MRNA expression analysis of undifferentiated Dicer -/- (27H10) embryonic stem cells after miRNA transfection
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE8503 | GEO | 2008/01/01
SECONDARY ACCESSION(S): PRJNA101611
REPOSITORIES: GEO
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Project description:We have analyzed the transcript expression levels in Dicer knock-out embryonic stem (ES) cells 24 hours after transfection with either control siRNA agains Renilla luciferase or miRNA Mimics (Dharmacon) of mmu-miR-290 cluster members in order to identify primary targets of miR-290 cluster miRNAs. Experiment Overall Design: Cell were analysed 24h after transfections in an undifferentiated state. Triplicates of both transfections were analyzed.
2008-06-16 | E-GEOD-8503 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target. Three replicates of mouse embryonic stem cells with Dicer (CTRL) compared to three replicates of mouse embryonic stem cells lacking Dicer (CRE)
2011-06-18 | E-GEOD-30012 | biostudies-arrayexpress
Project description:Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays, and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-Sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs, and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes, and upregulating pluripotency-associated genes such as Lin28. For RNA-IP experiments, cells were transfected with 300 pmol of miR-294 or cel-239b control miRNA (Dharmacon) and harvested 12-16 hr after transfection for immunoprecipitation. Following RNA-immunoprecipitation of Ago2-myc, RNA from the INPUT (total RNA) and IP were subjected to library preparation and sequenced by SOLiD. There are 10 samples from Dicer1-null mouse ES cells, which are essentially 5 sample pairs of INPUT (A) and its corresponding RNA-IP (B): Ago2-myc transfected with miR-294 (sampe 1), 2 replicates of Ago2-myc-MUT (catalytically inactive) transfected with miR-294 (sample 2 & 5), 2 replicates of Ago2-myc transfected with cel-239b (sample 3 & 4).
2010-02-27 | E-GEOD-20199 | biostudies-arrayexpress
Project description:We investigated functions of miRNAs at the level of the whole transcriptome of primary neurons. We transfected mouse E17.5 forebrain primary neuronal cultures (at four to six days of in vitro development) with miRNA mimics and inhibitors. After approximately 48 h post transfection we profiled the effect of these transfections on gene expression with Illumina mRNA microarrays. Cultures were transfected with mimics and inhibitors of five mouse miRNAs (mmu-miR-124, mmu-miR-434-3p, mmu-miR-143, mmu-miR-145 and mmu-miR-25) and with a mimic of a non-mouse miRNA (cel-miR-67). Direct widespread inhibition of gene expression by the perturbed miRNAs was evident when gene expression in cultures transfected with miRNA mimics was compared to those transfected with the inhibitors (or to matched mock transfected cultures): 3-prime UTRs of downregulated transcripts were significantly enriched in seed matching sites for the perturbed miRNAs. Interestingly, analysis of differential gene expression in mock transfected cells (identified through comparison of mock transfected cultures with matched untreated cultures) revealed that genes inhibited by miRNAs were enriched in genes upregulated in mock transfected cultures. This inhibition was the most efficient by the two neuronal miRNAs under investigation (mmu-miR-124 and mmu-miR-434-3p). To investigate if miRNA mediated inhibition of stress induced genes (i.e. stress associated with transfections) was also the case in other stresses, we profiled gene expression changes triggered by chronic neuronal depolarisation. For this, we treated the cultures with KCl (15 mM, 48 h) and compared them to matched untreated cultures. We found that genes upregulated by KCl had a significant intersection with those upregulated by the mock transfection. Moreover, we also found that genes upregulated by KCl had a significant intersection with genes inhibited by mmu-miR-124 and mmu-miR-434-3p. Therefore we concluded that neuronal miRNAs stabilise the neuronal transcriptome by inhibiting stress inducible genes.
2011-12-31 | E-MTAB-686 | biostudies-arrayexpress
Project description:Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with decreased expression of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferases (Dnmts), and methylation levels can be recovered by their overexpression. We identify the retinoblastoma-like 2 protein (Rbl2) as responsible for decreased Dnmt expression in Dicer1-null cells, suggesting the existence of Dicer-dependent small RNAs that target Rbl2. We identify the miR-290 cluster as being downregulated in Dicer1-deficient cells and show that it silences Rbl2, thereby controlling Dnmt expression. These results identify a pathway by which miR-290 directly regulates Rbl2-dependent Dnmt expression, indirectly affecting telomere-length homeostasis. Experiment Overall Design: Two-condition experiments. 6 comparisons. One dye-swap technical replicate for all samples. 2-color platform.
