Search

Background: Sepsis represents a complex disease with the dysregulated inflammatory response and high mortality rate. The goal of this study was to identify potential transcriptomic markers in developing pediatric sepsis by a co-expression module analysis of the transcriptomic dataset.Methods: Using the R software and Bioconductor packages, we performed a weighted gene co-expression network analysis to identify co-expression modules significantly associated with pediatric sepsis. Functional interpretation (gene ontology and pathway analysis) and enrichment analysis with known transcription factors and microRNAs of the identified candidate modules were then performed. In modules significantly associated with sepsis, the intramodular analysis was further performed and "hub genes" were identified and validated by quantitative real-time PCR (qPCR) in this study.Results: 15 co-expression modules in total were detected, and four modules ("midnight blue", "cyan", "brown", and "tan") were most significantly associated with pediatric sepsis and suggested as potential sepsis-associated modules. Gene ontology analysis and pathway analysis revealed that these four modules strongly associated with immune response. Three of the four sepsis-associated modules were also enriched with known transcription factors (false discovery rate-adjusted P < 0.05). Hub genes were identified in each of the four modules. Four of the identified hub genes (MYB proto-oncogene like 1, killer cell lectin like receptor G1, stomatin, and membrane spanning 4-domains A4A) were further validated to be differentially expressed between septic children and controls by qPCR.Conclusions: Four pediatric sepsis-associated co-expression modules were identified in this study. qPCR results suggest that hub genes in these modules are potential transcriptomic markers for pediatric sepsis diagnosis. These results provide novel insights into the pathogenesis of pediatric sepsis and promote the generation of diagnostic gene sets. [ABSTRACT FROM AUTHOR]

Background: Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Methods: Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Results: Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. Conclusions: We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence. [ABSTRACT FROM AUTHOR]

Background: Surgical stress initiates a series of host hormone, metabolism and immune responses, which predominantly affect the homeostatic mechanism of patients with major surgery. B7-H3 is a co-stimulatory molecule and has been shown to participate in both adaptive and innate immune responses. In this study we evaluated the clinical significance of plasma B7-H3 levels in pediatric patients with different types of operation and degrees of surgical stress.Methods: A total of 48 children received pediatric general and cardiac surgery were recruited into this study. Based on the surgical stress scoring, children were divided into moderate stress (n = 14) and severe stress (n = 34) groups. Plasma B7-H3 levels were assessed at selected time points: before surgery, immediately after surgery, at day 1, day 3, and day 7 after surgery. Correlations between plasma B7-H3 levels and surgical stress scores were also examined.Results: Plasma B7-H3 levels were significantly decreased in all 48 pediatric patients after surgery compared to the B7-H3 level before surgery (p < 0.01). Children with general surgery showed significant decreases in plasma B7-H3 immediately after surgery, and at day 3 and day 7 after surgery (p < 0.05, p < 0.01), whereas children with cardiac surgery showed reduced plasma B7-H3 immediately after surgery and at day 3 after surgery (p < 0.05). Plasma B7-H3 in cardiac surgery group was dropped much lower than that in general surgery group at day 1 (p < 0.05) and day 3 (p < 0.01) after surgery. Significantly reduced plasma B7-H3 was observed in the severe stress group, but not in the moderate stress group, immediately after surgery and at day 3 after surgery (p < 0.05), and severe stress group had significantly lower plasma B7-H3 levels than moderate stress group at day 1, day 3, and day 7 after surgery (p < 0.05). Furthermore, plasma B7-H3 levels at day 1 (p = 0.01) and day 3 (p = 0.025) after surgery correlated negatively with surgical stress scores.Conclusions: Plasma B7-H3 levels were decreased significantly in children subjected to pediatric general and cardiac surgery, which is closely associated with the severity of surgical stress. The negative correlation of plasma B7-H3 levels at day 1 and day 3 after surgery with surgical stress scoring implicates that the plasma B7-H3 level might be a useful biomarker for monitoring stress intensity during pediatric surgery. [ABSTRACT FROM AUTHOR]

