Gene expression profiling of miR-regulated genes in proliferating C2C12
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ABSTRACT: We used microarrays to detail the global programme of gene expression upon the over-expression of seven different differentiation-associated, E1A-regulated microRNAs. seven different microRNAs (miR-1, miR-34a, miR-22, miR-365, miR-29a, miR-145 and Let-7b) and a negative control (AllStar, Qiagen) were over-expressed at 50nM for 36hrs in proliferating myoblasts. Three independent biological experiments were performed.
Project description:We used microarrays to detail the global programme of gene expression upon the over-expression of seven different differentiation-associated, E1A-regulated microRNAs.
Project description:miR-29a/b1 was reported to be involved in the regulation of reproductive function in female mice, but the underlying molecular mechanisms were not clear. In this study, female mice lacking miR-29a/b1 showed a delay in vaginal opening, irregular estrus cycles, ovulation disorder and infertility. However, the development of egg was normal in mutant mice and the ovulation disorder could be rescued by the superovulation treatment. The plasma level of luteinizing hormone (LH) was significantly lower in the mutant mice. Using iTRAQ coupled with LC-MS/MS, we found that the deficiency of miR-29a/b1 in mice resulted in an abnormal expression of a number of proteins involved in vesicular transport and secretion in the pituitary gland. The miR-29a/b1 targeting gene Dnmt3a and Hdac4 were up-regulated in the pituitary of miR-29a/b1 knockout mice suggesting that these two epigenetic writers may be the upstream causes for these phenotype changes due to miR-29a/b1 deficiency. These findings demonstrated that miR-29a/b1 is indispensable for the function of the reproductive axis through regulating LH secretion in the pituitary gland.
Project description:The human microRNA hsa-miR-29a is a member of the miR-29 family of microRNAs, which is expressed in the adipogenic lineage. To identify putative direct target mRNAs of the miR-29 family, and to get an idea about potential functions of the miR-29 family in adipocyte development, we transfected human Multipotent Adipose-Derived Stem (hMADS) cells with miR-29a mimics (leading to elevation of intracellular levels of mature miR-29a). Subsequently, gene expression profiling was performed for hMADS cells at day 0 (48 h after transfection) and at day 3, i.e. 3 days after adipocyte differentiation was induced by a chemically defined medium.
Project description:Visual cortical circuits show profound plasticity during early life and are later stabilized by molecular "brakes" limiting excessive circuit rewiring beyond a critical period. How the appearance of these factors is coordinated during the transition from development to adulthood remains unknown. We analyzed the role of miR-29a, a miRNA targeting factors involved in several important pathways for plasticity such as extracellular matrix and chromatin regulation. We found that visual cortical miR-29a expression in the visual cortex dramatically increases with age, but it is not experience-dependent. Precocious high levels of miR-29a induced by targeted intracortical injections of a miR-29a mimic blocked ocular dominance plasticity and caused an early appearance of perineuronal nets. Conversely, inhibition of miR-29a in adult mice using LNA antagomirs activated ocular dominance plasticity, reduced perineuronal net intensity and number, and changed their chemical composition restoring permissive low chondroitin 4-O-sulfation levels characteristic of juvenile mice. Activated adult plasticity had the typical functional and proteomic signature of juvenile plasticity. Transcriptomic and proteomic studies indicated that miR-29a manipulation regulates the expression of plasticity factors acting at different cellular levels, from chromatin regulation to synaptic organization and extracellular matrix remodeling. Intriguingly, the projection of miR-29a regulated gene dataset onto cell-specific transcriptomes revealed that parvalbumin-positive interneurons and oligodendrocytes were the most affected cells. Overall, miR29a is a master regulator of the age-dependent plasticity brakes promoting stability of visual cortical circuits.
Project description:Visual cortical circuits show profound plasticity during early life and are later stabilized by molecular "brakes" limiting excessive circuit rewiring beyond a critical period. How the appearance of these factors is coordinated during the transition from development to adulthood remains unknown. We analyzed the role of miR-29a, a miRNA targeting factors involved in several important pathways for plasticity such as extracellular matrix and chromatin regulation. We found that visual cortical miR-29a expression in the visual cortex dramatically increases with age, but it is not experience-dependent. Precocious high levels of miR-29a induced by targeted intracortical injections of a miR-29a mimic blocked ocular dominance plasticity and caused an early appearance of perineuronal nets. Conversely, inhibition of miR-29a in adult mice using LNA antagomirs activated ocular dominance plasticity, reduced perineuronal net intensity and number, and changed their chemical composition restoring permissive low chondroitin 4-O-sulfation levels characteristic of juvenile mice. Activated adult plasticity had the typical functional and proteomic signature of juvenile plasticity. Transcriptomic and proteomic studies indicated that miR-29a manipulation regulates the expression of plasticity factors acting at different cellular levels, from chromatin regulation to synaptic organization and extracellular matrix remodeling. Intriguingly, the projection of miR-29a regulated gene dataset onto cell-specific transcriptomes revealed that parvalbumin-positive interneurons and oligodendrocytes were the most affected cells. Overall, miR29a is a master regulator of the age-dependent plasticity brakes promoting stability of visual cortical circuits.
