RESUMO
Bipotent megakaryocyte/erythroid progenitors (MEPs) give rise to progeny limited to the megakaryocyte (Mk) and erythroid (E) lineages. We developed a novel dual-detection functional in vitro colony-forming unit (CFU) assay for single cells that differentiates down both the Mk and E lineages (CFU-Mk/E), which allowed development and validation of a novel purification strategy for the identification and quantitation of primary functional human MEPs from granulocyte colony-stimulating factor-mobilized peripheral blood and bone marrow. Applying this assay to fluorescence-activated cell sorter-sorted cell populations, we found that the Lin(-)CD34(+)CD38(mid)CD45RA(-)FLT3(-)MPL(+)CD36(-)CD41(-) population is much more highly enriched for bipotent MEPs than any previously reported subpopulations. We also developed purification strategies for primary human lineage-committed Mk and E progenitors identified as CFU-Mk and burst forming unit-E. Comparative expression analyses in MEP, MkP, and ErP populations revealed differential expression of MYB We tested whether alterations in MYB concentration affect the Mk-E fate decision at the single cell level in MEPs and found that short hairpin RNA-mediated MYB knockdown promoted commitment of MEPs to the Mk lineage, further defining its role in MEP lineage fate. There are numerous applications for these novel enrichment strategies, including facilitating mechanistic studies of MEP lineage commitment, improving approaches for in vitro expansion of Mk and E cells, and developing improved therapies for benign and malignant hematologic disease.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD34/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Adulto , Linhagem da Célula , Separação Celular , Ensaio de Unidades Formadoras de Colônias , Células Eritroides/citologia , Células Eritroides/metabolismo , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/citologia , Fenótipo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Receptores de Trombopoetina/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
BACKGROUND: Sex hormone-related diseases, encompassing a wide range of conditions from reproductive disorders to certain cancers, pose significant health challenges worldwide. Recent scientific investigations have highlighted the intricate interplay between the gut microbiome and sex hormone regulation, indicating the potential for microbiota-targeted interventions in the management of such diseases. Although individual studies have elucidated the influence of the gut microbiome on sex hormones, a comprehensive cross-sectional examination of the population-wide prevalence of probiotic intake and its correlation with sex hormones is still lacking. OBJECTIVES: This study aimed to evaluate the association of probiotic ingestion with sex hormones in pre- and post-menopausal women. METHODS: We conducted an observational cohort study comprising a nationally representative sample of adults who participated in the National Health and Nutrition Examination Survey between 2013 and 2016. Probiotic ingestion was considered when a subject reported yogurt or probiotic supplement consumption during the 24-h dietary recall or during the Dietary Supplement Use 30-Day questionnaire. A survey-weighted generalized linear model was used to analyze the association between probiotic intake and female/male sex hormones. To reduce selection bias, we used propensity score matching (PSM). RESULTS: This study included 2,699 women, with 537 of them consuming yogurt and/or dietary supplements containing probiotics, while the remaining 2,162 women did not consume any probiotics. The findings indicated that there were associations between probiotic intake and sex hormone levels in premenopausal and postmenopausal women. For premenopausal women, probiotic intake was positively associated with estradiol (E2) levels. On the contrary, in postmenopausal women, probiotic intake was inversely associated with total testosterone (TT) levels. CONCLUSIONS: This study indicated that probiotic consumption was associated with higher E2 level in premenopausal women and lower TT level in postmenopausal women. Probiotic intake might be a sensible strategy for preventing sex hormone-related diseases.
