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Mitochondrial and oxidative stress genes are differentially expressed in neutrophils of sJIA patients treated with tocilizumab: a pilot microarray study.

ABSTRACT: Various pathways involved in the pathogenesis of sJIA have been identified through gene expression profiling in peripheral blood mononuclear cells (PBMC), but not in neutrophils. Since neutrophils are important in tissue damage during inflammation, and are elevated as part of the acute phase response, we hypothesised that neutrophil pathways could also be important in the pathogenesis of sJIA. We therefore studied the gene profile in both PBMC and neutrophils of sJIA patients treated with tocilizumab.We studied the transcriptomes of peripheral blood mononuclear cells (PBMC) and neutrophils from eight paired samples obtained from 4 sJIA patients taken before and after treatment, selected on the basis that they achieved ACR90 responses within 12 weeks of therapy initiation with tocilizumab. RNA was extracted and gene expression profiling was performed using Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray platform. A longitudinal analysis using paired t-test (p?

SUBMITTER: Omoyinmi E

PROVIDER: S-EPMC4746827 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Mitochondrial and oxidative stress genes are differentially expressed in neutrophils of sJIA patients treated with tocilizumab: a pilot microarray study.

Omoyinmi Ebun E Hamaoui Raja R Bryant Annette A Jiang Mike Chao MC Athigapanich Trin T Eleftheriou Despina D Hubank Mike M Brogan Paul P Woo Patricia P

Pediatric rheumatology online journal 20160209 1

<h4>Background</h4>Various pathways involved in the pathogenesis of sJIA have been identified through gene expression profiling in peripheral blood mononuclear cells (PBMC), but not in neutrophils. Since neutrophils are important in tissue damage during inflammation, and are elevated as part of the acute phase response, we hypothesised that neutrophil pathways could also be important in the pathogenesis of sJIA. We therefore studied the gene profile in both PBMC and neutrophils of sJIA patients ...[more]

PMID: 26861863

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Project description:ObjectiveTo identify specific miRNAs involved in sepsis-induced AKI and to explore their targeting pathways.MethodsThe expression profiles of miRNAs in serum from patients with sepsis-induced AKI (n = 6), sepsis-non AKI (n = 6), and healthy volunteers (n = 3) were investigated by microarray assay and validated by quantitative PCR (qPCR). The targets of the differentially expressed miRNAs were predicted by Target Scan, mirbase and Miranda. Then the significant functions and involvement in signaling pathways of gene ontology (GO) and KEGG pathways were analyzed. Furthermore, eight miRNAs were randomly selected out of the differentially expressed miRNAs for further testing by qPCR.ResultsqPCR analysis confirmed that the expressions levels of hsa-miR-23a-3p, hsa-miR-4456, hsa-miR-142-5p, hsa-miR-22-3p and hsa-miR-191-5p were significantly lower in patients with sepsis compared with the healthy volunteers, while hsa-miR-4270, hsa-miR-4321, hsa-miR-3165 were higher in the sepsis patients. Statistically, miR-4321; miR-4270 were significantly upregulated in the sepsis-induced AKI compared with sepsis-non AKI, while only miR-4321 significantly overexpressed in the sepsis groups compared with control groups. GO analysis showed that biological processes regulated by the predicted target genes included diverse terms. They were related to kidney development, regulation of nitrogen compound metabolic process, regulation of cellular metabolic process, cellular response to oxidative stress, mitochondrial outer membrane permeabilization, etc. Pathway analysis showed that several significant pathways of the predicted target genes related to oxidative stress. miR-4321 was involved in regulating AKT1, mTOR and NOX5 expression while miR-4270 was involved in regulating PPARGC1A, AKT3, NOX5, PIK3C3, WNT1 expression. Function and pathway analysis highlighted the possible involvement of miRNA-deregulated mRNAs in oxidative stress and mitochondrial dysfunction.ConclusionThis study might help to improve understanding of the relationship between serum miRNAs and sepsis-induced AKI, and laid an important foundation for further identification of the potential mechanisms of sepsis-induced AKI and oxidative stress and mitochondrial dysfunction.