Background: Wilms' tumor (WT) is one of the most common malignant neoplasms of the urinary tract in children. Anaplastic histology (unfavorable histology) accounts for about 10% of whole WTs, and it is the single most important histologic predictor of treatment response and survival in patients with WT; however, until now the molecular basis of this phenotype is not very clearly. Methods: A real-time polymerase chain reaction (PCR) array was designed and tested. Next, the gene expression profile of pediatric anaplastic histology WT and normal adjacent tissues were analyzed. These expression data were anlyzed with Multi Experiment View (MEV) cluster software further. Datasets representing genes with altered expression profiles derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool (IPA). Results: 88 real-time PCR primer pairs for quantitative gene expression analysis of key genes involved in pediatric anaplastic histology WT were designed and tested. The gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal controls; we identified 15 genes that are up-regulated and 16 genes that are down-regulated in the former. To investigate biological interactions of these differently regulated genes, datasets representing genes with altered expression profiles were imported into the IPA for further analysis, which revealed three significant networks: Cancer, Hematological Disease, and Gene Expression, which included 27 focus molecules and a significance score of 43. The IPA analysis also grouped the differentially expressed genes into biological mechanisms related to Cell Death and Survival 1.15E-12, Cellular Development 2.84E-11, Cellular Growth and Proliferation 2.84E-11, Gene Expression 4.43E-10, and DNA Replication, Recombination, and Repair 1.39E-07. The important upstream regulators of pediatric anaplastic histology WT were TP53 and TGFβ1 signaling (P = 1.15E-14 and 3.79E-13, respectively). Conclusions: Our study demonstrates that the gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal tissues with real-time PCR array. We identified some genes that are dysregulated in pediatric anaplastic histology WT for the first time, such as HDAC7, and IPA analysis showed the most important pathways for pediatric anaplastic histology WT are TP53 and TGFβ1 signaling. This work may provide new clues into the molecular mechanisms behind pediatric anaplastic histology WT. [ABSTRACT FROM AUTHOR]

Background: The aims of this study are to evaluate the relationship between early blood glucose concentrations and mortality and to define a 'safe range' of blood glucose concentrations during the first 24 h after pediatric intensive care unit (PICU) admission with the lowest risk of mortality. We further determine whether associations exist between PICU mortality and early hyperglycemia and hypoglycemia occurring within 24 h of PICU admission, even after adjusting for illness severity assessed by the pediatric risk of mortality III (PRISM III) score. Methods: This retrospective cohort study included patients admitted to PICU between July 2008 and June 2011 in a tertiary teaching hospital. Both the initial admission glucose values and the mean glucose values over the first 24 h after PICU admission were analyzed. Results: Of the 1349 children with at least one blood glucose value taken during the first 24 h after admission, 129 died during PICU stay. When analyzing both the initial admission and mean glucose values during the first 24 h after admission, the mortality rate was compared among children with glucose concentrations ≤65, 65-90, 90-110, 110-140, 140-200, and >200 mg/dL (≤3.6, 3.6-5.0, 5.0-6.1, 6.1-7.8, 7.8-11.1, and >11.1 mmol/L). Children with glucose concentrations ≤65 mg/dL (3.6 mmol/L) and >200 mg/dL (11.1 mmol/L) had significantly higher mortality rates, indicating a U-shaped relationship between glucose concentrations and mortality. Blood glucose concentrations of 110-140 mg/dL (6.1-7.8 mmol/L), followed by 90-110 mg/dL (5.0-6.1 mmol/L), were associated with the lowest risk of mortality, suggesting that a 'safe range' for blood glucose concentrations during the first 24 h after admission in critically ill children exists between 90 and 140 mg/dL (5.0 and 7.8 mmol/L). The odds ratios of early hyperglycemia (>140 mg/dL [7.8 mmol/L]) and hypoglycemia (≤65 mg/dL [3.6 mmol/L]) being associated with increased risk of mortality were 4.13 and 15.13, respectively, compared to those with mean glucose concentrations of 110-140 mg/dL (6.1-7.8 mmol/L) (p <0.001). The association remained significant after adjusting for PRISM III scores (p <0.001). Conclusions: There was a U-shaped relationship between early blood glucose concentrations and PICU mortality in critically ill children. Both early hyperglycemia and hypoglycemia were associated with mortality, even after adjusting for illness severity. [ABSTRACT FROM AUTHOR]

