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Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.
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The outcomes of children with acute lymphoblastic leukemia (ALL) have been incrementally improved with risk-directed chemotherapy but therapy responses remain heterogeneous. Parameters with added prognostic values are warranted to refine the current risk stratification system and inform appropriate therapies. CD9, implicated by our prior single-center study, holds promise as one such parameter. To determine its precise prognostic significance, we analyzed a nationwide, multicenter, uniformly treated cohort of childhood ALL cases, where CD9 status was defined by flow cytometry on diagnostic samples of 3781 subjects. CD9 was expressed in 88.5% of B-ALL and 27.9% of T-ALL cases. It conferred a lower 5-year EFS and a higher CIR in B-ALL but not in T-ALL patients. The prognostic impact of CD9 was most pronounced in the intermediate/high-risk arms and those with minimal residual diseases, particularly at day 19 of remission induction. The adverse impact of CD9 was confined to specific cytogenetics, notably BCR::ABL1+ rather than KMT2A-rearranged leukemia. Multivariate analyses confirmed CD9 as an independent predictor of both events and relapse. The measurement of CD9 offers insights into patients necessitating intervention, warranting its seamless integration into the diagnostic marker panel to inform risk level and timely introduction of therapeutic intervention for childhood ALL.
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Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Niño , Humanos , Pronóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Neoplasia Residual/diagnóstico , China , Tetraspanina 29RESUMEN
Despite the expanding portfolio of targeted therapies for adults with acute myeloid leukemia (AML), direct implementation in children is challenging due to inherent differences in underlying genetics. Here we established the pharmacologic profile of pediatric AML by screening myeloblast sensitivity to approved and investigational agents, revealing candidates of immediate clinical relevance. Drug responses ex vivo correlated with patient characteristics, exhibited age-specific alterations, and concorded with activities in xenograft models. Integration with genomic data uncovered new gene-drug associations, suggesting actionable therapeutic vulnerabilities. Transcriptome profiling further identified gene-expression signatures associated with on- and off-target drug responses. We also demonstrated the feasibility of drug screening-guided treatment for children with high-risk AML, with two evaluable cases achieving remission. Collectively, this study offers a high-dimensional gene-drug clinical data set that could be leveraged to research the unique biology of pediatric AML and sets the stage for realizing functional precision medicine for the clinical management of the disease. SIGNIFICANCE: We conducted integrated drug and genomic profiling of patient biopsies to build the functional genomic landscape of pediatric AML. Age-specific differences in drug response and new gene-drug interactions were identified. The feasibility of functional precision medicine-guided management of children with high-risk AML was successfully demonstrated in two evaluable clinical cases. This article is highlighted in the In This Issue feature, p. 476.
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Leucemia Mieloide Aguda , Medicina de Precisión , Niño , Adulto , Humanos , Medicina de Precisión/métodos , Farmacogenética , Leucemia Mieloide Aguda/tratamiento farmacológico , Perfilación de la Expresión Génica/métodos , TranscriptomaRESUMEN
G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors. They are involved in almost every physiologic process and consequently have a pivotal role in an extensive number of pathologies, including genetic, neurologic, and immune system disorders. Indeed, the vast array of GPCRs mechanisms have led to the development of a tremendous number of drug therapies and already account for about a third of marketed drugs. These receptors mediate their downstream signals primarily via G proteins. The regulators of G-protein signaling (RGS) proteins are now in the spotlight as the critical modulatory factors of active GTP-bound Gα subunits of heterotrimeric G proteins to fine-tune the biologic responses driven by the GPCRs. Also, they possess noncanonical functions by multiple mechanisms, such as protein-protein interactions. Essential roles and impacts of these RGS proteins have been revealed in physiology, including hematopoiesis and immunity, and pathologies, including asthma, cancers, and neurologic disorders. This review focuses on the largest subfamily of R4 RGS proteins and provides a brief overview of their structures and G-proteins selectivity. With particular interest, we explore and highlight, their expression in the hematopoietic system and the regulation in the engraftment of hematopoietic stem/progenitor cells (HSPCs). Distinct expression patterns of R4 RGS proteins in the hematopoietic system and their pivotal roles in stem cell trafficking pave the way for realizing new strategies for enhancing the clinical performance of hematopoietic stem cell transplantation. Finally, we discuss the exciting future trends in drug development by targeting RGS activity and expression with small molecules inhibitors and miRNA approaches.
