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Cited by 41MicroRNA in disease regulationNeurogenesis and neuroplasticity mechanismsGlioma Diagnosis and Treatment

MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation.

Open access
Howe, James R., Li, Emily S., Streeter, Sarah E., Rahme, Gilbert J., Chipumuro, Edmond, Russo, Grace B., Litzky, Julia F., Hills, L. Benjamin +3 more·2017·PLoS ONE
DOI 10.1371/journal.pone.0177661PMC PMC5426787
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Abstract

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.

AI snapshot

Written from the paper’s full text.

TL;DR

miR-338-3p is highly expressed in the adult mouse dentate gyrus and increases with neuronal maturity, where it regulates granule-cell dendrite number/orientation. Its knockdown causes abnormal neuronal morphology and in vivo neoplasia, while overexpression suppresses glioblastoma cell proliferation and low expression is associated with worse GBM patient survival.

Key findings

  • In adult mouse dentate gyrus, miR-338-3p was among the most highly expressed of 10 assayed miRNAs: mean mCherry sensor expression was 3.68 ± 1.04% (n = 4) of cells versus GFP control, compared with miR-137-3p at 3.11 ± 0.53% (n = 3).
  • miR-338-3p sensor labeling decreased with maturity markers relative to control: 1.69 ± 0.27-fold decrease in NeuN co-labeling (p < 0.05) and 3.01 ± 1.61-fold decrease in DCX co-labeling (p < 0.01), which the authors interpret as highest expression in mature neurons.
  • miR-338-3p knockdown altered granule neuron morphology: mean branching angle was 155.8 ± 1.7° versus 163.9 ± 2.2° in controls (mean difference 8.0 ± 2.8°, p < 0.01), and primary dendrites/neuron increased to 1.28 ± 0.04 versus 1.07 ± 0.04 (p < 0.001).
  • Knockdown produced cellular neoplasia in vivo (6/6 lentiviral sponge+sensor mice at 7 DPI; no neoplasia with miR-132-3p sponge, n = 4), and miR-338-3p overexpression slowed GBM cell proliferation: U251 18.7 ± 1.55 × 10^5 vs 34.1 ± 4.95 × 10^5 control cells at 12 DPI (doubling times 19.3 vs 16.9 h); SF295 7.525 ± 0.275 × 10^5 vs 13.05 ± 1.38 × 10^5 at 10 DPI (14.7 vs 12.6 h; p < 0.0001). In TCGA, low miR-338-3p in 157 GBM patients was associated with decreased overall and disease-free survival.

Methods

  • In vivo: lentiviral sensors for 10 miRNAs and a miR-338-3p sponge/overexpressor were injected into C57BL/6J mice; adults (7–8 weeks) were used for expression/knockdown and neonates (postnatal day 7) for retroviral morphology. Immunohistochemistry used Nestin, DCX, NeuN, GFAP, and BrdU; morphology was analyzed with ImageJ/NeuroLucida.
  • In vitro: U251 and SF295 GBM cell lines were infected with miR-338-3p overexpressor or control virus at MOI 20; proliferation was counted every 24 h after 7 days post-infection.
  • Analysis: one-way ANOVA with Tukey post-hoc, t-tests, Poisson regression/Pearson chi-squared for proliferation, and log-rank tests for TCGA survival; TCGA low expression was defined as Z ≤ -0.4.

Limitations

  • Stated: the mechanisms by which miR-338-3p regulates neuronal maturation, proliferation, and tumor suppression are not fully characterized; the observed cell-nonautonomous proliferation pattern is unexpected and warrants further study.
  • Stated: the discrepancy with Wang et al. (no effect on U251 proliferation) may reflect different time courses (96 h vs 7 days) and vector designs.
  • Evident: the TCGA finding is correlational, not causal; in vitro proliferation uses only two GBM lines; in vivo morphology and neoplasia rely on viral manipulation in mice with small sample sizes; sex differences were not analyzed despite mixed-sex groups.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0177661

Cites

54 of 54 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 61 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    miR-338-3p knockdown alters the branching angle of primary dendrites in granule neurons.

    “The granule cells expressing only the control vector had a mean branching angle of 163.9 ± 2.2° (n = 5), while the granule cells expressing the miR-338-3p sponge had a mean branching angle 155.8 ± 1.7° (n = 5), with a mean difference in branching angle of 8.0 ± 2.8°, indicating a significant deviation from the mean branching angle in miR-338-3p knockdown neurons (Fig 4D, S5 Table; p<0.01).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The miR-338-3p sponge specifically knocks down miR-338-3p without affecting other miRNAs or cellular processes.
    • Rests on an assumption: Retroviral infection specifically labels actively proliferating cells that become dentate gyrus granule neurons.
  • Findingown data

    Few cells expressing the miR-338-3p sponge were BrdU-positive in observed neoplasms.