Background Histone acetyltransferase (HAT) inhibitors can inhibit proliferation and induce apoptosis in cancer cell lines. The novel cell-permeable p300/CREB-binding protein (CBP)-selective HAT inhibitor HATi II can reduce histone H3 acetylation and induce chromatin condensation in HeLa cells. Here, we examined the effects and mechanism of action of HATi II in glioma cell lines. Methods Cell viability was assessed using the CCK-8 assay. Cell cycle analysis was performed using flow cytometry. Apoptosis was evaluated using Annexin V staining and flow cytometry, Hoechst 33342 staining and the TUNEL assay. Expression and cleavage of caspase-3, caspase-9 and poly ADP-ribose polymerase (PARP) were assessed by Western blotting. Statistical analysis was performed using two-tailed Student's t-tests. The gene expression profiles of U251 glioma cells treated with HATi II or DMSO were analyzed using the Arraystar Human 8 x 60 K LncRNA/mRNA expression array; data was analyzed using MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profiles (≥2-fold) derived from the cluster analyses were subjected to gene ontology and pathway analysis. Results HATi II inhibited the proliferation of U251, U87, HS683 and SHG44 cells in a dosedependent manner. HATi II induced cell cycle arrest at the G2/M phase, and induced significant levels of apoptosis, apoptotic body formation and DNA fragmentation in HATi IItreated U251 and SHG44 cells. HATi II induced cleavage of caspase-3, caspase-9 and PARP in U251 and SHG44 cells. In HATi II-treated U251 cells, 965 genes were upregulated, 984 genes were downregulated and 3492/33327 lncRNAs were differentially expressed. GO analysis showed the differentially expressed genes with known functions are involved in a variety of processes; alcoholism, p53 signaling pathway, cytokine-cytokine receptor interaction and transcriptional mis-regulation in cancer were the four most significant pathways. Upregulation of p53 signaling pathway-related genes in HATi II-treated cells was confirmed by quantitative RT-PCR and Western blotting. Conclusions HATi II inhibits proliferation and induces apoptosis via the caspase-dependent pathway in human glioma cell lines, possibly by activating the p53 signaling pathway. HATi II deserves further investigation as a novel treatment for glioma. [ABSTRACT FROM AUTHOR]

Background: Hyperlactatemia upon admission is a documented risk factor for mortality in critically ill adult patients. However, the predictive significance of a single lactate measurement at admission for mortality in the general population of critically ill children remains uncertain. This study evaluated the predictive value of blood lactate levels at admission and determined the cut-off values for predicting in-hospital mortality in the critically ill pediatric population. Methods: We enrolled 1109 critically ill children who were admitted to a pediatric intensive care unit between July 2008 and December 2010. Arterial blood samples were collected in the first 2 hours after admission, and the lactate levels were determined. The Pediatric Risk of Mortality III (PRISM III) scores were calculated during the first 24 hours after admission. Results: Of the 1109 children admitted, 115 (10.4%) died in the hospital. The median (interquartile range) blood lactate level in critically ill children was 3.2 mmol/l (2.2-4.8). Among the children, 859 (77.5%) had a lactate concentration >2.0 mmol/l. The blood lactate level upon admission was significantly associated with mortality (odds ratio [OR] = 1.38; 95% confidence interval [CI], 1.30-1.46; p <0.001), even after adjustment for age, gender, and illness severity assessed by PRISM III (OR = 1.27; p <0.001). Multivariate regression analysis showed that a high blood lactate level (OR = 1.17; 95% CI, 1.07-1.29; p = 0.001), a high PRISM III score (OR = 1.15; 95% CI, 1.11-1.20; p <0.001), and a low serum albumin (OR =0.92; 95% CI, 0.88-0.96; p <0.001) were independent risk factors for mortality in critically ill children. Blood lactate achieved an area under-the-receiver-operating-characteristic curve (AUC) of 0.79 (p <0.001) for predicting mortality that was similar to that of PRISM III (AUC = 0.82; p <0.001). The p-value for a comparison of both AUCs was 0.318. Blood lactate displayed a sensitivity of 61% and a specificity of 86% in predicting mortality at the optimal cut-off value of 5.55 mmol/l, and the positive and negative likelihood ratios were 4.5 and 0.45, respectively. Conclusions: A high blood lactate level at admission is independently associated with and predictive of in-hospital mortality in the general population of critically ill children. [ABSTRACT FROM AUTHOR]

