A Strategic Expression Method of MiR-29b and Its Anti-Fibrotic Effect Based on RNA-Sequencing Analysis
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ABSTRACT:
PROVIDER: PRJNA666655 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12748273_1.fastq.gz | Fastqsanger.gz | |||
| SRR12748273_2.fastq.gz | Fastqsanger.gz | |||
| SRR12748274_1.fastq.gz | Fastqsanger.gz | |||
| SRR12748274_2.fastq.gz | Fastqsanger.gz | |||
| SRR12748275_1.fastq.gz | Fastqsanger.gz |
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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.
2020-12-04 | GSE158823 | GEO
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 tissue fibrosis. The microRNA-29 (miR-29) family has been demonstrated as anti-fibrotic microRNAs. Our recent work showed that dysregulation of miR-29 contributes to the formation of cardiac fibrosis in animal models of uremic cardiomyopathy, whereas replenishing miR-29 attenuated cardiac fibrosis in these animals. However, excessive overexpression of miR-29 is a concern because microRNAs usually have multiple targets, which could result in unknown and unexpected side effect. In the current study, we constructed a novel Col1a1-miR-29b vector using collagen 1a1 (Col1a1) promoter, which can strategically express miR-29b-3p (miR-29b) in response to increased collagen synthesis and reach a dynamic balance between collagen and miR-29b. Our experimental results showed that in mouse embryonic fibroblasts (MEF cells) transfected with Col1a1-miR-29b vector, the miR-29b expression is about 1000 times less than that in cells transfected with CMV-miR-29b vector, which uses cytomegalovirus (CMV) as a promoter for miR-29b expression. Moreover, TGF-β treatment increased the miR-29b expression by about 20 times in cells transfected with Col1a1-miR-29b, suggesting a dynamic response to fibrotic stimulation. Western blot using cell lysates and culture media demonstrated that transfection of Col1a1-miR-29b vector significantly reduced TGF-β induced collagen synthesis and secretion, and the effect was as effective as the CMV-miR-29b vector. Using RNA-sequencing analysis, we found that 249 genes were significantly altered (180 upregulated and 69 downregulated, at least 2-fold change and adjusted p-value <0.05) after TGF-β treatment in MEF cells transfected with empty vector. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using GAGE R-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. As expected, transfection of Col1a1-miR-29b or CMV-miR-29b vector partially reversed the activation of these pathways. We also analyzed the expression pattern of the top 100 miR-29b targeting genes in these cells using the RNA-sequencing data. We identified that miR-29b targeted a broad spectrum of ECM genes, but the inhibition effect is mostly moderate. In summary, our work demonstrated that the Col1a1-miR-29b vector can be used as a dynamic regulator of collagen and other ECM protein expression in response to fibrotic stimulation, which could potentially reduce unnecessary side effect due to excessive miR-29b levels while remaining an effective potential therapeutic approach for fibrosis.
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Project description:Overexpression and inhibition of miR-29 (pre-miR and anti-miR to miR-29b) in murine aortic smooth muscle cells, analysis of their secretome (conditioned media after serum starvation), n=3 for all four groups (pre-miR control, pre-miR-29b, anti-miR control, anti-miR-29b).
2016-05-06 | PXD003938 | Pride
Project description: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 human. 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
2018-10-03 | GSE120763 | GEO
Project description: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
2018-10-03 | GSE120764 | GEO
Project description:Dendritic cells (DCs) are among the major components of multiple myeloma (MM)-associated bone-marrow microenvironment and are involved in MM progression, growth and chemo-resistance. We observed that after 24h co-culture with different MM cell lines, DCs downregulate miR-29b expression. To gain insight on the effects of miR-29b, we performed a transcriptome analysis of DCs after transient transfection of a negative control or miR-29b mimics and 24h co-culture with U266 cell line.
2018-10-10 | GSE104831 | GEO
Project description:KRAS activation drives DNA methylation and silencing of specific tumor suppressor genes (TSGs). We previously showed that the ERK pathway induces transcriptional repression of TET1, which results in conversion of TSG promoters from a hydroxymethylated, active state to a hypermethylated and silenced state. Here we identified miR-29b as a KRAS-induced molecule that represses TET1 expression. In KRAS-transformed cells, ectopic miR-29b inhibition restores expression of TET1, thereby reactivating TSGs by reducing methylation and restoring hydroxymethylation. Mining gene expression data of lung cancer cell lines identified additional TSGs suppressed by KRAS signaling whose expression was restored by inhibition of miR-29b and re-expression of TET1. Because KRAS changes TSG promoters from hydroxymethylated to hypermethylated with miR-29b-dependent silencing of TET1, we demonstrate a model in which DNMT1 is present on target promoters prior to KRAS transformation. We also propose miR-29b as a potential circulating biomarker and target for rational treatment of specific malignancies.
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2011-03-16 | E-TABM-944 | biostudies-arrayexpress