdown. The knockdown of c-KIT also blocked the activity of the c-Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK/c-Jun signal pathway and reduced further the level of HEY1 (hes-related family bHLH transcription factor with YRPW motif 1), a repressor of GATA1 (GATA-binding protein 1) transcription, which finally up-regulated the expression of GATA1, a central regulator of erythroid and megakaryocytic differentiation. In conclusion the results of the present study demonstrate that ZNF16 plays an important role in erythropoiesis and megakaryocytopoiesis via its regulation of the c-Kit/c-Raf/MEK/ERK/c-Jun/HEY1/GATA1 cascade.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Eritropoyesis/fisiología , Regulación de la Expresión Génica/fisiología , Megacariocitos/citología , Proteínas Proto-Oncogénicas c-kit/genética , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Cartilla de ADN , Humanos , Células K562 , Reacción en Cadena en Tiempo Real de la Polimerasa , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Although microRNAs (miRNAs) are increasingly linked to various physiologic processes, including hematopoiesis, their function in the myeloid development is poorly understood. We detected up-regulation of miR-29a and miR-142-3p during myeloid differentiation in leukemia cell lines and CD34(+) hematopoietic stem/progenitor cells. By gain-of-function and loss-of-function experiments, we demonstrated that both miRNAs promote the phorbol 12-myristate 13-acetate-induced monocytic and all-trans-retinoic acid-induced granulocytic differentiation of HL-60, THP-1, or NB4 cells. Both the miRNAs directly inhibited cyclin T2 gene, preventing the release of hypophosphorylated retinoblastoma and resulting in induction of monocytic differentiation. In addition, a target of miR-29a, cyclin-dependent kinase 6 gene, and a target of miR-142-3p, TGF-ß-activated kinase 1/MAP3K7 binding protein 2 gene, are involved in the regulation of both monocytic and granulocytic differentiation. A significant decrease of miR-29a and 142-3p levels and an obvious increase in their target protein levels were also observed in blasts from acute myeloid leukemia. By lentivirus-mediated gene transfer, we demonstrated that enforced expression of either miR-29a or miR-142-3p in hematopoietic stem/progenitor cells from healthy controls and acute myeloid leukemia patients down-regulated expression of their targets and promoted myeloid differentiation. These findings confirm that miR-29a and miR-142-3p are key regulators of normal myeloid differentiation and their reduced expression is involved in acute myeloid leukemia development.
Asunto(s)
Diferenciación Celular/genética , Leucemia Mieloide Aguda/genética , MicroARNs/fisiología , Células Mieloides/fisiología , Antineoplásicos/farmacología , Carcinógenos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/fisiología , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , MicroARNs/genética , MicroARNs/metabolismo , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transfección , Tretinoina/farmacologíaRESUMEN
Hypoxia-inducible factor-1 (HIF-1) can activate expression of a broad range of genes in response to hypoxia. It has been shown that the levels of peroxisome proliferator-activated receptor γ (PPARγ) are influenced by changes in oxygen tension, and PPARγ plays a critical role in metabolism regulation and cancers. In this research, we observed an increased PPARγ mRNA and protein levels in company with increased HIF-1 protein levels in HepG2 cells in hypoxia as compared with in normoxia. Enforced expression of HIF-1α induced PPARγ1 and PPARγ2 expression, while knockdown of HIF-1α by small interference RNA deduced PPARγ1 and PPARγ2 expression in HepG2 cells under hypoxic conditions. By dual-luciferase reporter assay and chromatin immunoprecipitation assay we confirmed a functional hypoxic response element (HRE) localized at 684bp upstream of the transcriptional start site (TSS) of PPARγ1 and a functional HRE localized at 204bp downstream of the TSS of PPARγ2 in HepG2 cells. Additionally we observed an increase and co-presence of PPARγ and HIF-1α, and a highly positive correlation between PPARγ expression and HIF-1α expression (r=0.553, p<0.0001), in the same tumor tissue areas of hepatocellular carcinoma patients. Our data suggested a new mechanism of hepatocellular carcinoma cells response to hypoxia.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Hipoxia de la Célula , Células Hep G2 , Humanos , Elementos de Respuesta/genética , Regulación hacia ArribaRESUMEN