Project description:BackgroundMicroarray experiments are often performed with a small number of biological replicates, resulting in low statistical power for detecting differentially expressed genes and concomitant high false positive rates. While increasing sample size can increase statistical power and decrease error rates, with too many samples, valuable resources are not used efficiently. The issue of how many replicates are required in a typical experimental system needs to be addressed. Of particular interest is the difference in required sample sizes for similar experiments in inbred vs. outbred populations (e.g. mouse and rat vs. human).ResultsWe hypothesize that if all other factors (assay protocol, microarray platform, data pre-processing) were equal, fewer individuals would be needed for the same statistical power using inbred animals as opposed to unrelated human subjects, as genetic effects on gene expression will be removed in the inbred populations. We apply the same normalization algorithm and estimate the variance of gene expression for a variety of cDNA data sets (humans, inbred mice and rats) comparing two conditions. Using one sample, paired sample or two independent sample t-tests, we calculate the sample sizes required to detect a 1.5-, 2-, and 4-fold changes in expression level as a function of false positive rate, power and percentage of genes that have a standard deviation below a given percentile.ConclusionsFactors that affect power and sample size calculations include variability of the population, the desired detectable differences, the power to detect the differences, and an acceptable error rate. In addition, experimental design, technical variability and data pre-processing play a role in the power of the statistical tests in microarrays. We show that the number of samples required for detecting a 2-fold change with 90% probability and a p-value of 0.01 in humans is much larger than the number of samples commonly used in present day studies, and that far fewer individuals are needed for the same statistical power when using inbred animals rather than unrelated human subjects.

Project description:Osteoarthritis (OA) is the most prevalent chronic degenerative disease that affects the health of the elderly. The present study aimed to identify significant genes involved in OA via bioinformatics analysis. A gene expression dataset (GSE104793) was downloaded from the Gene Expression Omnibus. Bioinformatics analysis was then performed in order to identify differentially expressed genes (DEGs) between untreated chondrocytes and chondrocytes cultured with interleukin-1β (IL-1β) for 24 h. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using Metascape. A protein-protein interaction network of DEGs was constructed using the Search Tool for the Retrieval of Interacting Genes. Gene set enrichment analysis (GSEA) was performed using GSEA software. Furthermore, chondrocytes were extracted and treated with IL-1β (10 ng/ml) for 24 h, and reverse-transcription quantitative PCR was used to confirm differential expression of hub genes. Patient samples were also collected to verify the bioinformatic analysis results. Based on the cut-off criteria used for determination of the DEGs, a total of 844 DEGs, including 498 upregulated and 346 downregulated DEGs, were identified. The DEGs were mainly enriched in the GO terms and KEGG pathways 'inflammatory response', 'negative regulation of cell proliferation', 'ossification', 'taxis', 'blood vessel morphogenesis', 'extracellular structure organization', 'mitotic cell cycle process' and 'TNF signaling pathway'. The majority of the PCR results, namely the differential expression of kininogen 2, complement C3, cyclin B1, cell division cycle 20, cyclin A2, 1-phosphatidylinositol 4-kinase, BUB1 mitotic checkpoint serine/threonine kinase, kinesin family member 11, cyclin B2 and BUB1 mitotic checkpoint serine/threonine kinase B were consistent with the bioinformatics results. Collectively, the present observations provided a regulation network of IL-1β-stimulated chondrocytes, which may provide potential targets of OA therapy.

Dataset Information

Mitochondrial and oxidative stress genes are differentially expressed in neutrophils of sJIA patients treated with tocilizumab: a pilot microarray study.

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Mitochondrial and oxidative stress genes are differentially expressed in neutrophils of sJIA patients treated with tocilizumab: a pilot microarray study.

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