Assuntos
Pós-Menopausa , Probióticos , Adulto , Humanos , Masculino , Feminino , Inquéritos Nutricionais , Pós-Menopausa/fisiologia , Estudos Transversais , Estudos de Coortes , Hormônios Esteroides Gonadais , Estradiol , Ingestão de Alimentos , Globulina de Ligação a Hormônio Sexual , TestosteronaRESUMO
Ovarian cancer (OC), the third common gynecologic malignancy, contributes to the most cancer-caused mortality in women. However, 70% of patients with OC are diagnosed at an advanced stage, of which the 5-year survival is less than 30%. Long noncoding RNAs (long ncRNAs or lncRNA), a type of RNA with exceeding 200 nucleotides in length but no protein-coding capability, have been demonstrated to involve the pathogenesis of various cancers and show considerable potential in the diagnosis of OC. In this study, we found that the LINC00909 expression in tumor and serum specimens of OC patients was elevated, determined by real-time quantitative, and droplet digital PCR. In receiver operating characteristic (ROC) analysis, our results revealed that serum LINC00909 distinguished cancers from normal ovarian tissue with 87.8% of sensitivity and 69.6% of specificity (AUC, 81.2%) and distinguished serous ovarian cancer from normal ovarian tissue with 90.0% of sensitivity and 75.9% of specificity (AUC, 84.5%). Furthermore, we observed that the tumor and serum LINC00909 level was positively associated with the International Federation of Gynecology and Obstetrics (FIGO) stage and the Eastern Cooperative Oncology Group (ECOG) score (reflecting patients' performance status). Also, patients with low serum LINC00909 level showed a longer overall (hazard ratio, HR = 1.874, p = 0.0004) and progression-free (HR = 1.656, p = 0.0017) survival. Functional assays indicated that the elevation of LINC00909 expression contributes to cell proliferation, migration, and invasion capability of ovarian cancer cells. Besides, we demonstrated that LINC00909 functions as a competing endogenous RNA (ceRNA) of MRC2 mRNA by sponging miR-23-3p, and thereby promotes epithelial-to-mesenchymal transition (EMT) of ovarian cancer cells. Therefore, we highlight that the LINC00909/miR-23b-3p/MRC2 axis is implicated in the pathogenesis of ovarian cancer, and serum LINC00909 may be a promising biomarker for the diagnosis of OC.
Assuntos
MicroRNAs/metabolismo , Neoplasias Ovarianas/genética , RNA Longo não Codificante/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Feminino , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, and chemotherapy remains the cornerstone for ovarian cancer management. Due to the unsatisfactory prognosis, a better understanding of the underlying molecular carcinogenesis is urgently required. METHODS: Assays for determining cell growth, cell motility, and apoptosis were employed to evaluate the potential antitumor effects of metformin against ovarian cancer cells. Molecular biological methods were employed to explore the underlying mechanism. Human ovarian cancer samples and Gene Expression Profiling Interactive Analysis (GEPIA) dataset were used for uncovering the clinical significances of mesothelin (MSLN) on ovarian cancer. RESULTS: In the present work, we found that metformin treatment led to cell growth and cell migration inhibition, and induced cell apoptosis. Metformin administration also impaired cancer cell stemness and the capillary-like structure formation capacity of SKOV3 cells. On mechanism, metformin treatment remarkably reduced mesothelin (MSLN) expression, downregulated IL-6/STAT3 signaling activity, subsequently resulted in VEGF and TGFß1 expression. We also observed an oncogenic function of MSLN on ovarian cancer. CONCLUSIONS: Collectively, our findings suggested that metformin exerts anticancer effects by suppressing ovarian cancer cell malignancy, which attributed to MSLN inhibition mediated IL6/STAT3 signaling and VEGF and TGFß1 downregulation.
Assuntos
Hipoglicemiantes/uso terapêutico , Interleucina-6/metabolismo , Metformina/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Fator de Transcrição STAT3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Transdução de SinaisRESUMO
Cadherins play a major role in mediating cell-cell adhesion, which shares many parallels with platelet-platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet function and/or platelet production is currently unknown. To assess the role of E-cadherin in platelet production and function in vitro and in vivo, we utilized a megakaryocyte-specific E-cadherin knockout mouse model. Loss of E-cadherin in megakaryocytes does not affect megakaryocyte maturation, platelet number or size. However, platelet dysfunction in the absence of E-cadherin is revealed when conditional knockout mice are challenged with acute antibody-mediated platelet depletion. Unlike wild-type mice that recover fully, knockout mice die within 72 hours post-antibody administration, likely from haemorrhage. Furthermore, conditional knockout mice have prolonged tail bleeding times, unstable clot formation, reduced clot retraction and reduced fibrin deposition in in vivo injury models. Murine platelet aggregation in vitro in response to thrombin and thrombin receptor activating peptide is compromised in E-cadherin null platelets, while aggregation in response to adenosine diphosphate (ADP) is not significantly different. Consistent with this, in vitro aggregation of primary human platelets in response to thrombin is decreased by an inhibitory E-cadherin antibody. Integrin activation and granule secretion in response to ADP and thrombin are not affected in E-cadherin null platelets, but Akt and glycogen synthase kinase 3ß (GSK3ß) activation are attenuated, suggesting a that E-cadherin contributes to aggregation, clot stabilization and retraction that is mediated by phosphoinositide 3-kinase/Akt/GSK3ß signalling. In summary, E-cadherin plays a salient role in platelet aggregation and clot stability.
