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1.  Regulation of epithelial—mesenchymal and mesenchymal—epithelial transitions by microRNAs 
Current opinion in cell biology  2013;25(2):200-207.
Epithelial–mesenchymal transition (EMT) and the reverse process, mesenchymal–epithelial transition (MET), are essential during development and in the regulation of stem cell pluripotency, yet these processes are also activated in pathological contexts, such as in fibrosis and cancer progression. In EMT and MET, diverse signaling pathways cooperate in the initiation and progression of the EMT and MET programs, through regulation at transcriptional, post-transcriptional, translational, and post-translational levels. MicroRNAs recently emerged as potent regulators of EMT and MET, with their abilities to target multiple components involved in epithelial integrity or mesenchymal traits. By affecting EMT and MET processes, microRNAs are involved in the regulation of stem cell pluripotency and the control of tumor progression.
PMCID: PMC4240277  PMID: 23434068
2.  miR-19, miR-345, miR-519c-5p Serum Levels Predict Adverse Pathology in Prostate Cancer Patients Eligible for Active Surveillance 
PLoS ONE  2014;9(6):e98597.
Serum microRNAs hold great promise as easily accessible and measurable biomarkers of disease. In prostate cancer, serum miRNA signatures have been associated with the presence of disease as well as correlated with previously validated risk models. However, it is unclear whether miRNAs can provide independent prognostic information beyond current risk models. Here, we focus on a group of low-risk prostate cancer patients who were eligible for active surveillance, but chose surgery. A major criteria for the low risk category is a Gleason score of 6 or lower based on pre-surgical biopsy. However, a third of these patients are upgraded to Gleason 7 on post surgical pathological analysis. Both in a discovery and a validation cohort, we find that pre-surgical serum levels of miR-19, miR-345 and miR-519c-5p can help identify these patients independent of their pre-surgical age, PSA, stage, and percent biopsy involvement. A combination of the three miRNAs increased the area under a receiver operator characteristics curve from 0.77 to 0.94 (p<0.01). Also, when combined with the CAPRA risk model the miRNA signature significantly enhanced prediction of patients with Gleason 7 disease. In-situ hybridizations of matching tumors showed miR-19 upregulation in transformed versus normal-appearing tumor epithelial, but independent of tumor grade suggesting an alternative source for the increase in serum miR-19a/b levels or the release of pre-existing intracellular miR-19a/b upon progression. Together, these data show that serum miRNAs can predict relatively small steps in tumor progression improving the capacity to predict disease risk and, therefore, potentially drive clinical decisions in prostate cancer patients. It will be important to validate these findings in a larger multi-institutional study as well as with independent methodologies.
PMCID: PMC4043973  PMID: 24893170
3.  MicroRNA-based discovery of barriers to dedifferentiation of fibroblasts to pluripotent stem cells 
Nature structural & molecular biology  2013;20(10):1227-1235.
Individual microRNAs (miRNAs) can target hundreds of messenger RNAs forming networks of presumably cooperating genes. To test this presumption, we functionally screened miRNAs and their targets in the context of de-differentiation of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Along with the miR-302/miR-294 family, the miR-181 family arose as a novel enhancer of the initiation phase of reprogramming. Endogenous miR-181 miRNAs were transiently elevated with introduction of Oct4, Sox2, and Klf4 (OSK), and their inhibition diminished iPSC colony formation. We tested the functional contribution of 114 individual targets of the two families, revealing twenty-five genes that normally suppress initiation. Co-inhibition of targets cooperatively promoted both the frequency and kinetics of OSK reprogramming. These data establish two of the largest functionally defined networks of miRNA-mRNA interactions, elucidating novel relationships among genes that act together to suppress early stages of reprogramming.
PMCID: PMC3955211  PMID: 24037508
4.  MiR-294/-302 promotes proliferation, suppresses G1-S restriction point, and inhibits embryonic stem cell differentiation through separable mechanisms 
Cell reports  2013;4(1):99-109.