    “In observed neoplasms, few cells expressing the miR-338-3p sponge were BrdU-positive.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: BrdU incorporation accurately reflects cellular proliferation in this tissue.
  • Findingown data

    When mice are injected with a virus expressing both the miR-338-3p sensor and the sponge, 101.5 ± 6.57 cells/section were labeled.

    “In contrast, when mice are injected with a virus expressing both the miR-338-3p sensor and the sponge, 101.5 ± 6.57 cells/section (n = 8) were labeled (S3 Table).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The level of mCherry fluorescence from the lentiviral sensor quantitatively reflects the endogenous level or activity of the target miRNA.
  • Findingown data

    miR-338-3p is among the most highly expressed of 10 assayed miRNAs in the dentate gyrus of adult mice.

    “In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus.”
    • Also stated in: § MiR-338-3p is expressed at high levels in the dentate gyrus · plain
      “We determined miR-137-3p and mir-338-3p were the most highly-expressed assayed miRNAs in the dentate gyrus, based on mean mCherry expression levels of 3.11 ± 0.53% (n = 3) and 3.68 ± 1.04% (n = 4) of cells, respectively, compared to GFP (Fig 1E, S1 Table).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The level of mCherry fluorescence from the lentiviral sensor quantitatively reflects the endogenous level or activity of the target miRNA.
  • Findingown data

    Low miR-338-3p expression is associated with decreased overall and disease-free survival in glioblastoma patients.

    “Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma.”
    • Also stated in: § MiR-338-3p expression is associated with overall and disease-free survival in GBM patients · plain
      “Using data from 157 GBM patients with miRNA expression profiling of their tumors, we show that low expression of miR-338-3p corresponded to a decrease in overall and disease-free survival (Fig 8).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The TCGA GBM patient data are representative of glioblastoma patients generally.
  • May combine two claims. 29
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 33

  • May combine two claims.

    Findingown data

    The majority of labeled cells were either infected by the sponge or co-infected with both the sponge and the GFP control, and only a small minority was infected solely with the GFP control.

    “The majority of labeled cells were either infected by the sponge or co-infected with both the sponge and the GFP control, and only a small minority was infected solely with the GFP control (Fig 4A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    NeuN was observed at a much lower level than in the uninfected neurons surrounding the neoplasm, but some NeuN-positive cells were sporadically observed within regions of neoplasia.

    “NeuN was observed at a much lower level than in the uninfected neurons surrounding the neoplasm. However, we did sporadically observe some NeuN-positive cells within regions of neoplasia (Fig 5C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The miR-338-3p sensor labels 3.0 ± 2.19 cells/section when infected alone, while control mCherry labels 182.1 ± 12.81 cells/section.

    “When infected with the miR-338-3p sensor, the average number of cells labeled per tissue section is 3.0 ± 2.19 cells/section (n = 6), while an equal titer of a control virus expressing only mCherry labeled 182.1 ± 12.81 cells/section, (n = 8) indicating strong regulation of the sensor by endogenous miR-338-3p (S3 Table).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The level of mCherry fluorescence from the lentiviral sensor quantitatively reflects the endogenous level or activity of the target miRNA.

  • May combine two claims.

    Findingown data

    Qualitatively, Nestin expression seemed to be almost entirely localized to the neoplasm's margins, while mostly absent from the neoplasm's center.

    “Qualitatively, Nestin expression seemed to be almost entirely localized to the neoplasm’s margins, while mostly absent from the neoplasm’s center (Fig 5A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Most cells labeled by the mCherry control were also labeled with the miR-338-3p sponge virus, but the control virus labeled a higher proportion of mature-appearing granule neurons compared to the sponge.

    “We found that most cells labeled by the mCherry control were also labeled with the miR-338-3p sponge virus, but the control virus labeled a higher proportion of mature-appearing granule neurons compared to the sponge (Fig 3C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The U251 and SF295 cells highly expressed mCherry compared to GFP when co-infected with miR-338-3p sensor and control, indicating low endogenous miR-338-3p expression.

    “The cells highly expressed mCherry compared to GFP, indicating low endogenous miR-338-3p expression (Fig 7B and 7D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The level of mCherry fluorescence from the lentiviral sensor quantitatively reflects the endogenous level or activity of the target miRNA.

  • May combine two claims.

    Findingown data

    The majority of neurons with multiple primary dendrites had two, a minority had three, but none had four or greater.

