RESUMO
Here we investigated in primary human erythroid tissues a downstream element of the heterochronic let-7 miRNA pathway, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), for its potential to affect the hemoglobin profiles in human erythroblasts. Comparison of adult bone marrow to fetal liver lysates demonstrated developmental silencing in IGF2BP1. Erythroid-specific overexpression of IGF2BP1 caused a nearly complete and pancellular reversal of the adult pattern of hemoglobin expression toward a more fetal-like phenotype. The reprogramming of hemoglobin expression was achieved at the transcriptional level by increased gamma-globin combined with decreased beta-globin transcripts resulting in gamma-globin rising to 90% of total beta-like mRNA. Delta-globin mRNA was reduced to barely detectable levels. Alpha-globin levels were not significantly changed. Fetal hemoglobin achieved levels of 68.6 ± 3.9% in the IGF2BP1 overexpression samples compared with 5.0 ± 1.8% in donor matched transduction controls. In part, these changes were mediated by reduced protein expression of the transcription factor BCL11A. mRNA stability and polysome studies suggest IGF2BP1 mediates posttranscriptional loss of BCL11A. These results suggest a mechanism for chronoregulation of fetal and adult hemoglobin expression in humans.
Assuntos
Proteínas de Transporte/metabolismo , Eritroblastos/metabolismo , Hemoglobina Fetal/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Medula Óssea/metabolismo , Células HEK293 , Proteína HMGA2/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fígado/embriologia , Fenótipo , RNA Mensageiro/metabolismo , Proteínas Repressoras , Globinas beta/metabolismo , gama-Globinas/metabolismoRESUMO
BACKGROUND: The relationship between the tissue injury healing response and development of heterotopic ossification (HO) is poorly understood. Here we compare a rat blast model and human traumatized muscle from a blast injury to study the early signatures of osteogenesis and fibrosis during the formation of HO. METHODS: Rat and human tissues were characterized using histology, scanning electron microscopy, immunohistochemistry, as well as gene and protein expression analysis. Additionally, animals and humans were assessed radiographically for HO formation following injury. RESULTS: Markers of bone formation were dramatically increased in tissue samples from both humans and rats, and both displayed increased fibroproliferative regions within the injured tissues and elevated expression of markers of tissue fibrosis such as TGF-ß1, Fibronectin, SMAD3 and PAI-1. Markers of inflammation and fibrosis (ACTA, TNFα, BMP1 and BMP3) were elevated at the RNA level in both rat and human samples. By day 42, bone formation in the rat blast model appeared similar in radiographs compared to human patients who progressed to develop post-traumatic HO. CONCLUSIONS: Our data demonstrates that a similar early fibrotic response is evident in both the rat blast model and the human tissues following a traumatic injury and demonstrates the relevance of this animal model for future translational studies.
Assuntos
Traumatismos por Explosões/metabolismo , Músculos/lesões , Ossificação Heterotópica , Animais , Biomarcadores/metabolismo , Traumatismos por Explosões/fisiopatologia , Desenvolvimento Ósseo , Modelos Animais de Doenças , Fêmur/diagnóstico por imagem , Fêmur/crescimento & desenvolvimento , Fibrose , Perfilação da Expressão Gênica , Humanos , Inflamação , Masculino , Músculos/metabolismo , Ratos , Ratos Sprague-Dawley , Pesquisa Translacional Biomédica , Cicatrização , Microtomografia por Raio-XRESUMO
BACKGROUND: In humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19-40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts. METHODS: The expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins. RESULTS: The expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged. CONCLUSIONS: Overall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , Adulto , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proliferação de Células/genética , Células Cultivadas , Hemoglobina Fetal , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , MicroRNAs/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras , Reticulócitos/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismoRESUMO
Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and ß-thalassemia. Transcription of ß-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult ß-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on ß-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult ß-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of ß-hemoglobinopathies.
