Cited by 1,578MicroRNA in disease regulationDevelopmental Biology and Gene RegulationNeurogenesis and neuroplasticity mechanisms

The DOI matches its Crossref record; 50 of the 51 references it cites were found in a registry or an index; 20 of the 42 sources its claims cite were checked for support.

Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation

Open access

Abstract

BACKGROUND: The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18 to 25 nucleotides) with probable roles in the regulation of gene expression. In Caenorhabditis elegans, lin-4 and let-7 miRNAs control the timing of fate specification of neuronal and hypodermal cells during larval development. lin-4, let-7 and other miRNA genes are conserved in mammals, and their potential functions in mammalian development are under active study. RESULTS: In order to identify mammalian miRNAs that might function in development, we characterized the expression of 119 previously reported miRNAs in adult organs from mouse and human using northern blot analysis. Of these, 30 miRNAs were specifically expressed or greatly enriched in a particular organ (brain, lung, liver or skeletal muscle). This suggests organ- or tissue-specific functions for miRNAs. To test if any of the 66 brain-expressed miRNAs were present in neurons, embryonal carcinoma cells were treated with all-trans-retinoic acid to promote neuronal differentiation. A total of 19 brain-expressed miRNAs (including lin-4 and let-7 orthologs) were coordinately upregulated in both human and mouse embryonal carcinoma cells during neuronal differentiation. The mammalian ortholog of C. elegans lin-28, which is downregulated by lin-4 in worms via 3' untranslated region binding, was also repressed during neuronal differentiation of mammalian embryonal carcinoma cells. Mammalian lin-28 messenger RNAs contain conserved predicted binding sites in their 3' untranslated regions for neuron-expressed miR-125b (a lin-4 ortholog), let-7a, and miR-218. CONCLUSIONS: The identification of a subset of brain-expressed miRNAs whose expression behavior is conserved in both mouse and human differentiating neurons implicates these miRNAs in mammalian neuronal development or function.

Overview

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Written from the paper’s full text.

TL;DR

Northern blot profiling of 119 mammalian microRNAs in adult mouse and human organs identified conserved organ-specific and organ-enriched expression, including a prominent brain-expressed subset. Several brain-expressed miRNAs were induced during retinoic-acid-driven neuronal differentiation of mouse P19 and human NT2/D1 embryonal carcinoma cells, and LIN-28 was downregulated with conserved predicted miRNA binding sites.

Key findings

  • Of 119 miRNAs analyzed, 92 were previously cloned and 27 were computationally predicted; 21 of 27 predicted miRNAs were detected by northern blot. Seventeen miRNAs were detected exclusively in one mouse organ, including seven brain-specific miRNAs (miR-9, -124a, -124b, -135, -153, -183, -219), six lung-specific, two spleen-specific, one liver-specific, and one heart-specific miRNA. Among 75 miRNAs detected in two or more mouse organs, 14 were enriched at least two-fold in one organ, including seven brain-enriched miRNAs (miR-9*, -125a, -125b, -128, -132, -137, -139), three skeletal muscle-enriched, two kidney-enriched, and one spleen-enriched miRNA.
  • Hierarchical clustering identified at least six major groups of miRNAs with similar expression profiles, including a group of 17 miRNAs expressed in mouse and human brain; about 50% of expressed miRNAs were assigned to a specific or restricted set of organs.
  • Retinoic acid treatment induced 19 miRNAs in both P19 and NT2/D1 cells, including four brain-specific, four brain-enriched, and 11 brain-nonenriched miRNAs; induction in P19 cells typically occurred by day four after RA treatment.
  • LIN-28 protein levels began to decrease around day five and almost disappeared by day eight in P19 cells; in NT2/D1 cells, LIN-28 decreased slightly by day 14 and was still detectable at day 21. This kinetics paralleled upregulation of miR-125b, let-7a, and miR-218, and mouse and human Lin-28 mRNAs contain conserved predicted binding sites for these miRNAs.

Methods

  • Northern analysis of 119 miRNAs using StarFire probes on total RNA from adult mouse organs (six-week-old C57BL/6 mice, pooled organs from two to four mice) and human organs, plus P19 and NT2/D1 embryonal carcinoma cells treated with 1 μM and 10 μM retinoic acid, respectively.
  • Northern blot signals were quantified with ImageQuant and clustered with GeneTraffic 6.2 using Pearson hierarchical clustering; western analysis was used to measure LIN-28 protein.
  • miRNA::target mRNA predictions were made by testing 20-nucleotide 3′ UTR segments for RNA secondary structure with the RNAfold program of the Vienna RNA package, applying binding, free-energy, and mouse-human conservation filters and a numerical scoring formula.