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Productos Biológicos , Proteínas de Unión al GTP Heterotriméricas , MicroARNs , Proteínas RGS , Guanosina Trifosfato , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM) microenvironment are tightly regulated by the chemokine stromal cell-derived factor-1 (SDF-1) and its G-protein-coupled receptor C-X-C motif chemokine receptor 4 (CXCR4), which on engagement with G-protein subunits, trigger downstream migratory signals. Regulators of G-protein signaling (RGS) are GTPase-accelerating protein of the Gα subunit and R4 subfamily members have been implicated in SDF-1-directed trafficking of mature hematopoietic cells, yet their expression and influence on HSPCs remain mostly unknown. Here, we demonstrated that human CD34+ cells expressed multiple R4 RGS genes, of which RGS1, RGS2, RGS13, and RGS16 were significantly upregulated by SDF-1 in a CXCR4-dependent fashion. Forced overexpression of RGS1, RGS13, or RGS16 in CD34+ cells not only inhibited SDF-1-directed migration, calcium mobilization, and phosphorylation of AKT, ERK, and STAT3 in vitro, but also markedly reduced BM engraftment in transplanted NOD/SCID mice. Genome-wide microarray analysis of RGS-overexpressing CD34+ cells detected downregulation of multiple effectors with established roles in stem cell trafficking/maintenance. Convincingly, gain-of-function of selected effectors or ex vivo priming with their ligands significantly enhanced HSPC engraftment. We also constructed an evidence-based network illustrating the overlapping mechanisms of RGS1, RGS13, and RGS16 downstream of SDF-1/CXCR4 and Gαi. This model shows that these RGS members mediate compromised kinase signaling and negative regulation of stem cell functions, complement activation, proteolysis, and cell migration. Collectively, this study uncovers an essential inhibitory role of specific R4 RGS proteins in stem cell engraftment, which could potentially be exploited to develop improved clinical HSPC transplantation protocols.
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Trasplante de Células Madre Hematopoyéticas , Proteínas RGS , Animales , Antígenos CD34 , Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas RGS/genética , Receptores CXCR4/genéticaRESUMEN
We previously demonstrated that microRNA(miR)-223 is overexpressed in intestinal tissue of infants with necrotizing enterocolitis (NEC). The objective of the current study was to identify the target gene of miR-223 and to investigate the role of the miR-223/nuclear factor I-A (NFIA) axis in cellular functions that underpin the pathophysiology of NEC. The target gene of miR-223 was identified by in silico target prediction bioinformatics, luciferase assay, and western blotting. We investigated downstream signals of miR-223 and cellular functions by overexpressing the miRNA in Caco-2 and FHs74 cells stimulated with lipopolysaccharide or lipoteichoic acid (LTA). NFIA was identified as a target gene of miR-223. Overexpression of miR-223 significantly induced MYOM1 and inhibited NFIA and RGN in Caco-2 cells, while costimulation with LTA decreased expression of GNA11, MYLK, and PRKCZ. Expression levels of GNA11, MYLK, IL-6, and IL-8 were increased, and levels of NFIA and RGN were decreased in FHs74 cells. These potential downstream genes were significantly correlated with levels of miR-223 or NFIA in primary NEC tissues. Overexpression of miR-223 significantly increased apoptosis of Caco-2 and FHs74 cells, while proliferation of FHs74 was inhibited. These results suggest that upon binding with NFIA, miR-223 regulates functional effectors in pathways of apoptosis, cell proliferation, G protein signaling, inflammation, and smooth muscle contraction. The miR-223/NFIA axis may play an important role in the pathophysiology of NEC by enhancing inflammation and tissue damage.