Assuntos
Hemoglobina Fetal/biossíntese , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Região de Controle de Locus Gênico , gama-Globinas/genética , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Diferenciação Celular , Proteínas de Ligação a DNA/sangue , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Eritropoese , Antígenos de Histocompatibilidade , Humanos , Técnicas In Vitro , Proteínas com Domínio LIM/sangue , Modelos Biológicos , Regiões Promotoras Genéticas , Quinazolinas/farmacologia , Fatores de Transcrição/sangue , Talassemia beta/sangue , Talassemia beta/tratamento farmacológico , Talassemia beta/genéticaRESUMO
Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and ß-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Eritroblastos/citologia , Eritrócitos/citologia , Hemoglobina Fetal/metabolismo , Regulação da Expressão Gênica , Antígenos CD34/metabolismo , Anidrase Carbônica I/metabolismo , Técnicas de Cultura de Células , Sangue Fetal/citologia , Hemoglobina A/metabolismo , Humanos , MicroRNAs/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fenótipo , Proteínas de Ligação a RNARESUMO
Cancer affects millions of people and understanding the molecular mechanisms related to disease development and progression is essential to manage the disease. Post-translational modification (PTM) processes such as ubiquitination and neddylation have a significant role in cancer development and progression by regulating protein stability, function, and interaction with other biomolecules. Both ubiquitination and neddylation are analogous processes that involves a series of enzymatic steps leading to the covalent attachment of ubiquitin or NEDD8 to target proteins. Neddylation modifies the CRL family of E3 ligase and regulates target proteins' function and stability. The DCUN1D1 protein is a regulator of protein neddylation and ubiquitination and acts promoting the neddylation of the cullin family components of E3-CRL complexes and is known to be upregulated in several types of cancers. In this review we compare the PTM ubiquitination and neddylation. Our discussion is focused on the neddylation process and the role of DCUN1D1 protein in cancer development. Furthermore, we provide describe DCUN1D1 protein and discuss its role in pathogenesis and signalling pathway in six different types of cancer. Additionally, we explore both the neddylation and DCUN1D1 pathways as potential druggable targets for therapeutic interventions. We focus our analysis on the development of compounds that target specifically neddylation or DCUN1D1. Finally, we provide a critical analysis about the challenges and perspectives in the field of DCUN1D1 and neddylation in cancer research. KEY POINTS: Neddylation is a post-translational modification that regulates target proteins' function and stability. One regulator of the neddylation process is a protein named DCUN1D1 and it is known to have its expression deregulated in several types of cancers. Here, we provide a detailed description of DCUN1D1 structure and its consequence for the development of cancer. We discuss both the neddylation and DCUN1D1 pathways as potential druggable targets for therapeutic interventions and provide a critical analysis about the challenges and perspectives in the field of DCUN1D1 and neddylation in cancer research.
RESUMO
Most patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) have hippocampal sclerosis on the postoperative histopathological examination. Although most patients with MTLE do not refer to a family history of the disease, familial forms of MTLE have been reported. We studied surgical specimens from patients with MTLE who had epilepsy surgery for medically intractable seizures. We assessed and compared gene expression profiles of the tissue lesion found in patients with familial MTLE (n = 3) and sporadic MTLE (n = 5). In addition, we used data from control hippocampi obtained from a public database (n = 7). We obtained expression profiles using the Human Genome U133 Plus 2.0 (Affymetrix) microarray platform. Overall, the molecular profile identified in familial MTLE differed from that in sporadic MTLE. In the tissue of patients with familial MTLE, we found an over-representation of the biological pathways related to protein response, mRNA processing, and synaptic plasticity and function. In sporadic MTLE, the gene expression profile suggests that the inflammatory response is highly activated. In addition, we found enrichment of gene sets involved in inflammatory cytokines and mediators and chemokine receptor pathways in both groups. However, in sporadic MTLE, we also found enrichment of epidermal growth factor signaling, prostaglandin synthesis and regulation, and microglia pathogen phagocytosis pathways. Furthermore, based on the gene expression signatures, we identified different potential compounds to treat patients with familial and sporadic MTLE. To our knowledge, this is the first study assessing the mRNA profile in surgical tissue obtained from patients with familial MTLE and comparing it with sporadic MTLE. Our results clearly show that, despite phenotypic similarities, both forms of MTLE present distinct molecular signatures, thus suggesting different underlying molecular mechanisms that may require distinct therapeutic approaches.