Background: Bronchopulmonary dysplasia (BPD) is the most common serious pulmonary morbidity in premature infants. The score for neonatal acute physiology (SNAP) is a physiologic severity index for neonatal intensive care and correlates well with neonatal mortality and clinical outcomes. The prognostic value of the SNAP score for BPD in preterm infants remains to be clarified. The aim of the study was to determine whether SNAP can predict the development of BPD or death, and to investigate the contribution of SNAP to the predictive accuracy of other potential perinatal risk factors for the adverse outcome in critically ill preterm infants. Methods: We conducted a study in 160 critically ill preterm infants with less than 33 gestational weeks. The original SNAP score was prospectively calculated based on 28 items collected during the first 24 hours of admission. The potential perinatal risk factors were assessed during the first 72 hours of life. Major outcome measures were BPD and mortality before the time of BPD screening. Results: Of the 160 infants, 17 died and 41 developed BPD. The SNAP score was significantly associated with BPD or death (odds ratio [OR] =1.28; 95% confidence interval [CI], 1.16-1.41; p <0.001), even after adjustment for gestational age (OR =1.27; 95% CI, 1.13-1.41; p <0.001). High SNAP score was an independent predictor of BPD or death (area under the curve [AUC] =0.78; 95% CI, 0.70-0.85; p <0.001), with additional predictive value when combined with other perinatal risk factors. Multivariate regression analysis resulted in a final model, including SNAP, gestational age, apnea of prematurity, patent ductus arteriosus, and surfactant use as independent risk factors, with a higher predictive accuracy compared with individual components (AUC =0.92; 95% CI, 0.87-0.96; p <0.001). Conclusions: SNAP is associated with adverse outcome of BPD or death. High SNAP scores are predictive of BPD or death in critically ill preterm infants, and add prognostic value to other perinatal risk factors. [ABSTRACT FROM AUTHOR]

Background: There is growing evidence supporting a role for microRNAs (miRNA) as targets in aberrant mechanisms of DNA hypermethylation. Epigenetic silencing of tumor suppressor miRNAs, including miR-663, which has recently been reported to be inactivated by hypermethylation in several cancers, may play important roles in pediatric acute myeloid leukemia (AML). However, expression of miR-663 and its promoter methylation remain status unclear in childhood leukemia., Methods: Promoter methylation status of miR-663 was investigated by methylation specific PCR (MSP) and bisulfate genomic sequencing (BGS). Transcriptional expression of miR-663 was evaluated by semi-quantitative and real-time PCR, and the relationship between expression of miR-663 and promoter methylation was confirmed using 5-aza-2'-deoxycytidine (5-Aza) demethylation reagent., Results: MiR-663 was aberrantly methylated in 45.5% (5/11) leukemia cell lines; BGS showed that the promoter was significantly methylated in three AML cell lines; methylation of miR-663 was significantly higher in Chinese pediatric AML patients [41.4% (29/70)] compared to normal bone marrow (NBM) control samples [10.0% (3/30)]. These results were confirmed by both BGS and 5-Aza demethylation analysis. In addition, miR-663 transcript expression was significantly lower in AML patients, both with and without miR-663 methylation, compared to controls; however, there were no significant differences in clinical features or French-American-British (FAB) classification between patients with and without miR-663 methylation., Conclusions: Expression of miR-663 was significantly lower in pediatric AML cells compared to NBM controls; furthermore, a high frequency of miR-663 promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Inactivation of miR-663 by promoter hypermethylation could be affected by 5-Aza demethylation. These findings suggest that hypermethylation of the miR-663 promoter may be an early event in the development of pediatric AML.

Background: Leukemia, a heterogeneous clonal disorder of hematopoietic progenitor cells, presents a world-wide health problem, especially in childhood. F1F0 ATPase, an inner mitochondrial enzyme, is expressed on the plasma membrane of tumor cells, and its inhibition induces both anti-angiogenic and anti-tumorigenic activity., Methods: Monoclonal Antibody (McAb) against ATPase was produced by polyethylene glycol-mediated fusions and screened by ELISA. Proliferation, cell cycle and apoptosis of cells were analyzed when the surface ATPase of cells was blockaded with McAb., Results: We detected cell-membrane expression of the F1F0 ATPase β subunit on 0.1% to 56% of the 11 cell lines derived from leukemia, including acute myeloid leukemia (AML). We produced a monoclonal antibody, McAb7E10, which recognizes both the native and recombinant ATPase β subunit, with a dissociation constant (KD) of 3.26E-10. We demonstrate that McAb7E10 binds to ATPase at the cell surface, where it is able to inhibit ATP synthesis. McAb7E10 significantly inhibited proliferation of AML cell lines in vitro: the relative inhibitory rates of 50 μg/mL McAb7E10 treated MV4-11and HL-60 cells were 69.6% and 81.9% respectively. Cell cycle analysis indicated that McAb7E10 significantly induced apoptosis in MV4-11 and HL-60 cells: the relative rates of apoptosis in 5, 10 and 50ug/mL McAb7E10 treated MV4-11 cells was 3.6 ± 0.83%, 8.4 ± 1.69% and 17.3 ± 2.56% compared to 1.5% ± 0.85% in mouse IgG treated cells (p < 0.01). The relative rate of apoptosis in 5, 10 and 50ug/mL McAb7E10 treated HL-60 cells was 5.5 ± 2.37%, 11.3 ± 3.62% and 19.9 ± 3.31% compared to 1.56% ± 0.97% in mouse IgG treated cells (p < 0.01). Annexin V staining demonstrated that the relative apoptotic rates in 50 μg/mL McAb7E10 treated MV4-11 and HL-60 cells were 50.5% ± 7.04% and 32.9% ± 4.52%, respectively, significantly higher than IgG control antibody treated cells were 21.9% ± 3.11% and 15.3% ± 3.95%, p < 0.01., Conclusions: These findings indicate that ectopic expression of ATPase β subunit may be a tumor-associated antigen in hematological malignancies. The F1F0 ATPase β subunit provides a potential target for immunotherapy in AML and hematological malignancies.