    “The majority of neurons with multiple primary dendrites had two, a minority had three, but none had four or greater.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    miR-338-3p knockdown does not significantly change dendritic spine density, length, head area, soma size/length, dendritic nodes, termini, total dendrite length, mean dendrite length, or Sholl analyses.

    “We did not observe any statistically significant changes in dendritic spine density, length, or head area (p>0.05, S7 Table).”
    • Also stated in: § Granule cell morphology is altered by miR-338-3p knockdown · plain
      “Analyses of soma size and length, number of dendritic nodes, number of dendritic termini, total dendrite length, and mean dendrite length all returned non-significant results (p>0.05, S8 Table).”
    • Also stated in: § Granule cell morphology is altered by miR-338-3p knockdown · plain
      “Sholl analyses of dendrite intersections and dendrite length yielded similarly non-significant results as well (p>0.05, S8 Table).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Most BrdU-positive cells in the neoplasm are on the edge of the neoplasm, with few in histologically normal regions.

    “We found most BrdU-positive cells on the edge of the neoplasm, with few in histologically normal regions, suggesting the cells on the margins of the neoplasm, adjacent to the sponge-infected cells on the margin, proliferate at an abnormally high rate (Fig 6A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: BrdU incorporation accurately reflects cellular proliferation in this tissue.

  • May combine two claims.

    Findingown data

    The two most obvious differences between wild type and miR-338-3p-deficient neurons are their increased likelihood to sprout multiple primary dendrites and subsequently branch off at deviant angles with respect to the soma.

    “The two most obvious differences between wild type and miR-338-3p-deficient neurons are their increased likelihood to sprout multiple primary dendrites and subsequently branch off at deviant angles with respect to the soma.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    The miR-338-3p sensor expression in DCX-positive cells is decreased 3.01-fold compared to control.

    “Of all cells expressing the miR-132-3p sensor, there was a 2.30 ± 1.16-fold decrease (p<0.05) in co-labeling of DCX and the sensor compared to the control, and a 3.01 ± 1.61-fold decrease (p<0.01) in the miR-338-3p sensor-expressing neurons (Fig 2I).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The level of mCherry fluorescence from the lentiviral sensor quantitatively reflects the endogenous level or activity of the target miRNA.
    • Rests on an assumption: Normalization to the mCherry control corrects for differential tropism biases in sensor expression across cell types.

  • May combine two claims.

    Findingown data

    BrdU-positive cells tended to co-express GFAP, but not all BrdU-positive cells were GFAP-positive, and these BrdU-expressing cells were a minority of GFAP-positive cells overall.

    “These BrdU-positive cells tended to co-express GFAP as well, but not all BrdU-positive cells were also GFAP-positive, and these BrdU-expressing cells were a minority of GFAP-positive cells overall (Fig 6B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: BrdU incorporation accurately reflects cellular proliferation in this tissue.

  • May combine two claims.

    Findingown data

    When GBM lack miR-338-3p, they become more aggressive and more lethal.

    “Our analysis of the TCGA database indicates that when GBM lack miR-338-3p, they become more aggressive and more lethal.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Not every neoplastic cell is miR-338-3p deficient; the core lacks miR-338-3p while cells at the periphery of the neoplasm maintain relatively normal levels of miR-338-3p expression.

    “Upon histological examination of the tumor, it is clear that not every neoplastic cell is miR-338-3p deficient, but rather the core lacks miR-338-3p while cells at the periphery of the neoplasm maintain relatively normal levels of miR-338-3p expression.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-338-3p regulates axonal outgrowth in cortical neurons through regulation of Robo2.

    “MiR-338-3p regulates axonal outgrowth in cortical neurons through regulation of Robo2, an axonal guidance protein [29].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] A Kos, 2014, MicroRNA-338 Attenuates Cortical Neuronal Outgrowth by Modulating the Expression of Axonal Guidance Genes, Mol Neurobiol

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    The process of neuronal polarization begins very early in the maturing neuron's life, which is when miR-338-3p expression is increasing.

    “The process of neuronal polarization begins very early in the maturing neuron’s life, which is when miR-338-3p expression is increasing [27].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] A Gartner, 2014, Distinct temporal hierarchies in membrane and cytoskeleton dynamics precede the morphological polarization of developing neurons, J Cell Sci source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    miRNAs play a key role in regulating neuronal gene expression, which can impact neurogenesis in the dentate gyrus.

    “Recent studies show microRNAs (miRNAs) play a key role in regulating neuronal gene expression, which can impact neurogenesis in the dentate gyrus [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] P Lau, 2010, MicroRNAs in neural cell differentiation, Brain Res source

  • May combine two claims.