Assuntos
Epilepsia do Lobo Temporal , Humanos , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/cirurgia , Epilepsia do Lobo Temporal/patologia , Transcriptoma/genética , Hipocampo/metabolismo , RNA Mensageiro/metabolismo , Imageamento por Ressonância MagnéticaRESUMO
BACKGROUND: The development of post-traumatic heterotopic ossification (HO) is a common, undesirable sequela in patients with high-energy (war-related) extremity injuries. While inflammatory and osteoinductive signaling pathways are known to be involved in the development and progression of post-traumatic HO, features of the structural microenvironment within which the ectopic bone begins to form remain poorly understood. Thus, increasing our knowledge of molecular and structural changes within the healing wound may help elucidate the pathogenesis of post-traumatic HO and aid in the development of specific treatment and/or prevention strategies. METHODS: In this study, we performed high-resolution microscopy and biochemical analysis of tissues obtained from traumatic war wounds to characterize changes in the structural microenvironment. In addition, using an electrospinning approach, we modeled this microenvironment to reconstitute a three-dimensional type I collagen scaffold with non-woven, randomly oriented nanofibers where we evaluated the performance of primary mesenchymal progenitor cells. RESULTS: We found that traumatic war wounds are characterized by a disorganized, densely fibrotic collagen I matrix that influences progenitor cells adhesion, proliferation and osteogenic differentiation potential. CONCLUSION: Altogether, these results suggest that the structural microenvironment present in traumatic war wounds has the potential to contribute to the development of post-traumatic HO. Our findings may support novel treatment strategies directed towards modifying the structural microenvironment after traumatic injury.
Assuntos
Células-Tronco Mesenquimais , Ossificação Heterotópica , Diferenciação Celular , Humanos , Ossificação Heterotópica/etiologia , Ossificação Heterotópica/patologia , Ossificação Heterotópica/prevenção & controle , Osteogênese , Células-TroncoRESUMO
BACKGROUND: Multipotent progenitor cells have been harvested from different human tissues, including the bone marrow, adipose tissue, and umbilical cord blood. Previously, we identified a population of mesenchymal progenitor cells (MPCs) isolated from the traumatized muscle of patients undergoing reconstructive surgery following a war-related blast injury. These cells demonstrated the ability to differentiate into multiple mesenchymal lineages. While distal radius fractures from a civilian setting have a much lower injury mechanism (low-energy trauma), we hypothesized that debrided traumatized muscle near the fracture site would contain multipotent progenitor cells with the ability to differentiate and regenerate the injured tissue. METHODS: The traumatized muscle was debrided from the pronator quadratus in patients undergoing open reduction and internal fixation for a distal radius fracture at the Walter Reed National Military Medical Center. Using a previously described protocol for the isolation of MPCs from war-related extremity injuries, cells were harvested from the low-energy traumatized muscle samples and expanded in culture. Isolated cells were characterized by flow cytometry and q-RT-PCRs and induced to adipogenic, osteogenic, and chondrogenic differentiation. Downstream analyses consisted of lineage-specific staining and q-RT-PCR. RESULTS: Cells isolated from low-energy traumatized muscle samples were CD73+, CD90+, and CD105+ that are the characteristic of adult human mesenchymal stem cells. These cells expressed high levels of the stem cell markers OCT4 and NANOG 1-day after isolation, which was dramatically reduced over-time in monolayer culture. Following induction, lineage-specific markers were demonstrated by each specific staining and confirmed by gene expression analysis, demonstrating the ability of these cells to differentiate into adipogenic, osteogenic, and chondrogenic lineages. CONCLUSIONS: Adult multipotent progenitor cells are an essential component for the success of regenerative medicine efforts. While MPCs have been isolated and characterized from severely traumatized muscle from high-energy injuries, here, we report that cells with similar characteristics and multipotential capacity have been isolated from the tissue that was exposed to low-energy, community trauma.
Assuntos
Células-Tronco Mesenquimais , Células-Tronco Multipotentes , Adulto , Diferenciação Celular , Células Cultivadas , Condrogênese , Humanos , Células-TroncoRESUMO
Animal studies are essential to biomedical research and the cornerstone is a reproducible animal model. While there are many reports on rodent peripheral nerve injury models, a large animal model is essential to confirm the effects of nerve regeneration over the longer distances of regeneration required in humans. Swine have long been used as a large animal model for other surgical and biomedical studies. This paper represents a novel neurovascular injury model in the Sus scrofa domesticus swine (American Yorkshire pig). This paper will describe our experience and recommendations with pre-operative, operative and post-operative protocols and our refinements to produce an effective model.