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Enterocolitis Necrotizante , MicroARNs , Células CACO-2 , Enterocolitis Necrotizante/genética , Enterocolitis Necrotizante/metabolismo , Humanos , Recién Nacido , Inflamación/genética , MicroARNs/genética , MicroARNs/metabolismo , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismoRESUMEN
OBJECTIVE: The objective of this study is to evaluate the usefulness of fecal microRNA (miR)-223 and miR-451a, as novel noninvasive biomarkers for early diagnosis of necrotizing enterocolitis (NEC) in preterm infants. METHODS: Among the top-listed target miRNAs in our previous differential microarray analysis, miR-223 and miR-451a were quantified in a pilot validation case-controlled study (NEC vs. non-NEC/nonsepsis infants; n = 6 in each group). A definitive prospective cohort study (n = 218) further assessed their clinical usefulness as noninvasive and specific diagnostic biomarkers. Fecal calprotectin was quantified in parallel for comparison. RESULTS: Of 43 proven NEC cases in the cohort study, 24 (55.8%) had fecal samples recovered within the first 3 days of clinical presentation. Fecal miRNA-223 (10.5 fold), miR-451a (4.5 fold), and calprotectin (2.1 fold) concentrations were significant higher in NEC compared with the non-NEC group (p < 0.009). Accepting a minimum sensitivity of 0.75, the positive predictive values (PPVs) ranged between 0.19 and 0.20. Combining fecal biomarkers and CRP (Day 1) could marginally increase the PPVs (0.31-0.34) but adversely lowered the sensitivity (0.54-0.63). CONCLUSIONS: Although fecal miRNA biomarkers and calprotectin concentrations were significantly higher in the NEC group, the considerable overlapping of concentrations between groups and low recovery of stool specimens within 72 h of clinical presentation rendered fecal noninvasive tests of limited clinical value in guiding diagnosis of NEC during the acute phase. A further study is underway to evaluate their roles in surveillance for predicting high-risk premature infants developing NEC.
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Enterocolitis Necrotizante , MicroARNs , Biomarcadores , Estudios de Cohortes , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Estudios ProspectivosRESUMEN
BACKGROUND: Genetic variations in glutathione (GSH)-related and metallothionein (MT) genes, which are involved in producing enzymes in the methylmercury (MeHg) metabolism pathway, have been proposed as one of the reasons for the individual variability in MeHg toxicokinetics. OBJECTIVE: To investigate the impact of genetic variations in MT and GSH-related genes on the association of fish consumption with body burden of MeHg, as measured by hair Hg concentrations among young children and women of childbearing age. METHODS: A total of 179 unrelated children and 165 mothers with either high or low fish consumption were recruited from the community. Their hair total Hg (tHg) and MeHg levels and genotypes for SNPs located on the GCLC, GCLM, GPX1, GSTA1, GSTP1, MT1A, MT2A, and MT4 genes were determined. Based on their 14-day food records, the amounts of fish consumed and their MeHg intakes were estimated. The impact of genetic variations on hair Hg concentrations was examined by using Mann-Whitney tests and multivariable linear regression analyses. RESULTS: The presence of minor alleles of GCLC-129 (rs17883901), GPX1-198 (rs1050450) and MT1M (rs9936741) were associated with significantly lower hair tHg levels in mothers whereas mothers with minor alleles of GSTP1-105(rs1695) and MT1M (rs2270836) have significantly higher hair tHg levels. After adjustment for fish consumption and other confounding factors, apart from MT1M (rs2270836), all of the above SNPs remain significant in the multivariable linear regression models. CONCLUSIONS: Our results in a group of children and women show that genetic variants of GSH-related and MT genes are associated with hair Hg concentrations. These genetic variations are likely to significantly affect MeHg metabolism and thus influence the accumulation of Hg in the human body.
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Mercurio , Compuestos de Metilmercurio , Animales , Niño , Preescolar , Femenino , Peces , Contaminación de Alimentos/análisis , Variación Genética , Glutatión , Humanos , Mercurio/análisis , Metalotioneína/genética , Compuestos de Metilmercurio/análisis , Proyectos PilotoRESUMEN
CD9 has been implicated in cancer progression but its prognostic relevance and therapeutic potential in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are largely unknown. In a cohort of pediatric BCP-ALL patients, we found that CD9+ cases had a significantly lower 5-year relapse-free survival rate than CD9- cases. Multivariate analysis demonstrated that CD9 positivity independently predicted inferior survival outcomes, and could be applied with established prognostic features, including prednisone response and cytogenetic status, to refine patient stratification. Administration of CD9 antibody substantially suppressed disease progression in NOD/SCID mice xenografted with CD9+ cell lines and primary leukemic blasts from patients with high-risk and refractory BCP-ALL, without compromising hematopoietic stem cell engraftment. Combination of anti-CD9 with conventional chemotherapy further reduced leukemic burden and prolonged animal survival. Mechanistically, CD9 blockade inhibited leukemic cell proliferation, induced G0/G1 cell cycle arrest, activated p38, and enhanced chemotherapeutic agent-induced apoptosis. Further, CD9 physically interacted with integrin very late antigen-4, regulated affinity to vascular cell adhesion molecule-1, and was involved in leukemia-stroma interaction. Collectively, our study established CD9 as a new prognostic marker, validated the preclinical efficacy of CD9 antibody, and laid the foundation for clinical development of CD9-targeted therapy for high-risk and refractory pediatric BCP-ALL.