Limitations

  • Stated: some cloned and predicted miRNAs were not detected, possibly because of low expression, expression limited to a small number of cells or a narrow developmental window, or lack of expression in the organs examined; the organ panel did not include all organs and tissues from which miRNAs were cloned, and human kidney, lung, and spleen were not examined.
  • Stated: glial expression was not directly examined, so the possibility that RA-induced miRNAs were also expressed in glial cells could not be eliminated; a luciferase reporter fused to the mouse Lin-28 3′ UTR was not downregulated in differentiating P19 cells, and endogenous Lin-28 mRNA decreased, suggesting that repression of native Lin-28 may be largely transcriptional and that miRNA regulation may be auxiliary.
  • Evident: the miRNA target predictions are computational and were not experimentally validated by loss-of-function experiments; the paper notes that miRNA loss-of-function experiments in these EC cells could prove useful for understanding their regulatory contribution.

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Integrity signals

Registry

Crossref · 10.1186/gb-2004-5-3-r13

Cites

50 of 50 references verified0 citation errors · 0 not checkable

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Claims the paper makes

37 Own result · 19 Own interpretation · 11 No full text of the sources · 6 Partly supported · 4 Own method · 3 Supported · 3 Not checked yet · 2 Supported so far · 2 Sources not found · accuracy not yet measured

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  • It may state two separate claims as one. 28
  • Probably not a substantial claim. 13
  • The quote only partly states this claim. 1
34 of 34
  • The paper relies on this without support.

    Closely linked miRNAs arranged in tandem often exhibit similar expression profiles, consistent with shared cis-regulatory elements and possibly a polycistronic precursor transcript.

    “Closely linked miRNAs that are arranged in tandem often exhibit similar expression profiles, consistent with shared cis-regulatory elements, and possibly a polycistronic precursor transcript.”
  • The paper relies on this without support.

    miRNAs with evolutionarily conserved roles in neurons should be induced during neuronal differentiation of both mouse and human EC cells.

    “We reasoned that miRNAs with evolutionarily conserved roles in neurons should be induced during neuronal differentiation of both mouse and human EC cells.”
  • The paper relies on this without support.

    The induction of miRNAs in RA-treated EC cells reflects their expression during neuronal differentiation in vivo.

    “To determine which of brain-expressed miRNAs were associated with neuronal differentiation, we examined their expression during neuronal differentiation of EC cells.”
3 of 3

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Citations

Cites

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Research works printed but not linked: 2

  1. Chen X: A microRNA as a translational repressor of APETALA2 inArabidopsisflower development.Science 2003, 101126/science.1088060.
  2. National Center for Biotechnology Information[http://www.ncbi.nlm.nih.gov/]

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Metadata

Authors
Sempere, L. F., Freemantle, S., Pitha-Rowe, I., Moss, E., Dmitrovsky, E., & Ambros, V.
Affiliations
Dartmouth College (US)
Corresponding author
Ambros, Victor
Venue
Genome Biology
Published
2004-02-16
Volume
5
Issue
3
Pages
R13-R13
ISSN
  • 1465-6906 (ISSN-L)
  • 1474-760X
OpenAlex
W2138954682
Cited by
1,578
References
51
Citations by year
2026: 13 · 2025: 33 · 2024: 36 · 2023: 39 · 2022: 55 · 2021: 68 · 2020: 56 · 2019: 62 · 2018: 65 · 2017: 62 · 2016: 78 · 2015: 89 · 2014: 106 · 2013: 108 · 2012: 90
FWCI
29.01
Type
journal article
Language
en
Open access
Locations
  • Genome biology (publishedVersion)
  • PubMed Central (publishedVersion, other-oa)
  • PubMed (publishedVersion)
Funders
  • Lance Armstrong Foundation
  • Armstrong Foundation
  • National Institutes of Health
  • National Cancer Institute (R01CA062275, T32CA009658, R01CA087546)
  • National Institute of General Medical Sciences (R01GM034028)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
Keywords
microRNA, neuronal differentiation, lin-4, let-7 microRNA, Lin28, miR-125b, miR-218, embryonal carcinoma cells, all-trans retinoic acid, 3' untranslated region, tissue-specific gene expression, neuronal development, miRNA expression profiling
MeSH terms
Adult, Animals, Brain, Cell Differentiation, Cell Line, Embryo, Mammalian, Humans, Genetic Linkage, Mice, Inbred C57BL, Neurons, Repressor Proteins, RNA, Messenger, Sequence Homology, Nucleic Acid, Stem Cells, Translocation, Genetic, Tretinoin, Down-Regulation, Gene Expression Regulation, Neoplastic, Carcinoma, Embryonal, Gene Expression Regulation, Developmental, Gene Expression Profiling, Caenorhabditis elegans Proteins, MicroRNAs, Cell Line, Tumor, Mice, Genes, Neoplasm

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