Assuntos
Artéria Femoral , Sus scrofa , Animais , Modelos Animais de Doenças , Artéria Femoral/cirurgia , Isquemia , Regeneração Nervosa , Nervo Isquiático , SuínosRESUMO
BACKGROUND: Heterotopic ossification (HO) is characterized by the abnormal growth of ectopic bone in soft tissues, frequently occurring within the military population because of extensive orthopaedic combat trauma. MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene expression. We hypothesized that a clinically relevant miRNA signature could be detected in patients following injury that progressed to form HO (HO+) or did not form HO (HO-). METHODS: Tissue samples were obtained from injured servicemembers during their initial surgical debridements, and miRNA profiling was performed using a real-time miRNA polymerase chain reaction (PCR) array. Primary mesenchymal progenitor cells (MPCs) were harvested from debrided traumatized human muscle tissue, and cells were isolated and cultured in vitro. Mimic miRNAs were transfected into MPCs, followed by downstream in vitro analyses. RESULTS: The investigation of the miRNA expression profile in the tissue of HO+ compared with HO- patients demonstrated a molecular signature that included the upregulation of miR-1, miR-133a, miR-133b, miR-206, miR-26a, and miR-125b. Transfection of each of these mature miRNAs into MPCs followed by osteogenic induction demonstrated that miR-1, miR-133a, miR-133b, and miR-206 enhanced osteogenic differentiation compared with control treatments. In silico and in vitro analyses identified the transcription factor SOX9 as a candidate downstream target of miR-1 and miR-206 miRNAs. CONCLUSIONS: Our data demonstrated a molecular signature of miRNAs in the soft tissue of wounded servicemembers that was associated with the development of HO, providing novel insights into the underlying molecular mechanisms associated with posttraumatic HO. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.
Assuntos
MicroRNAs/metabolismo , Ossificação Heterotópica/metabolismo , Transcriptoma , Cicatrização , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ferimentos e Lesões/metabolismo , Adulto JovemRESUMO
Type 2 diabetes mellitus (T2DM), dyslipidemia and periodontitis are frequently associated pathologies; however, there are no studies showing the peripheral blood transcript profile of these combined diseases. Here we identified the differentially expressed genes (DEGs) of circulating lymphocytes and monocytes to reveal potential biomarkers that may be used as molecular targets for future diagnosis of each combination of these pathologies (compared to healthy patients) and give insights into the underlying molecular mechanisms of these diseases. Study participants (n = 150) were divided into groups: (H) systemically and periodontal healthy (control group); (P) with periodontitis, but systemically healthy; (DL-P) with dyslipidemia and periodontitis; (T2DMwell-DL-P) well-controlled type 2 diabetes mellitus with dyslipidemia and periodontitis; and (T2DMpoorly-DL-P) poorly-controlled type 2 diabetes mellitus with dyslipidemia and periodontitis. We preprocessed the microarray data using the Robust Multichip Average (RMA) strategy, followed by the RankProd method to identify candidates for DEGs. Furthermore, we performed functional enrichment analysis using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis. DEGs were submitted to pairwise comparisons, and selected DEGs were validated by quantitative polymerase chain reaction. Validated DEGs verified from T2DMpoorly-DL-P versus H were: TGFB1I1, VNN1, HLADRB4 and CXCL8; T2DMwell-DL-P versus H: FN1, BPTF and PDE3B; DL-P versus H: DAB2, CD47 and HLADRB4; P versus H: IGHDL-P, ITGB2 and HLADRB4. In conclusion, we identified that circulating lymphocytes and monocytes of individuals simultaneously affected by T2DM, dyslipidemia and periodontitis, showed an altered molecular profile mainly associated to inflammatory response, immune cell trafficking, and infectious disease pathways. Altogether, these results shed light on novel potential targets for future diagnosis, monitoring or development of targeted therapies for patients sharing these conditions.
Assuntos
Periodontite Crônica/genética , Diabetes Mellitus Tipo 2/genética , Dislipidemias/genética , Linfócitos/metabolismo , Monócitos/metabolismo , Adulto , Periodontite Crônica/complicações , Periodontite Crônica/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Dislipidemias/complicações , Dislipidemias/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , TranscriptomaRESUMO
Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with beta-hemoglobin disorders. Previous studies showed that let-7 microRNAs (miRNAs) are highly regulated in erythroid cells during the fetal-to-adult developmental transition, and that targeting let-7 mediated the up-regulation of HbF to greater than 30% of the total globin levels in human adult cultured erythroblasts. HMGA2 is a member of the high-mobility group A family of proteins and a validated target of the let-7 family of miRNAs. Here we investigate whether expression of HMGA2 directly regulates fetal hemoglobin in adult erythroblasts. Let-7 resistant HMGA2 expression was studied after lentiviral transduction of CD34(+) cells. The transgene was regulated by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2-OE). HMGA2-OE caused significant increases in gamma-globin mRNA expression and HbF to around 16% of the total hemoglobin levels compared to matched control transductions. Interestingly, no significant changes in KLF1, SOX6, GATA1, ZBTB7A and BCL11A mRNA levels were observed. Overall, our data suggest that expression of HMGA2, a downstream target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in adult human erythroblasts.