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Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Tetraspanina 29/antagonistas & inhibidores , Animales , Ciclo Celular , Línea Celular Tumoral , Linaje de la Célula , Niño , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis Multivariante , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Pronóstico , Resultado del Tratamiento , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The level of microRNA (miR)-431 was found to be markedly up-regulated in intestinal tissue of necrotizing enterocolitis (NEC). The objective of this study was to identify the target gene of miR-431 and to investigate the role of the miR-431-FOXA1 axis in the pathophysiology of NEC. The target gene of miR-431 was identified by in silico target prediction bioinformatics, luciferase assay, and Western blotting. Effects of miR-431 on downstream expression signals, cell proliferation, and apoptosis were investigated by overexpression in Caco-2 cells upon stimulation by LPS or lipoteichoic acid (LTA). FOXA1 was identified as the target gene of miR-431. Overexpression of miR-431 in Caco-2 cells significantly inhibited FOXA1, ESRRG, and HNF4A and activated IL-6, LGR5, NFKB2, PLA2G2A, PRKCZ, and TNF. IL-8 and - 10 were enhanced when costimulated with LPS or LTA. These potential downstream genes were also significantly dysregulated in primary NEC tissues compared with surgical-control tissues. Overexpression of miR-431 significantly decreased proliferation and increased apoptosis of Caco-2 cells. A proposed network of miR-431-FOXA1 interaction with LPS and LTA receptors demonstrates dysregulation of transcription factors, inflammatory mediators, epithelium tight junction regulators, and cell proliferation and apoptosis signals. The miR-431-FOXA1 axis could in part be responsible for the intensification of the inflammatory response in NEC tissues and contribute to the proinflammatory pathophysiology.-Wu, Y. Z., Chan, K. Y. Y., Leung, K. T., Lam, H. S., Tam, Y. H., Lee, K. H., Li, K., Ng, P. C. Dysregulation of miR-431 and target gene FOXA1 in intestinal tissues of infants with necrotizing enterocolitis.
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Enterocolitis Necrotizante/metabolismo , Factor Nuclear 3-alfa del Hepatocito/metabolismo , MicroARNs/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Células CACO-2 , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Enterocolitis Necrotizante/genética , Citometría de Flujo , Células HEK293 , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Mucosa Intestinal/metabolismo , Lipopolisacáridos/farmacología , MicroARNs/genética , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Ácidos Teicoicos/farmacologíaRESUMEN
OBJECTIVE: To discover specific circulating microRNA (miRNA) biomarkers for the early differentiation of necrotizing enterocolitis (NEC) from neonatal sepsis and inflammatory conditions. STUDY DESIGN: The study comprised 3 distinct phases: differential microarray analysis to compare plasma miRNA expression profiles of NEC vs sepsis and non-NEC/nonsepsis cases, a case-control study to quantify dysregulated miRNAs as potential specific biomarkers of NEC, and a prospective cohort study to assess the diagnostic usefulness of the best miRNA biomarker(s). RESULTS: A distinct miRNA expression profile was observed in the NEC compared with the sepsis and non-NEC/nonsepsis groups. miR-1290, miR-1246, and miR-375 were discovered to be specific biomarkers of NEC in the case-control study. In the cohort study (n = 301), plasma miR-1290 (day 0; >220 copies/µL) provided the greatest diagnostic usefulness for identifying both mild medical and severe surgical NEC cases. Of 20 infants with miR-1290 >650 copies/µL, 15 were diagnosed with NEC. Incorporating C-reactive protein (day 1; >15.8 mg/L) for cases with intermediate levels (220-650 copies/µL) in a 2-stage algorithm further optimized the diagnostic profile with a sensitivity of 0.83, a specificity of 0.96, a positive predictive value of 0.75, and a negative predictive value of 0.98. Importantly, 7 of 36 infants with NEC (19.4%) could be diagnosed 7.8-32.2 hours earlier (median, 13.3 hours) using miR-1290. CONCLUSIONS: Plasma miR-1290 is a novel and specific biomarker that can effectively differentiate NEC cases from neonatal sepsis. miR-1290 facilitates neonatologists to confidently and timely reach a decision for early transfer of sick infants with NEC from community-based hospitals to tertiary surgical centers.