Background: The Real-time PCR Array System is the ideal tool for analyzing the expression of a focused panel of genes. In this study, we will analyze the gene expression profile of pediatric acute myeloid leukemia with real-time PCR arrays., Methods: Real-time PCR array was designed and tested firstly. Then gene expression profile of 11 pediatric AML and 10 normal controls was analyzed with real-time PCR arrays. We analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool., Results: We designed and tested 88 real-time PCR primer pairs for a quantitative gene expression analysis of key genes involved in pediatric AML. The gene expression profile of pediatric AML is significantly different from normal control; there are 19 genes up-regulated and 25 genes down-regulated in pediatric AML. To investigate possible biological interactions of differently regulated genes, datasets representing genes with altered expression profile were imported into the Ingenuity Pathway Analysis Tool. The results revealed 12 significant networks. Of these networks, Cellular Development, Cellular Growth and Proliferation, Tumor Morphology was the highest rated network with 36 focus molecules and the significance score of 41. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to hematological disease, cell death, cell growth and hematological system development. In the top canonical pathways, p53 and Huntington's disease signaling came out to be the top two most significant pathways with a p value of 1.5E-8 and2.95E-7, respectively., Conclusions: The present study demonstrates the gene expression profile of pediatric AML is significantly different from normal control; there are 19 genes up-regulated and 25 genes down-regulated in pediatric AML. We found some genes dyes-regulated in pediatric AML for the first time as FASLG, HDAC4, HDAC7 and some HOX family genes. IPA analysis showed the top important pathways for pediatric AML are p53 and Huntington's disease signaling. This work may provide new clues of molecular mechanism in pediatric AML.

Background: The purpose of this study was to investigate invasion and metastasis related genes in gastric cancer., Methods: The transwell migration assay was used to select a highly invasive sub-line from minimally invasive parent gastric cancer cells, and gene expression was compared using a microarray. MMP28 upregulation was confirmed using qRT-PCR. MMP28 immunohistochemistry was performed in normal and gastric cancer specimens. Invasiveness and tumor formation of stable cells overexpressing MMP28 were tested in vitro and in vivo., Results: MMP28 was overexpressed in the highly invasive sub-cell line. Immunohistochemistry revealed MMP28 expression was markedly increased in gastric carcinoma relative to normal epithelia, and was significantly associated with depth of tumor invasion, lymph node metastasis and poorer overall survival. Ectopic expression of MMP28 indicated MMP28 promoted tumor cell invasion in vitro and increased gastric carcinoma metastasis in vivo., Conclusions: This study indicates MMP28 is frequently overexpressed during progression of gastric carcinoma, and contributes to tumor cell invasion and metastasis. MMP28 may be a novel therapeutic target for prevention and treatment of metastases in gastric cancer.

Background: Differentiation of the acute myeloid leukemia (AML) cell line HL-60 can be induced by all trans-retinoic acid (ATRA); however, the mechanism regulating this process has not been fully characterized., Methods: Using bioinformatics and in vitro experiments, we identified the microRNA gene expression profile of HL-60 cells during ATRA induced granulocytic differentiation., Results: Six microRNAs were upregulated by ATRA treatment, miR-663, miR-494, miR-145, miR-22, miR-363* and miR-223; and three microRNAs were downregulated, miR-10a, miR-181 and miR-612. Additionally, miR-663 expression was regulated by ATRA. We used a lentivirus (LV) backbone incorporating the spleen focus forming virus (SFFV-F) promoter to drive miR-663 expression, as the CMV (Cytomegalovirus) promoter is ineffective in some lymphocyte cells. Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro., Conclusions: Our results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation. Lentivirus delivery of miR-663 could potentially be used directly as an anticancer treatment in hematological malignancies.