Assuntos
Eritroblastos/metabolismo , Hemoglobina Fetal/genética , Regulação da Expressão Gênica , Proteína HMGA2/metabolismo , Adulto , Diferenciação Celular/genética , Células Cultivadas , Eritroblastos/citologia , Eritropoese/genética , Hemoglobina Fetal/metabolismo , Expressão Gênica , Proteína HMGA2/genética , Humanos , MicroRNAs/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismoRESUMO
Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30-40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.
Assuntos
Eritroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas de Ligação a RNA/genética , gama-Globinas/genética , gama-Globinas/metabolismo , Adulto , Diferenciação Celular , Proliferação de Células , Eritroblastos/citologia , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Although early stages of clear cell renal cell carcinoma (ccRCC) are curable, survival outcome for metastatic ccRCC remains poor. We previously established a highly accurate signature of differentially expressed genes that distinguish ccRCC from normal kidney. The purpose of this study was to apply a new individualized bioinformatics analysis (IBA) strategy to these transcriptome data in conjunction with Gene Set Enrichment Analysis of the Connectivity Map (C-MAP) database to identify and reposition FDA-approved drugs for anticancer therapy. Here, we demonstrate that one of the drugs predicted to revert the RCC gene signature toward normal kidney, pentamidine, is effective against RCC cells in culture and in a RCC xenograft model. ccRCC-specific gene expression signatures of individual patients were used to query the C-MAP software. Eight drugs with negative correlation and P-value <0.05 were analyzed for efficacy against RCC in vitro and in vivo. Our data demonstrate consistency across most patients with ccRCC for the set of high-scoring drugs. Most of the selected high-scoring drugs potently induce apoptosis in RCC cells. Several drugs also demonstrate selectivity for Von Hippel-Lindau negative RCC cells. Most importantly, at least one of these drugs, pentamidine, slows tumor growth in the 786-O human ccRCC xenograft mouse model. Our findings suggest that pentamidine might be a new therapeutic agent to be combined with current standard-of-care regimens for patients with metastatic ccRCC and support our notion that IBA combined with C-MAP analysis enables repurposing of FDA-approved drugs for potential anti-RCC therapy.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Pentamidina/farmacologia , Animais , Antineoplásicos/farmacologia , Antiprotozoários/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Análise em MicrossériesRESUMO
Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.
Assuntos
Anemia Falciforme/genética , Eritrócitos Anormais/metabolismo , Hemoglobina Fetal/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Globinas beta/genética , Adolescente , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Anemia Falciforme/terapia , Diferenciação Celular , Forma Celular , Criança , Eritroblastos/metabolismo , Eritroblastos/patologia , Transfusão de Eritrócitos , Eritrócitos Anormais/patologia , Hemoglobina Fetal/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Masculino , MicroRNAs/metabolismo , Cultura Primária de Células , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transfecção , Globinas beta/metabolismoRESUMO
Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14-21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.
Assuntos
Antígenos CD34/metabolismo , Doadores de Sangue , Eritropoese , Saúde , Modelos Biológicos , Talassemia beta/patologia , Adulto , Apoptose/genética , Biomarcadores/metabolismo , Western Blotting , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Eritroblastos/citologia , Eritroblastos/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solubilidade , Talassemia beta/genéticaRESUMO
The reliability of gene expression studies by mRNA quantification is highly dependent upon several experimental procedures, including the choice of reference genes used for data normalization. In order to contribute to gene expression studies in mesial temporal lobe epilepsy (MTLE) we used microarray data, followed by real time quantitative PCR validation of selected housekeeping genes, to determine the most appropriate reference genes to be used in human hippocampal tissue gene expression studies. Our results unequivocally showed a significant impact of the reference gene chosen for normalization on the overall results of expression studies, clearly demonstrating the importance of adequate validation using stable reference genes. In addition, we found that HPRT, NSE, SDHA and SYP are suitable genes to be used as reference for normalization in expression studies of hippocampal tissue obtained from patients with MTLE.