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Enterocolitis Necrotizante/sangre , MicroARNs/sangre , Biomarcadores/sangre , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Diagnóstico Diferencial , Diagnóstico Precoz , Enterocolitis Necrotizante/diagnóstico , Enterocolitis Necrotizante/genética , Femenino , Edad Gestacional , Humanos , Lactante , Recién Nacido , Masculino , Análisis por Micromatrices , Sepsis Neonatal/diagnóstico , Valor Predictivo de las Pruebas , Estudios ProspectivosRESUMEN
Preterm infants are at high risk of developing severe sepsis. Circulating hematopoietic stem and progenitor cells (HSPCs; CD45+CD34+) have been suggested to play a vital role in the host immunological defense against invading pathogens. The objectives were to investigate the regulation of circulating HSPCs in preterm infants during infection episodes, and to assess the relationship of CD45+CD34+ cells with immunological mediators and differential leukocyte populations. First, we conducted a cross-sectional case-control study comparing these parameters among infected infants (n = 23), gestational and postnatal age-matched noninfected infants (n = 46), and "healthy" control (CTL) infants (n = 12). Second, we investigated the longitudinal change of CD45+CD34+ cell concentrations in infected infants before, during, and after an infection episode, and compared them with the other two groups. Our cross-sectional results showed that CD45+CD34+ cell count and percentage were significantly reduced in infected infants during systemic infection, compared with the noninfected or CTL infants. There were significant positive correlation between levels of CD45+CD34+ cells and lymphocytes or monocytes, and significant negative correlation between CD45+CD34+ cells and neutrophils or interleukin (IL)-6 in infected infants. Longitudinal analysis showed that changes of CD45+CD34+ cells at the onset of sepsis relative to levels 1 week prior and 1 week postsepsis in infected infants were significantly different from those changes in the corresponding time points for the other two groups. Our findings suggested that circulating HSPCs were dynamically regulated during septicemia and could play an important role in the defense mechanism, plausibly contributing to replenishment of leukocytes during sepsis in preterm infants.
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Movimiento Celular , Células Madre Hematopoyéticas/citología , Recien Nacido Prematuro/fisiología , Sepsis/patología , Antígenos CD/metabolismo , Estudios de Casos y Controles , Femenino , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Recién Nacido , Interleucina-6/metabolismo , Masculino , Sepsis/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The small intestine is the exclusive site of arginine synthesis in neonates. Low levels of circulating arginine have been associated with the occurrence of necrotizing enterocolitis (NEC) but the mechanism of arginine dysregulation has not been fully elucidated. We aimed to investigate (i) expressional changes of arginine synthesizing and catabolic enzymes in human intestinal tissues of NEC, spontaneous intestinal perforation (SIP) and noninflammatory surgical conditions (Surg-CTL) and to investigate the (ii) mechanisms of arginine dysregulation and enterocyte proliferation upon stimulation by bacterial components, arginine depletion, ARG1 overexpression and nitric oxide (NO) supplementation. Our results showed that expressions of arginine synthesizing enzymes ALDH18A1, ASL, ASS1, CPS1, GLS, OAT and PRODH were significantly decreased in NEC compared with Surg-CTL or SIP tissues. Catabolic enzyme ARG1 was increased (>100-fold) in NEC tissues and histologically demonstrated to be expressed by infiltrating neutrophils. No change in arginine metabolic enzymes was observed between SIP and Surg-CTL tissues. In CaCO2 cells, arginine metabolic enzymes were differentially dysregulated by lipopolysaccharide or lipoteichoic acid. Depletion of arginine reduced cell proliferation and this phenomenon could be partially rescued by NO. Overexpression of ARG1 also reduced enterocyte proliferation. We provided the first expressional profile of arginine metabolic enzymes at the tissue level of NEC. Our findings suggested that arginine homeostasis was severely disturbed and could be triggered by inflammatory responses of enterocytes and infiltrating neutrophils as well as bacterial components. Such reactions could reduce arginine and NO, resulting in mucosal damage. The benefit of arginine supplementation for NEC prophylaxis merits further clinical evaluation.