BACKGROUND: An embryonic toxicity of Rhizoma sparganii was observed in mice. This study was aimed to evaluate the anticancer effects of Grailsine-Al-glycoside, the bioactive component of Rhizoma sparganii, on estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) cancer cell lines. METHODS: After A549, HeLa, HepG-2 and MCF-7 cells were treated with Grailsine-Al-glycoside, cell proliferation was analyzed by MTT, cell cycle and apoptosis by flow cytometry, and morphology with an immunofluorescence microscope. RESULTS: Grailsine-Al-glycoside strongly suppressed cell proliferation in a dose-dependent fashion in A549, MCF-7, HepG2, and HeLa cells, though this growth inhibitory effect on HepG2 cells was not as strong and long lasting. Compared to the control, Grailsine-Al-glycoside caused a significant increase of apoptosis in A549, MCF-7 and Hela cells. A549 and MCF-7 cells were arrested at the G2/S phase whereas HepG2 cells were arrested at the G1 phase by a high concentration of Grailsine-Al-glycoside . Cell shapes were also changed by the presence of Grailsine-Al-glycoside. CONCLUSIONS: Grailsine-Al-glycoside from Rhizoma sparganii inhibited the proliferation of ER+ and some ER- cancer cells. Grailsine-Al-glycoside may be used as a chemotherapeutic agent against ER+ and ERRα-expressing ER- cancers.
Asunto(s)
Antineoplásicos/farmacología , Medicamentos Herbarios Chinos/farmacología , Rizoma/química , Typhaceae/química , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , RatonesRESUMEN
Considering the impact of differences in watershed characteristics on river water quality, with the Chaohu Lake Basin as the research object, based on the data of water quality, meteorology, topography, soil, and remote sensing images of the river monitoring points from October 2019 to September 2020, the watershed unit at each monitoring point was divided through digital terrain analysis, and the comprehensive landscape characteristics based on the watershed unit were explored through the comprehensive use of correlation analysis, redundancy analysis, and multiple regression analysis to investigate the influence of comprehensive landscape characteristics based on watershed units (including land use, climate, topography, soil, etc.) on the water quality of rivers around Chaohu Lake. The results showed that:â the water quality of rivers around Chaohu Lake had large spatial differences, with the main pollutants being total nitrogen and ammonia nitrogen. Most of the rivers had total nitrogen concentrations exceeding the Class V water quality standards, and the areas with serious nitrogen and phosphorus pollution were concentrated in the urban area of Hefei and the surrounding rivers, as well as in the middle and lower reaches of the Fengle and Hangbu Rivers. â¡ The comprehensive landscape characteristics of the watershed unit had a significant impact on the river water quality. Among them, the proportion of built-up land, the density of patches, the dispersion and juxtaposition index, and the Shannon diversity index were positively correlated with the water quality indicators, whereas the proportion of forest and grassland and the spreading index were negatively correlated with the water quality indicators. ⢠In different seasons, the effect of the integrated landscape characteristics of the watershed unit on river water quality was stronger in the wet season than in the dry season, which was mainly caused by the difference in precipitation in the dry and wet seasons.