The miR-294/miR-302 microRNAs promote the abbreviated G1 phase of the embryonic stem cell (ESC) cell cycle and suppress differentiation induced by let-7. Here we evaluated the role of the Retinoblastoma (Rb) family proteins in these settings. Under normal growth conditions miR-294 promoted the rapid G1-S transition independent of the Rb family. In contrast, miR-294 suppressed the further accumulation of cells in G1 in response to nutrient deprivation and cell-cell contact in an Rb dependent fashion. We uncovered five additional miRNAs (miRs-26a, -99b, -193, -199a-5p, and 218) that silenced ESC self-renewal in the absence of other miRNAs, all of which were antagonized by miR-294/302. Four of the six differentiation-inducing miRNAs induced an Rb-dependent G1 accumulation. However, all six still silenced self-renewal in the absence of the Rb proteins. These results show that the miR-294/miR-302 family acts through Rb dependent and independent pathways to regulate the G1 restriction point and the silencing of self-renewal respectively.
PMCID: PMC3740202  PMID: 23831024
5.  Dgcr8 is required in pyramidal neurons for normal inhibitory synaptic function 
Molecular and cellular neurosciences  2012;50(3-4):283-292.
MicroRNAs (miRNAs) are critical regulators of nervous system function, and in vivo knockout studies have demonstrated that miRNAs are necessary for multiple aspects of neuronal development and survival. However, the requirements of miRNA biogenesis in the formation and function of synapses in the cerebral cortex are only minimally understood. Here, we have generated and characterized a mouse line with a conditional neuronal deletion of Dgcr8, a miRNA biogenesis protein predicted to process miRNAs exclusively. Loss of Dgcr8 in pyramidal neurons of the cortex results in a non-cell-autonomous reduction in parvalbumin interneurons in the prefrontal cortex, accompanied by a severe deficit in inhibitory synaptic transmission and a corresponding reduction of inhibitory synapses. Together, these results suggest a vital role for miRNAs in governing essential aspects of inhibitory transmission and interneuron development in the mammalian nervous system. These results may be relevant to human diseases such as schizophrenia, where both altered Dgcr8 levels as well as aberrant inhibitory transmission in the prefrontal cortex have been postulated to contribute to the pathophysiology of the disease.
PMCID: PMC3613865  PMID: 22728723
microRNA; Dgcr8; inhibitory synaptic transmission; parvalbumin interneurons; prefrontal cortex
6.  Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells 
Nature biotechnology  2011;29(5):443-448.
The embryonic stem cell–specific cell cycle–regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells1. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore, these miRNAs repress multiple target genes, with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT), epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle2,3. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGFβ-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions.
PMCID: PMC3685579  PMID: 21490602
7.  DGCR8-Mediated Production of Canonical Micrornas Is Critical for Regulatory T Cell Function and Stability 
PLoS ONE  2013;8(5):e66282.
Regulatory T cells (Treg) are integral for immune homeostasis. Here we demonstrate that canonical microRNAs (miRNAs) are required for Treg function because mice with DGCR8-deficient Treg cells spontaneously develop a scurfy-like disease. Using genetic lineage marking we show that absence of miRNAs leads to reduced FoxP3 expression in Treg cells in vivo. In vitro culture of purified DGCR8-deficient Treg leads to a loss of FoxP3 expression. We conclude that canonical miRNAs are essential to maintain stable FoxP3 expression and Treg function. Thus, signals interfering with miRNA homeostasis might contribute to autoimmune diseases.
PMCID: PMC3669207  PMID: 23741528
8.  An integrated nano-scale approach to profile miRNAs in limited clinical samples 
Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; under accession no. GSE31030.
PMCID: PMC3538381  PMID: 23304658
microRNA (miRNA); asthma; helper T cell; microfluidic; qPCR arrays
9.  An integrated nano-scale approach to profile miRNAs in limited clinical samples 
Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; under accession no. GSE31030.
PMCID: PMC3538381  PMID: 23304658
microRNA (miRNA); asthma; helper T cell; microfluidic; qPCR arrays
10.  MicroRNA-29 Regulates T-Box Transcription Factors and Interferon-γ Production in Helper T Cells 
Immunity  2011;35(2):169-181.