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Arginina/metabolismo , Enterocolitis Necrotizante/enzimología , Intestinos/enzimología , Arginasa/genética , Arginina/biosíntesis , Células CACO-2 , Femenino , Humanos , Lactante , Recién Nacido , Intestinos/microbiología , MasculinoRESUMEN
BACKGROUND: Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are acute intestinal conditions which could result in mortality and severe morbidity in preterm infants. Our objective was to identify dysregulated micro-RNAs (miRNAs) in small bowel tissues of NEC and SIP, and their possible roles in disease pathophysiology. METHODS: We performed differential miRNA arrays on tissues of NEC (n = 4), SIP (n = 4) and surgical-control (Surg-CTL; n = 4), and validated target miRNAs by qPCR (n = 10 each group). The association of target miRNAs with 52 dysregulated mRNAs was investigated by bioinformatics on functional and base-pair sequence algorithms, and correlation in same tissue samples. RESULTS: We presented the first miRNA profiles of NEC, SIP and Surg-CTL intestinal tissues in preterm infants. Of 28 validated miRNAs, 21 were significantly different between NEC or SIP and Surg-CTL. Limited overlapping in the aberrant expression of miRNAs between NEC and SIP indicated their distinct molecular mechanisms. A proposed network of dysregulated miRNA/mRNA pairs in NEC suggested interaction at bacterial receptor TLR4 (miR-31, miR-451, miR-203, miR-4793-3p), mediated via key transcription factors NFKB2 (miR-203), AP-1/FOSL1 (miR-194-3p), FOXA1 (miR-21-3p, miR-431 and miR-1290) and HIF1A (miR-31), and extended downstream to pathways of angiogenesis, arginine metabolism, cell adhesion and chemotaxis, extracellular matrix remodeling, hypoxia/oxidative stress, inflammation and muscle contraction. In contrast, upregulation of miR-451 and miR-223 in SIP suggested modulation of G-protein-mediated muscle contraction. CONCLUSIONS: The robust response of miRNA dysregulation in NEC and SIP, and concerted involvement of specific miRNAs in the molecular networks indicated their crucial roles in mucosa integrity and disease pathophysiology.
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Enterocolitis Necrotizante/metabolismo , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Perforación Intestinal/metabolismo , Intestino Delgado/metabolismo , MicroARNs/biosíntesis , Enterocolitis Necrotizante/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Recién Nacido , Recien Nacido Prematuro , Mucosa Intestinal/patología , Perforación Intestinal/patología , Intestino Delgado/patología , MasculinoRESUMEN
Makorin-2 (MKRN2) is a highly conserved protein and yet its functions are largely unknown. We investigated the expression levels of MKRN2 and RAF1 in normal and malignant hematopoietic cells, and leukemia cell lines. We also attempted to delineate the role of MKRN2 in umbilical cord blood CD34+ stem/progenitor cells and K562 cell line by over-expression and inhibition of MKRN2 through lentivirus transduction and shRNA nucleofection, respectively. Our results provided the first evidence on the ubiquitous expression of MKRN2 in normal hematopoietic cells, embryonic stem cell lines, primary leukemia and leukemic cell lines of myeloid, lymphoid, erythroid and megakaryocytic lineages. The expression levels of MKRN2 were generally higher in primary leukemia samples compared with those in age-matched normal BM cells. In all leukemia subtypes, there was no significant correlation between expression levels of MKRN2 and RAF1. sh-MKRN2-silenced CD34+ cells had a significantly lower proliferation capacity and decreased levels of the early stem/progenitor subpopulation (CFU-GEMM) compared with control cultures. Over-expression of MKRN2 in K562 cells increased cell proliferation. Our results indicated possible roles of MKRN2 in normal and malignant hematopoiesis.