Assuntos
Plaquetas/fisiologia , Caderinas/metabolismo , Fígado/patologia , Megacariócitos/fisiologia , Trombose/metabolismo , Animais , Tempo de Sangramento , Coagulação Sanguínea , Caderinas/genética , Adesão Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Agregação Plaquetária , Transdução de Sinais , Trombina/metabolismoRESUMO
Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.
Assuntos
Plaquetas/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Plaquetas/citologia , Diferenciação Celular/fisiologia , Tamanho Celular , Estudos de Coortes , Feminino , Sangue Fetal , Regulação da Expressão Gênica , Humanos , Masculino , Volume Plaquetário Médio , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras GenéticasRESUMO
Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bß3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.
Assuntos
Plaquetas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/sangue , Proteínas rho de Ligação ao GTP/sangue , Proteína rhoA de Ligação ao GTP/sangue , Animais , Tempo de Sangramento , Plaquetas/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cadeias Leves de Miosina/sangue , Testes de Função Plaquetária , Fatores de Troca de Nucleotídeo Guanina Rho/antagonistas & inibidores , Fatores de Troca de Nucleotídeo Guanina Rho/deficiência , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Trombina/metabolismo , Trombina/farmacologia , Trombopoese/genética , Trombopoese/fisiologia , Tromboxanos/sangue , Tromboxanos/farmacologia , Proteínas rho de Ligação ao GTP/agonistas , Proteína rhoA de Ligação ao GTP/agonistasRESUMO
In cystic fibrosis (CF) patients, hyper-inflammation is a key factor in lung destruction and disease morbidity. We have previously demonstrated that macrophages drive the lung hyper-inflammatory response to LPS in CF mice, because of reduced levels of the scaffold protein CAV1 with subsequent uncontrolled TLR4 signalling. Here we show that reduced CAV1 and, consequently, increased TLR4 signalling, in human and murine CF macrophages and murine CF lungs, is caused by high microRNA-199a-5p levels, which are PI3K/AKT-dependent. Downregulation of microRNA-199a-5p or increased AKT signalling restores CAV1 expression and reduces hyper-inflammation in CF macrophages. Importantly, the FDA-approved drug celecoxib re-establishes the AKT/miR-199a-5p/CAV1 axis in CF macrophages, and ameliorates lung hyper-inflammation in Cftr-deficient mice. Thus, we identify the AKT/miR-199a-5p/CAV1 pathway as a regulator of innate immunity, which is dysfunctional in CF macrophages contributing to lung hyper-inflammation. In addition, we found that this pathway can be targeted by celecoxib.
Assuntos
Caveolina 1/metabolismo , Fibrose Cística/patologia , Inflamação/patologia , Pulmão/patologia , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Celecoxib/farmacologia , Fibrose Cística/enzimologia , Humanos , Pulmão/enzimologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
Polyploidization can precede the development of aneuploidy in cancer. Polyploidization in megakaryocytes (Mks), in contrast, is a highly controlled developmental process critical for efficient platelet production via unknown mechanisms. Using primary cells, we demonstrate that the guanine exchange factors GEF-H1 and ECT2, which are often overexpressed in cancer and are essential for RhoA activation during cytokinesis, must be downregulated for Mk polyploidization. The first (2N-4N) endomitotic cycle requires GEF-H1 downregulation, whereas subsequent cycles (>4N) require ECT2 downregulation. Exogenous expression of both GEF-H1 and ECT2 prevents endomitosis, resulting in proliferation of 2N Mks. Furthermore, we have shown that the mechanism by which polyploidization is prevented in Mks lacking Mkl1, which is mutated in megakaryocytic leukemia, is via elevated GEF-H1 expression; shRNA-mediated GEF-H1 knockdown alone rescues this ploidy defect. These mechanistic insights enhance our understanding of normal versus malignant megakaryocytopoiesis, as well as aberrant mitosis in aneuploid cancers.