MicroRNA (miRNA)-deficient helper T cells exhibit abnormal IFN-γ production and decreased proliferation. However, the contributions of individual miRNAs to this phenotype remain poorly understood. We conducted a screen for miRNA function in primary T cells and identified individual miRNAs that rescue the defects associated with miRNA deficiency. Multiple members of the miR-17 and miR-92 families enhanced miRNA-deficient T cell proliferation whereas miR-29 largely corrected their aberrant interferon-γ (IFN-γ) expression. Repression of IFN-γ production by miR-29 involved direct targeting of both T-bet and Eomes, two transcription factors known to induce IFN-γ production. Although not usually expressed at functionally relevant amounts in helper T cells, Eomes was abundant in miRNA-deficient cells and was upregulated after miR-29 inhibition in wild-type cells. These results demonstrate that miR-29 regulates helper T cell differentiation by repressing multiple target genes, including at least two that are independently capable of inducing the T helper 1 (Th1) cell gene expression program.
PMCID: PMC3361370  PMID: 21820330
11.  From microRNAs to targets: pathway discovery in cell fate transitions 
MicroRNAs (miRNAs) are 22 nt non-coding RNAs that regulate expression of downstream targets by messenger RNA (mRNA) destabilization and translational inhibition. A large number of eukaryotic mRNAs are targeted by miRNAs, with many individual mRNAs being targeted by multiple miRNAs. Further, a single miRNA can target hundreds of mRNAs, making these small RNAs powerful regulators of cell fate decisions. Such regulation by miRNAs has been observed in the maintenance of the embryonic stem cell (ESC) cell cycle and during ESC differentiation. MiRNAs can also promote the dedifferentiation of somatic cells to induced pluripotent stem cells. During this process they target multiple downstream genes, which represent important nodes of key cellular processes. Here, we review these findings and discuss how miRNAs may be used as tools to discover novel pathways that are involved in cell fate transitions using dedifferentiation of somatic stem cells to induced pluripotent stem cells as a case study.
PMCID: PMC3210035  PMID: 21636265
12.  Generation of Induced Pluripotent Stem Cells from the Prairie Vole 
PLoS ONE  2012;7(5):e38119.
The vast majority of animals mate more or less promiscuously. A few mammals, including humans, utilize more restrained mating strategies that entail a longer term affiliation with a single mating partner. Such pair bonding mating strategies have been resistant to genetic analysis because of a lack of suitable model organisms. Prairie voles are small mouse-like rodents that form enduring pair bonds in the wild as well as in the laboratory, and consequently they have been used widely to study social bonding behavior. The lack of targeted genetic approaches in this species however has restricted the study of the molecular and neural circuit basis of pair bonds. As a first step in rendering the prairie vole amenable to reverse genetics, we have generated induced pluripotent stem cell (IPSC) lines from prairie vole fibroblasts using retroviral transduction of reprogramming factors. These IPSC lines display the cellular and molecular hallmarks of IPSC cells from other organisms, including mice and humans. Moreover, the prairie vole IPSC lines have pluripotent differentiation potential since they can give rise to all three germ layers in tissue culture and in vivo. These IPSC lines can now be used to develop conditions that facilitate homologous recombination and eventually the generation of prairie voles bearing targeted genetic modifications to study the molecular and neural basis of pair bond formation.
PMCID: PMC3365000  PMID: 22675440
13.  microRNA induced transdifferentiation 
Recent months have seen rapid advances in the field of transdifferentiation, specifically in the conversion of fibroblasts to neurons. Most surprising is the observation that the ability to drive these transitions is not limited to transcription factors, but that they can be promoted by microRNAs as well. Indeed, in one case, microRNAs alone induced the transdifferentiation of fibroblasts to neuron-like cells, albeit at a low efficiency. Here, we review this rapidly advancing field, discuss possible mechanisms underlying microRNA-induced transdifferentiation and the potential for microRNAs to drive such transitions to any cell type of interest in vitro and in vivo.
PMCID: PMC3270586  PMID: 22312415
14.  Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients 
Cancer research  2010;71(2):550-560.
Recent prostate specific antigen (PSA) based screening trials indicate an urgent need for novel and non-invasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Non-coding microRNAs (miRNAs) in the serum and plasma have been shown to have potential as non-invasive markers for physiological and pathological conditions. To identify serum miRNAs that diagnose and correlate with prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR (qRT-PCR) method involving purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA - deficient) mouse ES cells (mESC) as the benchmark, we confirmed the validity of our technique, while uncovering a significant lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA (Cancer of the Prostate Risk Assessment) scores, we identified miRNA signatures that allow to diagnose cancer patients and correlate with prognosis. These serum signatures include oncogenic and tumor suppressive miRNAs suggesting functional roles in prostate cancer progression.
PMCID: PMC3022112  PMID: 21098088
microRNA; multiplex qRT-PCR; serum; prostate; cancer
15.  PML-RARα and Dnmt3a1 Cooperate in vivo to Promote Acute Promyelocytic Leukemia 
Cancer research  2010;70(21):8792-8801.
The PML-RARα oncogene is the central effector of acute promyelocytic leukemia (APL). PML-RARα physically interacts with epigenetic-modifying enzymes including DNA methyltransferases (Dnmt) to suppress critical downstream targets. Here, we show that increased expression of Dnmt3a1 cooperates with PML-RARα in vivo to promote early lethality secondary to myeloid expansion and dysfunction in primary mice. Bone marrow cells from these mice cause leukemogenesis with a shortened latency and a higher penetrance on transplantation into irradiated recipients. Furthermore, leukemic cells overexpressing PML-RARα and Dnmt3a1 display increased methylation at a target promoter compared with PML-RARα or Dnmt3a1 controls. Our findings show a cooperation between the PML-RARα oncogene and the Dnmt3a1 enzyme in vivo and that Dnmt levels can be rate limiting in APL progression.
PMCID: PMC3021794  PMID: 20861188
16.  Distinct requirements of microRNAs in NK cell activation, survival, and function 
MicroRNAs (miRNAs) are small noncoding RNAs that have recently emerged as critical regulators of gene expression within the immune system. In this study we used mice with conditional deletion of Dicer and Dgcr8 to dissect the roles of miRNAs in NK cell activation, survival, and function during viral infection. We developed a novel system for deletion of either Dicer or Dgcr8 in peripheral NK cells via drug-induced Cre activity. We found that Dicer- and Dgcr8- deficient NK cells were significantly impaired in survival and turnover, and had impaired function of the ITAM-containing activating NK cell receptors. We further demonstrated that both Dicer- and Dgcr8-dependent pathways were indispensable for the expansion of Ly49H+ NK cells during MCMV infection. Our data indicate similar phenotypes for Dicer- and Dgcr8- deficient NK cells, which strongly suggest that these processes are regulated by miRNAs. Thus, our findings indicate a critical role for miRNAs in controlling NK cell homeostasis and effector function, with implications for miRNAs regulating diverse aspects of NK cell biology.
PMCID: PMC2943981  PMID: 20805417
NK cells; microRNA; Dicer; Dgcr8; cytomegalovirus; NKG2D
17.  High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs 
The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.
PMCID: PMC3211115  PMID: 21847076
18.  MicroRNAs Control Hepatocyte Proliferation During Liver Regeneration 
Hepatology (Baltimore, Md.)  2010;51(5):1735-1743.
MicroRNAs (miRNAs) constitute a new class of regulators of gene expression. Among other actions, miRNAs have been shown to control cell proliferation in development and cancer. However, whether miRNAs regulate hepatocyte proliferation during liver regeneration is unknown. We addressed this question by performing 2/3 partial hepatectomy (2/3 PH) on mice with hepatocyte-specific inactivation of DiGeorge syndrome critical region gene 8 (DGCR8), an essential component of the miRNA processing pathway. Hepatocytes of these mice were miRNA-deficient and exhibited a delay in cell cycle progression involving the G1 to S phase transition. Examination of livers of wildtype mice after 2/3 PH revealed differential expression of a subset of miRNAs, notably an induction of miR-21 and repression of miR-378. We further discovered that miR-21 directly inhibits Btg2, a cell cycle inhibitor that prevents activation of forkhead box M1 (FoxM1), which is essential for DNA synthesis in hepatocytes after 2/3 PH. In addition, we found that miR-378 directly inhibits ornithine decarboxylase (Odc1), which is known to promote DNA synthesis in hepatocytes after 2/3 PH.
Our results show that miRNAs are critical regulators of hepatocyte proliferation during liver regeneration. Because these miRNAs and target gene interactions are conserved, our findings may also be relevant to human liver regeneration.
PMCID: PMC3108060  PMID: 20432256
19.  Monoallelic deletion of the microRNA biogenesis gene Dgcr8 produces deficits in the development of excitatory synaptic transmission in the prefrontal cortex 
Neural Development  2011;6:11.
Neuronal phenotypes associated with hemizygosity of individual genes within the 22q11.2 deletion syndrome locus hold potential towards understanding the pathogenesis of schizophrenia and autism. Included among these genes is Dgcr8, which encodes an RNA-binding protein required for microRNA biogenesis. Dgcr8 haploinsufficient mice (Dgcr8+/-) have reduced expression of microRNAs in brain and display cognitive deficits, but how microRNA deficiency affects the development and function of neurons in the cerebral cortex is not fully understood.
In this study, we show that Dgcr8+/- mice display reduced expression of a subset of microRNAs in the prefrontal cortex, a deficit that emerges over postnatal development. Layer V pyramidal neurons in the medial prefrontal cortex of Dgcr8+/- mice have altered electrical properties, decreased complexity of basal dendrites, and reduced excitatory synaptic transmission.
These findings demonstrate that precise microRNA expression is critical for the postnatal development of prefrontal cortical circuitry. Similar defects in neuronal maturation resulting from microRNA deficiency could represent endophenotypes of certain neuropsychiatric diseases of developmental onset.
PMCID: PMC3082233  PMID: 21466685
20.  miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN amplified neuroblastoma 
Nature medicine  2010;16(10):1134-1140.
Inactivation of the p53 tumor-suppressor pathway occurs in many human cancers, however some cancers such as neuroblastoma and normal stem cells maintain wild-type p53. Here we describe a microRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3′UTR. miR-380-5p is highly expressed in embryonic stem cells and neuroblastomas and high expression correlates with poor outcome in neuroblastomas with MYCN amplification. miR-380 overexpression cooperates with activated RAS to transform primary cells, form tumors in mice, and block oncogene induced senescence. In contrast, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of miR-380-5p targets including p53 and extensive apoptotic cell death. In vivo delivery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma.
PMCID: PMC3019350  PMID: 20871609
21.  MicroRNA Function Is Globally Suppressed in Mouse Oocytes and Early Embryos 
Current biology : CB  2010;20(3):271-277.
Dicer, which is required for the processing of both micro-RNAs (miRNAs) and small interfering RNAs (siRNAs), is essential for oocyte maturation [1, 2]. Oocytes express both miRNAs and endogenous siRNAs (endo-siRNAs) [3, 4]. To determine whether the abnormalities in Dicer knockout oocytes during meiotic maturation are secondary to the loss of endo-siRNAs and/or miRNAs, we deleted Dgcr8, which encodes an RNA-binding protein specifically required for miRNA processing. In striking contrast to Dicer, Dgcr8-deficient oocytes matured normally and, when fertilized with wild-type sperm, produced healthy-appearing offspring, even though miRNA levels were reduced to similar levels as Dicer-deficient oocytes. Furthermore, the deletion of both maternal and zygotic Dgcr8 alleles did not impair preimplantation development, including the determination of the inner cell mass and trophectoderm. Most surprisingly, the mRNA profiles of wild-type and Dgcr8 null oocytes were essentially identical, whereas Dicer null oocytes showed hundreds of misregulated transcripts. These findings show that miRNA function is globally suppressed during oocyte maturation and preimplantation development and that endo-siRNAs, rather than miRNAs, underlie the Dicer knockout phenotype in oocytes.
PMCID: PMC2872512  PMID: 20116247
22.  DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal 
Nature genetics  2007;39(3):380-385.
The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation.
PMCID: PMC3008549  PMID: 17259983
23.  Reprogramming Efficiency Following Somatic Cell Nuclear Transfer Is Influenced by the Differentiation and Methylation State of the Donor Nucleus 
Stem cells (Dayton, Ohio)  2006;24(9):2007-2013.
Reprogramming of a differentiated cell nucleus by somatic cell nuclear transplantation is an inefficient process. Following nuclear transfer, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modifications. These defects correlate with the low number of cloned embryos able to produce embryonic stem cells or develop into adult animals. Here, we show that the differentiation and methylation state of the donor cell influence the efficiency of genomic reprogramming. First, neural stem cells, when used as donors for nuclear transplantation, produce embryonic stem cells at a higher efficiency than blastocysts derived from terminally differentiated neuronal donor cells, demonstrating a correlation between the state of differentiation and cloning efficiency. Second, using a hypomorphic allele of DNA methyl-transferase-1, we found that global hypomethylation of a differentiated cell genome improved cloning efficiency. Our results provide functional evidence that the differentiation and epigenetic state of the donor nucleus influences reprogramming efficiency.
PMCID: PMC3000431  PMID: 16709876
Embryonic stem cells; DNA methylation; Nuclear transfer; Reprogramming
24.  Loss of cardiac microRNA-mediated regulation leads to dilated cardiomyopathy and heart failure 
Circulation research  2009;105(6):585-594.
Heart failure is a deadly and devastating disease that places immense costs on an aging society. In order to develop therapies aimed at rescuing the failing heart, it is important to understand the molecular mechanisms underlying cardiomyocyte structure and function.
microRNAs are important regulators of gene expression and we sought to define the global contributions made by microRNAs toward maintaining cardiomyocyte integrity.
First, we performed deep sequencing analysis to catalog the miRNA population in the adult heart. Secondly, we genetically deleted, in cardiac myocytes, an essential component of the machinery that is required to generate miRNAs. Deep sequencing of miRNAs from the heart revealed the enrichment of a small number of microRNAs with one, miR-1, accounting for 40% of all microRNAs. Cardiomyocyte-specific deletion of dgcr8, a gene required for microRNA biogenesis, revealed a fully penetrant phenotype that begins with left ventricular malfunction progressing to a dilated cardiomyopathy and premature lethality.
These observations reveal a critical role for microRNAs in maintaining cardiac function in mature cardiomyocytes and raise the possibility that only a handful of microRNAs maybe ultimately be responsible for the dramatic cardiac phenotype seen in the absence of dgcr8.
PMCID: PMC2828903  PMID: 19679836
25.  Opposing microRNA families regulate self-renewal in mouse embryonic stem cells 
Nature  2010;463(7281):621-626.
When embryonic stem cells (ESCs) differentiate, they must both silence the ESC self-renewal program as well as activate new tissue specific programs. In the absence of DGCR8 (Dgcr8 -/-), a protein required for microRNA (miRNA) biogenesis, mouse ESCs are unable to silence self-renewal. Here, we find that the introduction of let-7 miRNAs, a family of miRNAs highly expressed in somatic cells, can suppress self-renewal in Dgcr8 -/-, but not wild-type ESCs. Introduction of ESC cell cycle regulating (ESCC) miRNAs into the Dgcr8 -/- ESCs, blocks the capacity of let-7 to suppress self-renewal. Profiling and bioinformatic analyses show that let-7 inhibits while ESCC miRNAs indirectly activate numerous self-renewal genes. Furthermore, inhibition of the let-7 family promotes de-differentiation of somatic cells to induced pluripotent stem (iPS) cells. Together, these findings show how the ESCC and let-7 miRNAs act through common pathways to alternatively stabilize the self-renewing versus differentiated cell fates.
PMCID: PMC2894702  PMID: 20054295

Results 1-25 (32)