RESUMEN
In China, the traditional Chinese medicine "YiSui ShenXu Granule" has been used for treating ß-thalassemia over 20 years and known to be effective in clinic. Several purified components from "YiSui ShenXu Granule" are tested in K562 cells to reveal its effect on globin expression and erythroid differentiation, and one of the purified components, emodin, was demonstrated to increase the expression of α-, ε-, γ-globin, CD235a, and CD71 in K562 cells. Moreover, the increase of their expression is emodin concentration-dependent. The mRNA and microRNA (miRNA) expression profiles are further analyzed and 417 mRNAs and 35 miRNAs with differential expression between untreated and emodin-treated K562 cells were identified. Among them, two mRNAs that encode known positive regulators of erythropoiesis, ALAS2, and c-KIT respectively, increased during emodin-induced K562 erythroid differentiation, meanwhile, two negative regulators, miR-221 and miR-222, decreased during this process. These results indicate that emodin can improve the expression of globin genes in K562 cells and also induce K562 cells to erythroid differentiation possibly through up-regulating ALAS2 and c-KIT and down-regulating miR-221 and miR-222.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Emodina/farmacología , Células Eritroides/citología , Células Eritroides/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Globinas/genética , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Eritroides/metabolismo , Perfilación de la Expresión Génica , Globinas/metabolismo , Hemoglobinas/metabolismo , Humanos , Células K562 , Proteínas de Neoplasias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
Metabolism under hypoxia is significantly different from that under normoxia. It has been well elucidated that HIF-1 (hypoxia-inducible factor-1) plays a central role in regulating glucose metabolism under hypoxia; however, the role of HIF-1 in lipid metabolism has not yet been well addressed. In the present study we demonstrate that HIF-1 promotes LDL (low-density lipoprotein) and VLDL (very-LDL) uptake through regulation of VLDLR (VLDL receptor) gene expression under hypoxia. Increased VLDLR mRNA and protein levels were observed under hypoxic or DFO (deferoxamine mesylate salt) treatment in MCF7, HepG2 and HeLa cells. Using dual-luciferase reporter and ChIP (chromatin immunoprecipitation) assays we confirmed a functional HRE (hypoxia-response element) which is localized at +405 in exon 1 of the VLDLR gene. Knockdown of HIF1A (the α subunit of HIF-1) and VLDLR, but not HIF2A (the α subunit of HIF-2), attenuated hypoxia-induced lipid accumulation through affecting LDL and VLDL uptake. Additionally we also observed a correlation between HIF-1 activity and VLDLR expression in hepatocellular carcinoma specimens. The results of the present study suggest that HIF-1-mediated VLDLR induction influences intracellular lipid accumulation through regulating LDL and VLDL uptake under hypoxia.
Asunto(s)
Factor 1 Inducible por Hipoxia/fisiología , Hipoxia/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Receptores de LDL/biosíntesis , Línea Celular Tumoral , HumanosRESUMEN
Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.
Asunto(s)
Diferenciación Celular/genética , Células Eritroides/citología , Células Eritroides/metabolismo , Factor de Transcripción GATA1/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Secuencia de Bases , Hipoxia de la Célula/genética , Inmunoprecipitación de Cromatina , Citometría de Flujo , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Humanos , Células K562 , Células MCF-7 , Datos de Secuencia Molecular , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Elementos de Respuesta/genéticaRESUMEN
Calcium homeostasis is critical to amyloid beta precursor protein (APP) processing. Na(+)/Ca(2+) exchanger (NCX) proteins play an important role in maintaining intracellular Na(+) and Ca(2+) homeostasis in the brain under physiological and pathological conditions. We sequenced a hyper-variable region in intron 2 of the Na(+)/Ca(2+) exchanger 1 gene (NCX1), and investigated whether insertion/deletion variations in this region are associated with the occurrence for Alzheimer's disease (AD). Examining 413 AD patients and 361 healthy controls, we identified 3 insertion/deletion polymorphisms. No significant differences of the allele and genotype frequencies were observed between the AD cases and the controls for any of the three polymorphisms. However, among the AD patients whose age at onset (AAO) was 65 years or older (n = 299), carriers of a 14 bp insertion showed a lower average AAO (ins/ins and ins/del vs. del/del, 72.49 ± 5.17 vs. 74.28 ± 5.79, p = 0.016). It suggested that this 14 bp insertion/deletion polymorphism might modulate AAO in late-onset AD patients.
Asunto(s)
Enfermedad de Alzheimer/genética , Mutación INDEL , Polimorfismo Genético , Intercambiador de Sodio-Calcio/genética , Edad de Inicio , Anciano , Anciano de 80 o más Años , Alelos , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Intrones , Masculino , Persona de Mediana EdadRESUMEN
Expression profiling of microRNAs (miRNAs) in most diseases might be popular and provide the possibility for diagnostic implication, but few studies have accurately quantified the expression level of dysregulated miRNAs in acute myeloid leukemia (AML). In this study, we analyzed the peripheral blood mononuclear cells (PBMCs) from 10 AML patients (subtypes M1 to M5) and six normal controls by miRNA microarray and identified several differentially expressed miRNAs. Among them miR-29a and miR-142-3p were selectively encountered in Northern blot analysis and their significantly decreased expression in AML was further confirmed. Quantitative real-time PCR in 52 primarily diagnosed AML patients and 100 normal controls not only verified the expression properties of these 2 miRNAs, but also established that the expression level of miR-142-3p and miR-29a in PBMCs could be used as novel diagnostic markers. A better diagnostic outcome was achieved by combining miR-29a and miR-142-3p with about 90% sensitivity, 100% specificity, and an area under the ROC curve (AUC) of 0.97. Our results provide insights into the involvement of miRNAs in leukemogenesis, and offer candidates for AML diagnosis and therapeutic strategy.