2008-06-19 | E-GEOD-11229 | biostudies-arrayexpress
Project description:Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with decreased expression of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferases (Dnmts), and methylation levels can be recovered by their overexpression. We identify the retinoblastoma-like 2 protein (Rbl2) as responsible for decreased Dnmt expression in Dicer1-null cells, suggesting the existence of Dicer-dependent small RNAs that target Rbl2. We identify the miR-290 cluster as being downregulated in Dicer1-deficient cells and show that it silences Rbl2, thereby controlling Dnmt expression. These results identify a pathway by which miR-290 directly regulates Rbl2-dependent Dnmt expression, indirectly affecting telomere-length homeostasis. Keywords: Cell type comparison
2008-04-22 | GSE11229 | GEO
Project description:Liver is uniquely capable to repair itself after injury. Multiple molecular and biochemical processes initiated after partial hepatectomy, lead to proliferation of all cells within the liver. MicroRNAs (miRNAs) are a class of highly abundant non-coding RNA molecules that cause post-transcriptional gene repression and are involved in several biological processes including cell cycle regulation and differentiation. We examined the expression levels of miRNAs in liver tissue received from control mice (L0) and compared them with the corresponding levels in liver tissue 12 hours after liver regeneration induced by 2/3 partial hepatectomy (L12). MicroRNA expression was investigated using microRNA profiling. Further qPCR analysis was used for validation of the differentially expressed microRNAs at an early stage of liver regeneration, induced by 2/3 partial hepatectomy. TargetScan and Gene Ontology (GO) analysis was performed in order to identify the possible miRNA target genes and their ontology, respectively. A subset of miRNAs were found to be differentially expressed during liver regeneration. Mmu-miR-21 and mmu-miR-30b* showed the higher levels of up-regulation in liver tissue from the hepatectomized mice at the end of the experiment (L12) compared to the sham operated mice (L0). Mmu-miR-21 up-regulation was further confirmed by qPCR. In situ hybridization (ISH) revealed that mmu-miR-21 exhibited the higher levels of expression at 12 hours post hepatectomy. On the contrary, mmu-miR-34c*, mmu-miR-144, mmu-miR-207, mmu-miR-207, mmu-miR-451, mmu-miR-582-3p and mmu-miR-290-5p exhibited <0.5 down-regulation in liver tissue after partial hepatectomy in L12 vs. L0 mice. Microarrays and qPCR results were in good agreement (Pearson correlation = 0.881). Our results provide important information regarding how microRNAs are deferentially expressed in murine liver tissue before and after partial hepatectomy. The early up-regulation of mmu-miR-21 during the process of liver regeneration suggests a regulatory role in liver regeneration in vivo.
2012-01-18 | GSE35128 | GEO
Project description:MEFs were infected with Oct4, Sox2, Klf4 (+/- Sall4), sorted for miR-290/302 reporter expression at day 9 (OSK) or 12 (Sall4+OSK), and then profiled. Resulting iPSCs were also profiled. Mouse ES cells were differentiated and sorted for miR-290/302 reporter expression during Fgf/Activin differentiation (day 4 or day 7).
2014-03-31 | GSE54341 | GEO
Project description:Liver is uniquely capable to repair itself after injury. Multiple molecular and biochemical processes initiated after partial hepatectomy, lead to proliferation of all cells within the liver. MicroRNAs (miRNAs) are a class of highly abundant non-coding RNA molecules that cause post-transcriptional gene repression and are involved in several biological processes including cell cycle regulation and differentiation. We examined the expression levels of miRNAs in liver tissue received from control mice (L0) and compared them with the corresponding levels in liver tissue 12 hours after liver regeneration induced by 2/3 partial hepatectomy (L12). MicroRNA expression was investigated using microRNA profiling. Further qPCR analysis was used for validation of the differentially expressed microRNAs at an early stage of liver regeneration, induced by 2/3 partial hepatectomy. TargetScan and Gene Ontology (GO) analysis was performed in order to identify the possible miRNA target genes and their ontology, respectively. A subset of miRNAs were found to be differentially expressed during liver regeneration. Mmu-miR-21 and mmu-miR-30b* showed the higher levels of up-regulation in liver tissue from the hepatectomized mice at the end of the experiment (L12) compared to the sham operated mice (L0). Mmu-miR-21 up-regulation was further confirmed by qPCR. In situ hybridization (ISH) revealed that mmu-miR-21 exhibited the higher levels of expression at 12 hours post hepatectomy. On the contrary, mmu-miR-34c*, mmu-miR-144, mmu-miR-207, mmu-miR-207, mmu-miR-451, mmu-miR-582-3p and mmu-miR-290-5p exhibited <0.5 down-regulation in liver tissue after partial hepatectomy in L12 vs. L0 mice. Microarrays and qPCR results were in good agreement (Pearson correlation = 0.881). Our results provide important information regarding how microRNAs are deferentially expressed in murine liver tissue before and after partial hepatectomy. The early up-regulation of mmu-miR-21 during the process of liver regeneration suggests a regulatory role in liver regeneration in vivo. Twenty male wild type mice C57BL6J (8 weeks old) fed a standard diet were used for the liver regeneration experiment. Mice were housed in the animal facility of the University of Patras Medical School in temperature-, light- and humidity-controlled rooms with a 12-h light/dark cycle. All animal procedures were approved by the institutional review board of the University of Patras Medical School and were in accordance with EC Directive 86/609/EEC. A 2/3 partial hepatectomy (PH) was performed on 10 mice according to a recently published standardized protocol whereby the median and left lobes of the liver were removed and another 10 sham operated mice were used as the control group. The liver was allowed to regenerate and mice were sacrificed 12 hours after operation. Liver samples were collected from the sham operated mice (L0) and the hepatectomized mice at the end of the experiment (L12).
2012-01-18 | E-GEOD-35128 | biostudies-arrayexpress
Project description:Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays, and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-Sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs, and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes, and upregulating pluripotency-associated genes such as Lin28.
2010-02-27 | GSE20199 | GEO