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Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics - mouse

ABSTRACT: MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3cyanovinylcarbazole ( CNV K), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNV K-containing miR-29b ( CNV K-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, arehighly sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNV K-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and findrobust conservation of the focal adhesion and apoptotic target pathways in mouse and mouse. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNV K-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNV K-containing small RNA mimics may find applicability in other experimental models

ORGANISM(S): Mus musculus

PROVIDER: GSE120764 | GEO | 2018/10/03

REPOSITORIES: GEO

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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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Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics - mouse

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