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Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica/genética , Leucemia Mieloide Aguda/genética , MicroARNs/metabolismo , Área Bajo la Curva , Northern Blotting , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Leucocitos Mononucleares/metabolismo , Análisis por Micromatrices , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Hypoxia-inducible factor 1 (HIF1) is a heterodimeric basic helix-loop-helix transcription factor that regulates many key genes. δ-Aminolevulinate synthase (ALAS) catalyzes the first and rate-limiting reaction in the heme biosynthetic pathway. In this study, we show that hypoxia-induced expression of erythroid-specific ALAS2 is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased ALAS2 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells. Enforced HIF1α expression increased the level of ALAS2 expression, while HIF1α knockdown by RNA interference decreased the level of ALAS2 expression. In silico analysis revealed three potential hypoxia-response elements (HREs) that are located 611, 621, and 741 bp downstream of the ALAS2 gene. The results from reporter gene and mutation analysis suggested that these elements are necessary for a maximal hypoxic response. Chromatin immunoprecipitation and polymerase chain reaction showed that the HREs could be recognized and bound by HIF1α in vivo. These results demonstrate that the upregulation of ALAS2 during hypoxia is directly mediated by HIF1. We hypothesize that HIF1-mediated ALAS2 upregulation promotes erythropoiesis to satisfy the needs of an organism under hypoxic conditions. This may be accomplished via increased heme levels and an interaction between ALAS2 and erythropoietin.
Asunto(s)
5-Aminolevulinato Sintetasa/biosíntesis , Células Eritroides/enzimología , Factor 1 Inducible por Hipoxia/fisiología , 5-Aminolevulinato Sintetasa/genética , Secuencia de Bases , Sitios de Unión/genética , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Células Cultivadas , Inducción Enzimática/fisiología , Células Eritroides/metabolismo , Eritropoyesis/genética , Eritropoyesis/fisiología , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Células K562 , Modelos Biológicos , Especificidad de Órganos/genética , Unión Proteica , Elementos de Respuesta/genética , Elementos de Respuesta/fisiología , Estudios de Validación como AsuntoRESUMEN
BACKGROUND: A potential strategy for treatment of sickle cell disease (SCD) and ß-thalassemia in adults is reactivation of the ε- and γ-globin genes in the adult. We aimed to identify trans-activators of ε- and γ-globin expression and provide new candidate targets for effective treatment of sickle cell disease (SCD) and ß-thalassemia through activation of ε- and γ-globin genes in adults. RESULTS: We identified a CTD small phosphatase like 2 (CTDSPL2) gene that had higher transcription levels in umbilical cord blood (UCB) than in adult bone marrow (BM). Also, transcription of the CTDSPL2 gene increased significantly during erythroid differentiation. Further, we found that overexpression of CTDSPL2 could obviously improve the expression of ε- and γ-globin genes in K562 cells. Meanwhile, the repression of CTDSPL2 by RNA interference decreased expression of ε- and γ-globin genes but did not inhibit the increase of globin gene expression during K562 erythroid differentiation. In addition, the enforced expression of CTDSPL2 gene mediated by lentiviruses could also increase ε- and γ-globin gene expression during erythroid differentiation of CD34+ cells derived from UCB. CONCLUSION: CTDSPL2 gene can obviously improve the expression of ε- and γ-globin genes in K562 cells and CD34+ cells derived from UCB. Our study provides a new candidate target for effective treatment of SCD and ß-thalassemia.