Project description:During microRNA-biogenesis, stem-loop structured precursors are processed in two endonucleolytic steps, giving rise to mature microRNAs (miRNAs). This process is not only regulated at the level of transcription but also posttranscriptionally by RNA-binding proteins (RBPs). Here we have used a proteomics-based pull-down approach to map and characterize the interactome of 72 pre-miRNAs in eleven cell lines. We identify over 150 RBPs that interact specifically with distinct pre-miRNAs. To demonstrate their functional relevance, we used RNAi knockdown and CRISPR/Cas-mediated knockout experiments and analyzed changes in miRNA levels. Indeed, a large number of the investigated candidates have effects on miRNA-processing under our experimental conditions, including several C3H-zinc-finger proteins. Ultimately, we show that TRIM71/Lin41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. In summary, we provide an extended database of RBPs that interact with pre-miRNAs in different cell types helping to elucidate posttranscriptional regulation of miRNA biogenesis on a global scale.
Project description:Age-related hearing loss (ARHL) is the most common sensory degenerative disease and can significantly impact the quality of life in elderly people. A previous study using GeneChip miRNA microarray assays showed that the expression of miR-29a changes with age, however, its role in hearing loss is still unclear. In this study, we characterized the cochlear phenotype of miR-29a knockout (miR-29a-/-) mice and found that miR-29a-deficient mice had a rapid progressive elevation of the hearing threshold from 2 to 5 months of age compared with littermate controls as measured by the auditory brainstem response. Stereocilia degeneration, hair cell loss and abnormal stria vascularis were observed in miR-29a-/- mice at 4 months of age. Transcriptome sequencing results showed elevated extracellular matrix (ECM) gene expression in miR-29a-/- mice. Both GO annotation and KEGG pathway enrichment analysis revealed that the key differences were closely related to ECM. Further examination with a transmission electron microscope showed thickening of the basilar membrane in the cochlea of miR-29a-/- mice. Five Col4a genes (Col4a1-a5) and two laminin genes (Lamb2 and Lamc1) were validated as miR-29a direct targets by dual luciferase assays and miR-29a inhibition assays with a miR-29a inhibitor. Consistent with the target gene validation results, the expression of these genes was significantly increased in the cochlea of miR-29a-/- mice, as shown by RT-PCR and Western blot. These findings suggest that miR-29a plays an important role in maintaining cochlear structure and function by regulating the expression of collagen and laminin and that the disturbance of its expression could be a cause of progressive hearing loss.
Project description:To confirm the mechanism of miR-29a in liver fibrosis healing, we have employed whole genome microarray expression profiling as a discovery platform to identify genes. CCl4 and TAA liver fibrosis model mouse were used for this experiment. After five weeks liver fibrosis induction period, mouse have been observed for one week (1w) or two weeks (2w) and negative control nucleotide (N.C) or miR-29a were injected every 3 days on this period. We used CCl4 1w N.C (n = 1), 1w miR-29a (n = 1), 2w N.C (n = 1), 2w miR-29a (n = 1), and also used TAA model mouse (total n = 8) liver samples for microarray analysis. We can get only one gene (PDGF-c) as a target of miR-29a which relate to liver fibrosis and down-regulated more than 1.5 times in common miR-29a injected group than N.C group.
Project description:To confirm the mechanism of miR-29a in liver fibrosis healing, we have employed whole genome microarray expression profiling as a discovery platform to identify genes. CCl4 and TAA liver fibrosis model mouse were used for this experiment. After five weeks liver fibrosis induction period, mouse have been observed for one week (1w) or two weeks (2w) and negative control nucleotide (N.C) or miR-29a were injected every 3 days on this period. We used CCl4 1w N.C (n = 1), 1w miR-29a (n = 1), 2w N.C (n = 1), 2w miR-29a (n = 1), and also used TAA model mouse (total n = 8) liver samples for microarray analysis. We can get only one gene (PDGF-c) as a target of miR-29a which relate to liver fibrosis and down-regulated more than 1.5 times in common miR-29a injected group than N.C group. CCl4 and TAA liver fibrosis model mouse were used for this experiment. After five weeks liver fibrosis induction period, mouse have been obserbed for one week (1w) or two weeks (2w) and negative control nucleotide (N.C) or miR-29a were injected every 3 days on this period. We used CCl4 1w N.C (n = 1), 1w miR-29a (n = 1), 2w N.C (n = 1), 2w miR-29a (n = 1), and also used TAA model mouse (total n = 8) liver samples for microarray analysis.
Project description:To identify differentially expressed genes by anti cancer treatments (microRNAs, siRNAs and chemical compounds) in human cancer, several cell lines (prostate cancer, renal cell carcinoma and head and neck squamous cell carcinoma) were subjected to Agilent whole genome microarrays. Human cancer cell lines (PC3, DU145, LNCap, A498, 786-O, FaDu and SAS) were treated with miRNAs (miR-145, miR-375, miR-23b, miR-24, miR-27b and miR-29a), siRNAs (si-CAV2, si-LAMB3 and si-GOLM1) and chemical compunds (genistein, wogonin and CXCL10).