Unknown,Transcriptomics,Genomics,Proteomics

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MiRNA-29 regulation of gene expression in helper T cells

ABSTRACT: The goal of this study was to identify genes regulated by miRNA-29 in helper T cells. We compared gene expression in miRNA-deficient helper T cells (DGCR8-deficient) transfected with miR-29b versus control miRNA. We also compared gene expression in wildtype helper T cells transfected with miR-29 inhibitor to cells transfected with control inhibitor 3 biological replicates of each genotype (DGCR8-deficient or Wildtype C57BL/6 mice), with 2 conditions per genotype for 12 samples total. Cells from each DGCR8-deficient mouse were transfected with either miR-29b or control miRNA and cells from wildtype mice were transfected with either miR-29 inhibitors or control inhibitors

ORGANISM(S): Mus musculus

SUBMITTER: David Steiner

PROVIDER: E-GEOD-30525 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Tissue fibrosis is a significant health issue associated with organ dysfunction and failure. Increased deposition of collagen and other extracellular matrix (ECM) proteins in the interstitial area is a major process in the formation of tissue fibrosis. The microRNA-29 (miR-29) family has been demonstrated as anti-fibrotic microRNAs. Our recent work also showed that dysregulation of miR-29 contributes to the formation of cardiac fibrosis in animal models of uremic cardiomyopathy and replenish of miR-29 attenuated cardiac fibrosis in these animals. However, due to the multi-targeting effect, overexpression of miR-29 could result in unexpected side effect. In the current study, we constructed a novel col1a1-miR-29b vector using collagen 1a1 promoter, which can strategically express miR-29b-3p (miR-29b) in cells with high expression of collagen and thus minimize the side effect of excessive miR-29b. Because of the similarity between the col1a1 promoter in the plasmid and the endogenous col1a1 promoter in the cell, pro-fibrotic factors that stimulate endogenous collagen expression will simultaneously activate the col1a1 promoter in the plasmid vector and thus stimulate the anti-fibrotic miR-29b expression. The increased miR-29b will then contra-regulate the collagen synthesis and maintain a dynamic balance of collagen. To evaluate the anti-fibrotic effect of miR-29b overexpression, we performed RNA sequencing in MEF cells transfected with the col1a1-miR-29b vector with or without TGF-β treatment. A CMV-miR-29b vector was used as positive control. The RNA-sequencing data showed that TGF-β treatment upregulated a broad spectrum of extracellular matrix (ECM) genes. In accordance, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using GAGE package showed that the top 5 upregulated pathways after TGF-β treatment were mostly fibrosis-related, including focal adhesion, ECM reaction, and TGF-β signaling pathways, while transfection of miR-29b expression vectors attenuated the activation of these pathways, suggesting their anti-fibrotic effect. However, despite that the expression of miR-29b in CMV-miR-29b transfected cells were about 1000 times higher compared to the col1a1-miR-29b transfected cells, there was no significant difference in the anti-fibrotic effect in these cells. In summary, our work demonstrated that the col1a1-miR-29b vector expresses much lower miR-29b compared to the CMV-miR-29b vector, but it is as effective as the CMV-miR-29b vector in blocking the fibrosis-related signaling pathways in response to TGF-β treatment. Our results also suggest that miR-29b has a moderate effect on each of its targeting genes, and its anti-fibrotic effect may result from perturbation of a broad spectrum of fibrosis-related signaling pathways.

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MiRNA-29 regulation of gene expression in helper T cells

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