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Anemia de Células Falciformes/terapia , Proteínas Supresoras de Tumor/metabolismo , Talasemia beta/terapia , Globinas épsilon/genética , gamma-Globinas/genética , Adulto , Anemia de Células Falciformes/genética , Antígenos CD34/biosíntesis , Eritropoyesis/genética , Sangre Fetal/citología , Sangre Fetal/metabolismo , Terapia Genética , Humanos , Células K562 , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , ARN Interferente Pequeño/genética , Activación Transcripcional/genética , Transgenes/genética , Proteínas Supresoras de Tumor/genética , Talasemia beta/genéticaRESUMEN
Folate deficiency and elevated plasma homocysteine play important roles in pathogenesis of Alzheimer's disease (AD). The aim of this study was to test the association of folate metabolism-related genes, cystathionine beta-synthase gene (CbetaS) and 5, 10-methylenetetrahydrofolate dehydrogenase gene (MTHFD1), with sporadic AD. The CbetaS 844ins68 polymorphism was determined by PCR and the MTHFD1 G1958A single nucleotide polymorphism (rs2236225) by PCR-RFLP. No significant difference of allele and genotype contributions of the CbetaS polymorphism between AD cases and controls was detected, before and after stratification by APOE epsilon4-carrying status, age/age at onset and genders. No significant difference of allele and genotype contributions of the MTHFD1 polymorphism between AD cases and controls was detected in total samples. When stratified by age/at onset age, we found that A allele and AA genotype frequencies in cases were higher than in controls and the differences were close to significant [A vs. G, P = 0.032, Odds ratio (OR) 1.642, 95% CI 1.040-2.591; AA + GA vs. GG, P = 0.068, OR 1.665, 95% CI 0.961-2.885; AA vs. GG, P = 0.059, OR 3.458, 95% CI 0.894-13.369] in <65 years groups, which suggested that the MTHFD1 G1958A A allele might be a weak risk factor for early onset AD although it needs further confirmation.
Asunto(s)
Enfermedad de Alzheimer/genética , Pueblo Asiatico/genética , Cistationina betasintasa/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Polimorfismo Genético , Anciano , Anciano de 80 o más Años , Alelos , Apolipoproteína E4/genética , Estudios de Casos y Controles , China , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor , Polimorfismo de Nucleótido SimpleRESUMEN
The Tajik people in China have resided at high altitude for thousands of years. We analyzed the Pro12Ala (C > G) polymorphism in exon B and the 161C > T polymorphism in exon 6 of peroxisome proliferator activated receptor gamma gene (PPARG) in Chinese Tajik population living at high altitude and Chinese Han population living at low attitude. Significant higher frequencies of the CG and GG genotypes and G allele of the Pro12Ala (C > G) polymorphism were observed in the Tajik population than that in the Han population (P < 0.0001), which suggested the G allele was associated with high-altitude adaptation in the dominate model. The significant differences were remained in both of the male and female groups after stratified by gender, and the differences were more pronounced in men (G versus C, OR = 7.700) than in women (OR = 5.056). No significant difference was observed for the 161C > T polymorphism in the two populations. The frequencies of haplotypes GT (P < 0.0001) and GC (P < 0.05) were significantly higher, while the frequency of CT (P < 0.0001) was significantly lower in the Tajik population than that in the Han population. Our results suggest that PPARG is a candidate gene for high-altitude adaptation in the Chinese Tajik population.