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Expresión Génica , Hematopoyesis/genética , Leucemia/genética , Leucocitos Mononucleares/metabolismo , Ribonucleoproteínas/genética , Línea Celular Tumoral , Células Cultivadas , Células Madre Embrionarias/metabolismo , Silenciador del Gen , Células Madre Hematopoyéticas/metabolismo , Humanos , Células K562 , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ribonucleoproteínas/metabolismoRESUMEN
OBJECTIVE: To provide a comprehensive database of gene regulation and compare differentially regulated molecular networks in human tissues of necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP). BACKGROUND: Both NEC and SIP are devastating surgical emergencies associated with high morbidity and mortality in preterm infants. Their pathophysiology and molecular mechanisms remain unclear. METHODS: Differential whole genome microarray analysis was performed on intestinal tissues collected from NEC (n = 15) and SIP (n = 12) infants and compared with tissues collected from surgical-control patients with noninflammatory intestinal conditions (n = 14). Validation of 52 target gene expressions was performed by quantitative polymerase chain reaction. Regulatory networks of significantly affected genes were constructed according to functional pathways. RESULTS: Extensive and significant changes of gene expression were observed in NEC tissues, which comprised multiple pathways of angiogenesis, arginine metabolism, cell adhesion and chemotaxis, extracellular matrix remodeling, hypoxia and oxidative stress, inflammation, and muscle contraction. These dysregulated genes could be networked downstream of key receptors, TLR2, TLR4, and TREM1, and mediated via NF-κB, AP-1, and HIF1A transcription factor pathways, indicating predominant microbial and inflammatory involvement. In contrast, SIP tissues exhibited much milder and less diversified expressional changes, with target genes significantly associated with G-protein-mediated muscle contraction and extracellular matrix remodeling. CONCLUSIONS: The molecular evidence suggests that NEC and SIP are likely 2 different diseases caused by distinct etiology and pathophysiology. This first comprehensive database on differential gene expression profiles of human NEC and SIP tissues could lead to development of disease-specific diagnostic and prognostic biomarkers and new therapeutic strategies for improving outcomes.
Asunto(s)
Enterocolitis Necrotizante/genética , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Mucosa Intestinal/metabolismo , Perforación Intestinal/genética , ARN Mensajero/genética , Enterocolitis Necrotizante/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Enfermedades del Prematuro/genética , Enfermedades del Prematuro/metabolismo , Perforación Intestinal/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Estudios RetrospectivosRESUMEN
In newborn infants, the innate cellular system plays a crucial role in the first line of defense against pathogens. Neutrophils are the most abundant leukocytes, and their response to the commonly encountered nosocomial bacterial (Gram positive) infection in newborns remains largely unclear. In this study, a genome-wide expression array analysis was performed on CB neutrophils after challenge by PGN in vitro and compared with neutrophils in CTL cultures without PGN. We investigated responses of neutrophils to PGN and LPS, with respect to cytokine synthesis, chemotaxis, ROS production, cell death, and pathways of HSP response. Our results provide the first comprehensive expressional profile of neonatal neutrophils stimulated by PGN. mRNA levels of 16 up-regulated genes and 6 down-regulated genes were validated by qPCR. Their regulatory networks were identified downstream of TLR-2 and NOD-2, which work in concert toward signals of death, cytoprotection, inflammation, and stress responses. Members of the HSP family were significantly up-regulated in PGN-stimulated neutrophils, compared with those in LPS-stimulated cells. We confirmed protein co-precipitation of HSPA1A and OLR1 in stimulated neutrophils, and their transcription, induced by NF-κB but not by MAPK signals. We found increased CD11b, chemotaxis, TNF-α, and IL-8 in neutrophils stimulated by PGN or LPS. PGN, but not LPS, increased ROS production. We conclude that neonatal neutrophils are capable of vigorous molecular and functional responses to PGN and suggest that HSP plays a critical role in the host defense mechanism, possibly involving proinflammatory OLR1 and CD11b-facilitated chemotaxis.
