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Taqman miRNA Array on Murine livers with varying amounts of carbon tetrachloride induced liver fibrosis

ABSTRACT: Abstract from Knabel et al. PLoS One (2015): Fibrosis refers to the accumulation of excess extracellular matrix (ECM) components and represents a key feature of many chronic inflammatory diseases. Unfortunately, no currently available treatments specifically target this important pathogenic mechanism. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress target gene expression and the development of miRNA-based therapeutics is being actively pursued for a diverse array of diseases. Because a single miRNA can target multiple genes, often within the same pathway, variations in the level of individual miRNAs can potently influence disease phenotypes. Members of the miR-29 family, which include mir29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs. We observed downregulation of miR-29a/b/c in fibrotic livers of carbon tetrachloride (CCl4) treated mice as well as in isolated human hepatocytes exposed to the pro-fibrotic cytokine TGF-β. Importantly, we demonstrate that a single systemic injection of a miR-29a expressing adeno-associated virus (AAV) can prevent and even reverse histologic and biochemical evidence of fibrosis despite continued exposure to CCl4. The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases. Our data therefore demonstrate that delivery of mir-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders.

ORGANISM(S): Mus musculus

PROVIDER: GSE66278 | GEO | 2015/02/25

SECONDARY ACCESSION(S): PRJNA276310

REPOSITORIES: GEO

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Project description:Abstract from Knabel et al. PLoS One (2015): Fibrosis refers to the accumulation of excess extracellular matrix (ECM) components and represents a key feature of many chronic inflammatory diseases. Unfortunately, no currently available treatments specifically target this important pathogenic mechanism. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress target gene expression and the development of miRNA-based therapeutics is being actively pursued for a diverse array of diseases. Because a single miRNA can target multiple genes, often within the same pathway, variations in the level of individual miRNAs can potently influence disease phenotypes. Members of the miR-29 family, which include mir29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs. We observed downregulation of miR-29a/b/c in fibrotic livers of carbon tetrachloride (CCl4) treated mice as well as in isolated human hepatocytes exposed to the pro-fibrotic cytokine TGF-β. Importantly, we demonstrate that a single systemic injection of a miR-29a expressing adeno-associated virus (AAV) can prevent and even reverse histologic and biochemical evidence of fibrosis despite continued exposure to CCl4. The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases. Our data therefore demonstrate that delivery of mir-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders. The Taqman MicroRNA Array (Taqman Array Rodent MicroRNA A Cards v2.0, ABI) was run, using manufacturer's protocols, on 3 groups of mice with varying amounts fibrotic injury. The groups include carbon tetrachloride intraperitinially injected bi-weekly for 1, 4, and 8 weeks with n=4, 3, and 2 respectively for each group. The RNA was isolated from whole liver using the MiRvana miRNA Isolation Kit (Ambion) according to manufacturer's protocols. The fold_change.txt contains the following data columns; ID_Ref: mmu-miR being quantified not-treated (FC): Fold Change compared to not-treated group using Geometric mean normalization 1 week (FC): Fold Change compared to not-treated group using Geometric mean normalization 4 weeks (FC): Fold Change compared to not-treated group using Geometric mean normalization 8 weeks (FC): Fold Change compared to not-treated group using Geometric mean normalization

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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Taqman miRNA Array on Murine livers with varying amounts of carbon tetrachloride induced liver fibrosis

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