Asunto(s)
Altitud , Etnicidad/genética , PPAR gamma/genética , Polimorfismo de Nucleótido Simple/genética , Adaptación Fisiológica/genética , Sustitución de Aminoácidos/genética , Secuencia de Bases , China , Femenino , Frecuencia de los Genes/genética , Haplotipos/genética , Humanos , Desequilibrio de Ligamiento/genética , MasculinoRESUMEN
BACKGROUND: The Tibetan people in China have lived at high altitude for thousands of years, raising the possibility that the Tibetans are genetically adapted to high altitude. In this study we analyzed the Pro12Ala (C>G) polymorphism in exon 2 and the 161C>T polymorphism in exon 6 of peroxisome proliferator-activated receptor gamma gene (PPARγ) in a Tibetan population and a Han population. MATERIAL/METHODS: We recruited 142 Tibetan volunteers who are permanent inhabitants in Qingzang plateau (higher elevation) and 266 Han volunteers who are permanent inhabitants in the plain (lower elevation). PCR/RFLP method was applied to examine the 2 polymorphisms in the 2 populations. RESULTS: Significantly higher Pro12Ala (C>G) CC genotype frequency and 161C>T CC genotype frequency were observed in the Tibetan population compared to the Han population (p<0.001). When the samples were stratified by sex, significant differences were only observed in females. The haplotypes constructed by Pro12Ala (C>G) and 161C>T were also analyzed. The frequency of the haplotype CC (p<0.0001) was significantly higher, while the frequency of the haplotype CT (p<0.0001) and GT (p<0.01) was significantly lower in the Tibetan population than in the Han population. CONCLUSIONS: Our results suggested that PPARγ might be a candidate gene for high-altitude adaptation; the Pro12Ala (C>G) CC genotype and/or the 161C>T CC genotype are possibly advantageous factors in the female Tibetan population. Alternatively, the difference of the Pro12Ala (C>G) genotype distribution and /or the difference of the 161C>T genotype distribution in the 2 populations may be due to the racial difference.
Asunto(s)
Altitud , Pueblo Asiatico/genética , Genética de Población , PPAR gamma/genética , Polimorfismo Genético , Aclimatación , Adaptación Fisiológica , Alelos , China , Exones/genética , Femenino , Frecuencia de los Genes , Genotipo , Haplotipos , Humanos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético/genética , Polimorfismo de Longitud del Fragmento de Restricción/genética , Factores Sexuales , TibetRESUMEN
OBJECTIVE: To investigate the effect of microRNA-205 reduction by antagomirs on adhesion ability of normal human corneal epithelial keratinocytes (NHCEKs). METHODS: Antagomir-205, complementary and inhibitory to microRNA-205, was used to suppress endogenous microRNA-205 in NHCEKs. The adhesion ability of treated NHCEKs was then assessed by cell adhesion assay. Immunoblot and immunohistochemistry were conducted to determine the level of two focal adhesion-related proteins, focal adhesion kinase (FAK) and paxillin (Pax). Phalloidin staining was performed to measure the level of filamentous actin in antagomir-treated NHCEKs. RESULTS: Antagomir-205 markedly reduced the level of microRNA-205 in NHCEKs and significantly enhanced adhesion ability of NHCEKs (P<0.01). Further protein analysis validated that inhibition of microRNA-205 increased the number of phosphorylated FAK and phosphorylated Pax, and decreased filamentous actin. CONCLUSION: Our findings suggest that microRNA-205 has down-regulating effect on cell motility in NHCEKs.
Asunto(s)
Adhesión Celular/genética , Epitelio Corneal/citología , Queratinocitos/citología , MicroARNs/antagonistas & inhibidores , Secuencia de Bases , Células Cultivadas , Humanos , Sondas de OligonucleótidosRESUMEN
MicroRNAs (miRNAs) are thought to modulate a variety of cellular events. Several studies have revealed the functions of miR-223 in granulopoiesis. Here we analysed miR-223 expression in various human tissues, blood and leukaemia cells, and focused on its role in K562 erythroid and megakaryocytic differentiation. MiR-223 was detected not only in granulocytes but also in erythroid cells. In K562 cells, expression of miR-223 was down-regulated during haemin-induced erythroid differentiation but up-regulated during phorbol myristate acetate (PMA)-induced megakaryocytic differentiation. The overexpression of miR-223 resulted in significant decrease of gamma-globin mRNA and the fraction of benzidine-positive cells in K562 cells, suggesting a suppressive effect of miR-223 on erythroid differentiation. Peaks corresponding to 4N cells in stable transfectants overexpressing miR-223 were higher than that in control K562 cells during megakaryocytic differentiation, indicating that miR-223 increases megakaryocytic differentiation. The expression of LIM domain only 2 (LMO2) reporter was suppressed in NIH-3T3 when the expression of miR-223 was enforced by both the luciferase and fluorescence system. Furthermore, LMO2 mRNA and protein levels were significantly decreased in stable K562 transfectants overexpressing miR-223. These results indicate that LMO2 is a direct target of miR-223. Thus, our results suggest that miR-223 reversibly regulates erythroid and megakaryocytic differentiation of K562 cells via down-modulation of LMO2.