Asunto(s)
Sangre Fetal/citología , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Neutrófilos/inmunología , Neutrófilos/metabolismo , Peptidoglicano/inmunología , Receptores Depuradores de Clase E/genética , Transcriptoma , Antígeno CD11b/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Citocinas/biosíntesis , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Recién Nacido , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 4/metabolismo , FN-kappa B/metabolismo , Peptidoglicano/farmacología , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Receptores Depuradores de Clase E/metabolismo , Transducción de SeñalRESUMEN
Neonates possess a relatively "naive", yet inducible immune system. Our hypothesis is that upon strategic antigen exposure, cytokine priming and sensitization by accessory cells, natural killer (NK) cells could be activated to become a functional phenotype. We investigated the in vitro stimulation of cord blood (CB) and adult NK cells upon challenge with lipoteichoic acid (LTA), interleukin (IL)-15 and LTA-primed autologous macrophage-conditioned medium, using CD107a and CD69 phenotypes as indicators of activation. We also examined response of CB macrophages to LTA, in terms of P44/42 extracellular signal-regulated kinases (ERK1/2) activation and cytokine secretion. LTA significantly induced secretion of inflammatory cytokines tumor necrotic factor (TNF)-α, IL-6, IL-12 and activated the upstream signal of ERK1/2 phosphorylation in neonatal macrophages. The magnitude of responses to stimulation differed between neonatal and adult NK cells. Co-stimulation with IL-15 was critical for expansion of the CD69 and CD107a NK subpopulations in both neonatal and adult cells, upon a LTA challenge. NK cell activation could be enhanced by LTA-primed autologous macrophages through secretory factors. Our results indicated that neonatal macrophages and NK cells can evoke immunologic responses to a Gram-positive bacterial antigen. The combinatory priming strategy is relevant for development of novel protocols, such as IL-15 treatment, to compensate for the immaturity of the innate immune system in newborns against bacterial infections.
Asunto(s)
Interleucina-15/inmunología , Células Asesinas Naturales/inmunología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ácidos Teicoicos/inmunología , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sangre Fetal/citología , Humanos , Recién Nacido , Células Asesinas Naturales/efectos de los fármacos , Lectinas Tipo C/metabolismo , Lipopolisacáridos/farmacología , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Fosforilación/efectos de los fármacos , Ácidos Teicoicos/farmacología , Adulto JovenRESUMEN
BACKGROUND: Thrombopoietin (TPO) protects against heart damages by doxorubicin-induced cardiomyopathy in animal models. We aimed to investigate the therapeutic efficacy of TPO for treatment of myocardial infarction (MI) in a rat model and explored the mechanisms in terms of the genome-wide transcriptional profile, TPO downstream protein signals, and bone marrow endothelial progenitor cells (EPCs). METHODS: Sprague-Dawley rats were divided into 3 groups: Sham-operated, MI (permanent ligation of the left coronary artery) and MI+TPO. Three doses of TPO were administered weekly for 2 weeks, and outcomes were assessed at 4 or 8 weeks post-injury. RESULTS AND CONCLUSIONS: TPO treatment significantly improved left ventricular function, hemodynamic parameters, myocardium morphology, neovascularization and infarct size. MI damage upregulated a large cohort of gene expressions in the infarct border zone, including those functioned in cytoskeleton organization, vascular and matrix remodeling, muscle development, cell cycling and ion transport. TPO treatment significantly reversed these modulations. While phosphorylation of janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) was modified in MI animals, TPO treatment regulated phosphorylation of STAT3 and extracellular signal-regulated kinases (ERK), and bone morphogenetic protein 1 (BMP1) protein level. TPO also increased EPC colonies in the bone marrow of MI animals. Our data showed that TPO alleviated damages of heart tissues from MI insults, possibly mediated by multi-factorial mechanisms including suppression of over-reacted ventricular remodeling, regulation of TPO downstream signals and mobilization of endothelial progenitor cells. TPO could be developed for treatment of cardiac damages.
Asunto(s)
Modelos Animales de Enfermedad , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/patología , Trombopoyetina/uso terapéutico , Remodelación Ventricular/efectos de los fármacos , Remodelación Ventricular/fisiología , Animales , Masculino , Infarto del Miocardio/genética , Ratas , Ratas Sprague-Dawley , Trombopoyetina/farmacologíaRESUMEN
Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients.