[Show abstract][Hide abstract] ABSTRACT: Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.
[Show abstract][Hide abstract] ABSTRACT: Background:
Molecular mediators influencing the transition from acute to persistent musculoskeletal pain following common stress exposures such as motor vehicle collision (MVC) remain poorly understood. In this exploratory, proof of concept study, we compared circulating microRNA (miRNA) expression profiles in the early aftermath of MVC among individuals who did and did not subsequently develop persistent pain. Blood RNA samples were obtained from African American individuals (n = 53) who presented to the emergency department after MVC and were discharged to home after evaluation. The presence or absence of severe pain in the axial region, the most common and morbid region in which post-MVC pain occurs, was assessed 6 weeks following MVC via standardized questionnaire. miRNA expression was determined using miRNA-sequencing; nonparametric analyses were used to compare miRNA expression levels among individuals with and without persistent pain.
Thirty-two mature miRNA were differentially expressed (p < 0.05) in those with and without severe axial pain at 6 weeks. miR-135a-5p, a regulator of the serotonin receptor that is known to be stress-responsive, differed most significantly between groups (p = 3 × 10(-4)). This miRNA, and miR-3613-3p (p = 0.001) survived correction for multiple testing (FDR = 0.15) in this small sample. Interestingly, differentially expressed miRNA were enriched for X chromosome location. In secondary analyses, the eight X chromosome miRNA were (a) more significantly associated with axial pain in women than men, (b) expressed more highly in the peripheral blood of women than men, and (c) predicted in pathway analyses (DIANA miRPath v 2.0) to regulate neuronal and neuroendocrine pathways previously implicated in various pain pathologies.
These results show that circulating miRNA predict persistent severe axial pain after MVC and suggest that they may be involved in the pathogenesis of post-traumatic musculoskeletal pain. However, further studies are needed to determine if these miRNA play a direct causal role.
[Show abstract][Hide abstract] ABSTRACT: Interest in RNA dysfunction in amyotrophic lateral sclerosis (ALS) recently aroused upon discovering causative mutations in RNA-binding protein genes. Here, we show that extensive down-regulation of miRNA levels is a common molecular denominator for multiple forms of human ALS. We further demonstrate that pathogenic ALS-causing mutations are sufficient to inhibit miRNA biogenesis at the Dicing step. Abnormalities of the stress response are involved in the pathogenesis of neurodegeneration, including ALS. Accordingly, we describe a novel mechanism for modulating microRNA biogenesis under stress, involving stress granule formation and re-organization of DICER and AGO2 protein interactions with their partners. In line with this observation, enhancing DICER activity by a small molecule, enoxacin, is beneficial for neuromuscular function in two independent ALS mouse models. Characterizing miRNA biogenesis downstream of the stress response ties seemingly disparate pathways in neurodegeneration and further suggests that DICER and miRNAs affect neuronal integrity and are possible therapeutic targets.
No preview · Article · Sep 2015 · The EMBO Journal
[Show abstract][Hide abstract] ABSTRACT: The RNA transcriptome varies in response to cellular differentiation as well as environmental factors, and can be characterized by the diversity and abundance of transcript isoforms. Differential transcription analysis, the detection of differences between the transcriptomes of different cells, may improve understanding of cell differentiation and development and enable the identification of biomarkers that classify disease types. The availability of high-throughput short-read RNA sequencing technologies provides in-depth sampling of the transcriptome, making it possible to accurately detect the differences between transcriptomes. In this article, we present a new method for the detection and visualization of differential transcription. Our approach does not depend on transcript or gene annotations. It also circumvents the need for full transcript inference and quantification, which is a challenging problem because of short read lengths, as well as various sampling biases. Instead, our method takes a divide-and-conquer approach to localize the difference between transcriptomes in the form of alternative splicing modules (ASMs), where transcript isoforms diverge. Our approach starts with the identification of ASMs from the splice graph, constructed directly from the exons and introns predicted from RNA-seq read alignments. The abundance of alternative splicing isoforms residing in each ASM is estimated for each sample and is compared across sample groups. A non-parametric statistical test is applied to each ASM to detect significant differential transcription with a controlled false discovery rate. The sensitivity and specificity of the method have been assessed using simulated data sets and compared with other state-of-the-art approaches. Experimental validation using qRT-PCR confirmed a selected set of genes that are differentially expressed in a lung differentiation study and a breast cancer data set, demonstrating the utility of the approach applied on experimental biological data sets. The software of DiffSplice is available at http://www.netlab.uky.edu/p/bioinfo/DiffSplice.
Full-text · Article · Nov 2012 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Primitive erythropoiesis defines the onset of hematopoiesis in the yolk sac of the early embryo and is initiated by the emergence of progenitors assayed as colony-forming cells (EryP-CFCs). EryP-CFCs are detected for only a narrow window during embryonic development, suggesting that both their initiation and termination are tightly controlled. Using the embryonic stem differentiation system to model primitive erythropoiesis, we found that miR-126 regulates the termination of EryP-CFC development. Analyses of miR-126 null embryos revealed that this miR also regulates EryP-CFCs in vivo. We identified vascular cell adhesion molecule-1 (Vcam-1) expressed by a mesenchymal cell population as a relevant target of miR-126. Interaction of EryP-CFCs with Vcam-1 accelerated their maturation to ßh1-globin(+) and Ter119(+) cells through a Src family kinase. These findings uncover a cell nonautonomous regulatory pathway for primitive erythropoiesis that may provide insight into the mechanism(s) controlling the developmental switch from primitive to definitive hematopoiesis.
No preview · Article · Jun 2012 · Developmental Cell
[Show abstract][Hide abstract] ABSTRACT: Lin28 is critical for stem cell maintenance and is also associated with advanced human malignancies. Our recent genome-wide studies mark Lin28 as a master post-transcriptional regulator of a subset of messenger RNAs important for cell growth and metabolism. However, the molecular basis underpinning the selective mRNA target regulation is unclear. Here, we provide evidence that Lin28 recognizes a unique motif in multiple target mRNAs, characterized by a small but critical 'A' bulge flanked by two G:C base pairs embedded in a complex secondary structure. This motif mediates Lin28-dependent stimulation of translation. As Lin28 is also known to inhibit the biogenesis of a cohort of miRNAs including let-7, we propose that Lin28 binding to different RNA types (precursor miRNAs versus mRNAs) may facilitate recruitment of different co-factors, leading to distinct regulatory outcomes. Our findings uncover a putative yet unexpected motif that may constitute a mechanistic base for the multitude of functions regulated by Lin28 in both stem cells and cancer cells.
Preview · Article · Dec 2011 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. An emerging mechanism to control miRNA production is the addition of an oligo-uridine tail to the 3' end of the precursor miRNA. This has been demonstrated for the Let-7 family of miRNAs in embryonic cells. Additionally, nontemplated nucleotides have been found on mature miRNA species, though in most cases it is not known if nucleotide addition occurs at the precursor step or at the mature miRNA. To examine the diversity of nucleotide addition we have developed a high-throughput sequencing method specific for miRNA precursors. Here we report that nontemplated addition is a widespread phenomenon occurring in many miRNA families. As previously reported, Let-7 family members are oligo-uridylated in embryonic cells in a Lin28-dependent manner. However, we find that the fraction of uridylated precursors increases with differentiation, independent of Lin28, and is highest in adult mouse tissues, exceeding 30% of all sequence reads for some Let-7 family members. A similar fraction of sequence reads are modified for many other miRNA families. Mono-uridylation is most common, with cytidine and adenosine modification less frequent but occurring above the expected error rate for Illumina sequencing. Nucleotide addition in cell lines is associated with 3' end degradation, in contrast to adult tissues, where modification occurs predominantly on full-length precursors. This work provides an unprecedented view of the complexity of 3' modification and trimming of miRNA precursors.
[Show abstract][Hide abstract] ABSTRACT: The miR-17~92 cluster is a potent microRNA-encoding oncogene. Here, we show that miR-17~92 synergizes with loss of Rb family members to promote retinoblastoma. We observed miR-17~92 genomic amplifications in murine retinoblastoma and high expression of miR-17~92 in human retinoblastoma. While miR-17~92 was dispensable for mouse retinal development, miR-17~92 overexpression, together with deletion of Rb and p107, led to rapid emergence of retinoblastoma with frequent metastasis to the brain. miR-17~92 oncogenic function in retinoblastoma was not mediated by a miR-19/PTEN axis toward apoptosis suppression, as found in lymphoma/leukemia models. Instead, miR-17~92 increased the proliferative capacity of Rb/p107-deficient retinal cells. We found that deletion of Rb family members led to compensatory up-regulation of the cyclin-dependent kinase inhibitor p21Cip1. miR-17~92 overexpression counteracted p21Cip1 up-regulation, promoted proliferation, and drove retinoblastoma formation. These results demonstrate that the oncogenic determinants of miR-17~92 are context-specific and provide new insights into miR-17~92 function as an RB-collaborating gene in cancer.
Full-text · Article · Aug 2011 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: The execution of apoptosis is critical for proper development of the nervous system. However, it is equally important that neurons strictly inhibit apoptosis after development to ensure their survival throughout the lifetime of the organism. Here we show that a microRNA, miR-29b, is markedly induced with neuronal maturation and functions as a novel inhibitor of neuronal apoptosis. The prosurvival function of miR-29b is mediated by targeting genes in the proapoptotic BH3-only family. Our results identify a unique strategy evolved by maturing neurons that uses a single microRNA to inhibit the multiple, redundant BH3-only proteins that are key initiators of apoptosis.
Preview · Article · Jan 2011 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: Metastasis in breast cancer carries a disproportionately worse prognosis than localized primary disease. To identify microRNAs (miRNA) involved in metastasis, the expression of 254 miRNAs was measured across the following cell lines using microarray analysis: MDA-MB-231 breast cancer cells, cells that grew as a tumor in the mammary fat pad of nude mice (TMD-231), metastatic disease to the lungs (LMD-231), bone (BMD-231) and adrenal gland (ADMD-231). A brain-seeking variant of this cell line (231-BR) was used additionally in validation studies. Twenty miRNAs were upregulated and seven were downregulated in metastatic cancer cells compared with TMD-231 cells. The expression of the tumor suppressor miRNAs let-7 and miR-22 was consistently downregulated in metastatic cancer cells. These metastatic cells expressed higher levels of putative/proven miR-22 target oncogenes ERBB3, CDC25C and EVI-1. Introduction of miR-22 into cancer cells reduced the levels of ERBB3 and EVI-1 as well as phospho-AKT, an EVI-1 downstream target. The miR-22 primary transcript is located in the 5'-untranslated region of an open reading frame C17orf91, and the promoter/enhancer of C17orf91 drives miR-22 expression. We observed elevated C17orf91 expression in non-basal subtype compared with basal subtype breast cancers. In contrast, elevated expression of EVI-1 was observed in basal subtype and was associated with poor outcome in estrogen receptor-negative breast cancer patients. These results suggest that metastatic cancer cells increase specific oncogenic signaling proteins through downregulation of miRNAs. Identifying such metastasis-specific oncogenic pathways may help to manipulate tumor behavior and aid in the design of more effective targeted therapies.
[Show abstract][Hide abstract] ABSTRACT: Imatinib, a BCR-Abl inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). Despite the success of imatinib, multiple mechanisms of resistance remain a problem, including overexpression of Lyn kinase (Lyn) and Bcl-2 family antiapoptotic proteins. Profiling micro-RNA (miRNA) expression in a model of Lyn-mediated imatinib-resistant CML (MYL-R) identified approximately 30 miRNAs whose expression differed >2-fold compared with drug-sensitive MYL cells. In particular, the expression of the miR181 family (a-d) was significantly reduced (∼11- to 25-fold) in MYL-R cells. Incubation of MYL-R cells with a Lyn inhibitor (dasatinib) or nucleofection with Lyn-targeting short interfering RNA increased miR181b and miR181d expression. A similar Lyn-dependent regulation of miR181b and miR181d was observed in imatinib-resistant K562 CML cells. Sequence analysis of potential targets for miR181 regulation predicted myeloid cell leukemia-1 (Mcl-1), a Bcl-2 family member whose expression is increased in MYL-R cells and drug-resistant leukemias. Inhibition of Lyn or rescue of miR181b expression reduced Mcl-1 expression in the MYL-R cells. To further investigate the mechanism of Mcl-1 repression by miR181, a luciferase reporter construct incorporating the Mcl-1 3'-untranslated region was tested. Overexpression of miR181b reduced luciferase activity, whereas these effects were ablated by the mutation of the seed region of the miR181 target site. Finally, stimulation of Lyn expression by 1,25-dihydroxyvitamin D(3) treatment in HL-60 cells, a cell model of acute myelogenous leukemia, decreased miR181b expression and increased Mcl-1 expression. In summary, our results suggest that Lyn-dependent regulation of miR181 is a novel mechanism of regulating Mcl-1 expression and cell survival.
Full-text · Article · Nov 2010 · Molecular pharmacology
[Show abstract][Hide abstract] ABSTRACT: microRNAs are small regulatory RNAs that are processed from larger, genomically encoded transcripts. While the biochemical mechanism underlying microRNA processing is well understood, it was recently discovered that processing of one developmentally crucial group of microRNAs, the Let-7 family, is blocked by the protein Lin-28 in embryonic cells. This novel regulation of microRNA biogenesis may be very important for the maintenance of embryonic stem cell pluripotency as well as for the reprogramming of somatic cells to induce pluripotent stem cells. The studies leading to the discovery of the Let-7 block by Lin-28 and questions regarding the biochemical mechanism behind Lin-28-mediated microRNA silencing are discussed.
Full-text · Article · Aug 2010 · The international journal of biochemistry & cell biology
[Show abstract][Hide abstract] ABSTRACT: Recent studies have shown that microRNAs (miRNAs) play roles in tumorigenesis and are reliable classifiers of certain cancer types and subtypes. However, the role of miRNAs in the pathogenesis and diagnosis of small cell carcinoma (SCLC), the majority of which represent the most aggressive lung tumors, has not been investigated.
In order to explore miRNA involvement in the pathogenesis of small cell lung carcinoma (SCLC) and the potential role of miRNAs in SCLC diagnosis, we compared the miRNA expression profile of a set of SCLC cell lines to that of a set of non-small cell lung cancer (NSCLC) cell lines and normal immortalized human bronchial epithelial cells (HBECs) using microarray analysis.
Our results show that miRNA profiles reliably distinguish SCLC cell lines from NSCLC and HBEC cell lines. Further analysis of the miRNA expression profile of the two subtypes of lung cancer cell lines indicates that the expression levels of the majority of the miRNAs that are differentially expressed in SCLC cells relative to NSCLC cells and HBECs show a progressive trend from HBECs to NSCLC cells to SCLC cells.
The distinctive miRNA expression signature of SCLCs relative to NSCLCs and HBECs suggests that miRNA profiles have the potential to serve as a diagnostic marker of SCLC lung tumors. The progressive trend of miRNA profile changes from HBECs to NSCLCs to SCLCs suggests a possible pathological relationship between SCLCs and NSCLCs, and suggests that the increasing dysregulation of miRNA expression may play a role in lung tumor progression. The specific role of these miRNAs in lung tumor pathogenesis and differentiation need to be investigated further in future studies.
Full-text · Article · Jun 2010 · Journal of Experimental & Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) modulate a broad range of gene expression patterns during development and tissue homeostasis, and in the pathogenesis of disease. The exquisite spatio-temporal control of miRNA abundance is made possible, in part, by regulation of the miRNA biogenesis pathway. In this review, we discuss two emerging paradigms for post-transcriptional control of miRNA expression. One paradigm centers on the Microprocessor, the protein complex essential for maturation of canonical miRNAs. The second paradigm is specific to miRNA families, and requires interaction between RNA-binding proteins and cis-regulatory sequences within miRNA precursor loops.
Full-text · Article · Jun 2010 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: In an effort to understand the potential role of microRNAs (miRNAs) in mammary-gland stem or progenitor cells, miRNA microarrays were performed on subpopulations of the mouse mammary epithelial cell (MEC) line COMMA-DbetaGeo. This cell line contains a heterogeneous subpopulation of progenitors characterized by the expression of stem cell antigen 1 (Sca-1; encoded by Ly6a). Microarray analysis indicated that the Sca-1 subpopulations have distinct miRNA expression profiles. Functional studies were performed on miR-205, which was highly expressed in the Sca-1-positive (Sca-1(+)) cells. When miR-205 was overexpressed in vitro, the COMMA-DbetaGeo cells underwent several significant morphological and molecular changes. miR-205 overexpression led to an expansion of the progenitor-cell population, decreased cell size and increased cellular proliferation. In addition, the colony-forming potential of the two Sca-1 subpopulations was increased. Target prediction for miR-205 indicated that it might regulate the expression of the tumor-suppressor protein PTEN. Overexpression studies using reporter constructs confirmed that PTEN expression is regulated by miR-205. In addition to PTEN, several other putative and previously validated miR-205 targets were identified by microarray analysis, including the previously reported miR-205 targets ZEB1 and ZEB2. Additionally, in normal mouse MECs, high expression of miR-205 was observed in stem-cell-enriched cell populations isolated by FACS using established cell-surface markers.
Preview · Article · Feb 2010 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: The apicomplexan parasite Toxoplasma gondii can infect and replicate in virtually any nucleated cell in many species of warm-blooded animals; thus, it has evolved the ability to exploit well-conserved biological processes common to its diverse hosts. Here we have investigated whether Toxoplasma modulates the levels of host microRNAs (miRNAs) during infection.
Using microarray profiling and a combination of conventional molecular approaches we report that Toxoplasma specifically modulates the expression of important host microRNAs during infection. We show that both the primary transcripts for miR-17 approximately 92 and miR-106b approximately 25 and the pivotal miRNAs that are derived from miR-17 approximately 92 display increased abundance in Toxoplasma-infected primary human cells; a Toxoplasma-dependent up-regulation of the miR-17 approximately 92 promoter is at least partly responsible for this increase. The abundance of mature miR-17 family members, which are derived from these two miRNA clusters, remains unchanged in host cells infected with the closely related apicomplexan Neospora caninum; thus, the Toxoplasma-induced increase in their abundance is a highly directed process rather than a general host response to infection.
Altered levels of miR-17 approximately 92 and miR-106b approximately 25 are known to play crucial roles in mammalian cell regulation and have been implicated in numerous hyperproliferative diseases although the mechanisms driving their altered expression are unknown. Hence, in addition to the implications of these findings on the host-pathogen interaction, Toxoplasma may represent a powerful probe for understanding the normal mechanisms that regulate the levels of key host miRNAs.