Unknown,Transcriptomics,Genomics,Proteomics

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MicroRNA profiling in vitro activated primary hepatic stellate cells

ABSTRACT: pHSCs were isolated from Wistar rats and cultivated in cell culture flasks. The miRNA was isolated at day 0 and 10 and hybridized onto the Ncode Multi-species miRNA microarray V2 from Invitrogen 3 samples from different rats were analyzed on miRNA microarray performing dye swap

ORGANISM(S): Rattus norvegicus

SUBMITTER: jinmiao chen

PROVIDER: E-GEOD-19462 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by varying degrees of emphysematous lung destruction and small airway disease, each with distinct effects on clinical outcomes. There is little known about how microRNAs contribute specifically to the emphysema phenotype. We examined how microRNA expression is altered with regional emphysema severity within the lung and how these microRNAs regulate disease-associated gene-expression networks. Results: We profiled microRNAs in different regions in the lung with varying degrees of emphysema from 6 smokers with COPD and 2 controls (8 regions x 8 lungs = 64 samples). 63 microRNAs (p<0.05) were altered with regional emphysema severity as quantified by mean linear intercept (Lm). MicroRNA and gene expression data were then integrated in the same samples. A subset of microRNAs, including miR-638, miR-30c, and miR-181d, correlated with many of their predicted gene targets, suggesting a role in regulating the gene networks that underlie emphysematous lung destruction. Modulating miR-638 expression in primary human lung fibroblasts recapitulated the alterations in its targeted gene-expression network associated with emphysema progression. Pathway analysis revealed that genes involved in oxidative stress and accelerated aging were affected by miR-638 knock-down in fibroblasts. Many miR-638 gene targets in these pathways were amongst those negatively correlated with miR-638 expression in emphysematous lung tissue. Conclusions: Our findings demonstrate that microRNAs are altered with regional emphysema severity and modulate disease-associated gene expression networks. Furthermore, miR-638 may regulate gene expression associated with the oxidative stress response and aging in emphysematous lung tissue and fibroblasts. Paired samples were obtained from 8 regions at regular intervals between the apex and base of each explanted lung from six patients with severe COPD and two donors. The degree of emphysematous destruction was quantified in one sample from each region by mean linear intercept (Lm), while microRNA expression was profiled in the adjacent sample from the same region using the NCode Version 3 microRNA microarrays (Invitrogen, Carlsbad, CA). After quality control 60 samples were used for the analysis.

Project description:To determine whether DGCR8 is required for maturation of all miRNAs, we performed miRNA microarray analysis. Using RNA from wild-type ES cells as our reference sample, we observed a global loss of miRNAs in DGCR8 knockout cells, but normal levels of expression in DGCR8 heterozygous cells. The similarity in expression levels between wild-type and heterozygous cells suggests that DGCR8 is not limiting in the maintenance of steady-state levels of miRNAs in ES cells. Of the eighty-nine miRNA array probes that showed significant signals with wild-type RNA, eighty-two were drastically reduced in the DGCR8 knockout cells. The remaining seven were not significantly altered in the DGCR8 knockout cells, but at least four of these seven miRNAs appear to be due to unavoidable contamination with RNA from the mouse embryonic fibroblast (MEF) feeder cells used for ES cell culture prior to the isolation of RNA. These miRNAs were highly expressed in the MEFs and decreased when the ES cells were temporarily passaged on gelatin-coated plates without feeders. The remaining three showed similar levels of expression in the heterozygous, knockout and MEF feeder cells. Therefore, these signals may be small RNAs that are not processed with the help of the microprocessor complex. Alternatively, these signals may result from unavoidable degradation products within the purified small RNA population. In either case, our results show that DGCR8 is broadly required for miRNA processing with little evidence for redundancy or bypass mechanisms. Keywords: cell type comparison, genetic modification MicroRNAs extracted from wild-type, DGCR8 heterozygous knockout and DGCR8 homozygous knockout were analyzed by microarray. In each array, wild-type samples serve as reference. Ratios were normalized based on positive control RNAs on the array. To determine if some of signals appeared in DGCR8 knockout ES cells are due to mice embryonic fibroblast (MEF) feeder cell contamination (this is unavoidable because these ES cells were grown on MEFs and passaged off MEF before RNA extraction). Two independent DGCR8 knockout ES cell lines were analyzed. For each cell line, array hybridization was done in duplicates. For DGCR8 heterozygous knockout ES cells, two independent batches of RNA samples were prepared and analyzed.