    Study design and conductreasoning

    The lentiviral sensor system detects miRNAs via binding to complementary mRNA sequences, blocking translation and reducing mCherry fluorescence.

    “We designed a lentiviral sensor system to detect the miRNAs of interest via their binding to complementary mRNA sequences, which blocks translation.”
    • Also stated in: § MiR-338-3p is expressed at high levels in the dentate gyrus · plain
      “Thus, endogenous miRNAs will bind the mCherry transcript’s target sequences, blocking its translation and reducing the level of mCherry fluorescence in cells expressing the miRNA of interest.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The level of mCherry fluorescence from the lentiviral sensor quantitatively reflects the endogenous level or activity of the target miRNA.

  • May combine two claims.

    Study design and conductown data

    The miR-338-3p sponge increases the number of labeled cells compared to sensor alone, showing effective knockdown.

    “This increase in the number of labeled cells infected with the miR-338-3p sponge shows the sponge effectively knocks down endogenous miR-338-3p.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The miR-338-3p sponge specifically knocks down miR-338-3p without affecting other miRNAs or cellular processes.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Neurogenesis in the dentate gyrus has been linked to learning, memory, conditioning, and pattern separation.

    “Neurogenesis in the dentate gyrus has been linked to numerous phenomena, including but not limited to learning, memory, conditioning, and pattern separation [3].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] JB Aimone, 2014, Regulation and function of adult neurogenesis: from genes to cognition, Physiol Rev source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Successful integration of neurons into existing neurocircuits depends on the level of stimulation they receive.

    “Once these neurons begin forming synapses, successful integration into existing neurocircuits also depends on the level of stimulation they receive [18, 19].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [18] NR Campbell, 2010, Endogenous signaling through alpha7-containing nicotinic receptors promotes maturation and integration of adult-born neurons in the hippocampus, J Neurosci source
    • cites [19] S Ge, 2008, Synaptic integration and plasticity of new neurons in the adult hippocampus, J Physiol source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Some animal models of depression demonstrate reduced dentate gyrus neurogenesis; neurogenesis is necessary for certain antidepressants to be effective.

    “Some animal models of depression demonstrate reduced dentate gyrus neurogenesis; neurogenesis is necessary for certain antidepressants to be effective [4, 5].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] L Santarelli, 2003, Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants, Science source
    • cites [5] H Chen, 2006, Hippocampal cell proliferation regulation by repeated stress and antidepressants, Neuroreport source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-137 inhibits synaptic integration and dendritic spine growth when knocked down.

    “These miRNAs include miR-132, which is most highly expressed in mature neurons, and miR-137, which inhibits synaptic integration and dendritic spine growth when knocked down [12, 13].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [12] RD Smrt, 2010, MicroRNA miR-137 regulates neuronal maturation by targeting ubiquitin ligase mind bomb-1, Stem Cells source

      Not checked in the free run.

    • cites [13] BW Luikart, 2011, miR-132 Mediates the Integration of Newborn Neurons into the Adult Dentate Gyrus, PLoS ONE source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Proliferation of newborn neurons in the dentate gyrus increases in response to direct hippocampal stimulation and processes stimulating the hippocampus, such as exercise, spatial learning, and an enriched environment.

    “Proliferation of newborn neurons in the dentate gyrus increases in response to direct hippocampal stimulation as well as processes stimulating the hippocampus, such as exercise, spatial learning, and an enriched environment [14–16].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [14] J Brown, 2003, Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis, Eur J Neurosci source
    • cites [15] SS Stone, 2011, Stimulation of entorhinal cortex promotes adult neurogenesis and facilitates spatial memory, J Neurosci source

      Queued — the background check reaches it, usually within a day.

    • cites [16] E Gould, 1997, Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation, J Neurosci source

      Queued — the background check reaches it, usually within a day.

  • Classified as probably not a substantial claim.

    Premisecitation

    Earlier studies identified 10 activity-dependent miRNAs significantly upregulated following pilocarpine-induced seizures.

    “Earlier studies identified 10 activity-dependent miRNAs significantly upregulated following pilocarpine-induced seizures [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] BW Luikart, 2012, Dentate gyrus neurogenesis, integration and microRNAs, Behav Brain Res source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-132 is most highly expressed in mature neurons.

    “These miRNAs include miR-132, which is most highly expressed in mature neurons, and miR-137, which inhibits synaptic integration and dendritic spine growth when knocked down [12, 13].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [12] RD Smrt, 2010, MicroRNA miR-137 regulates neuronal maturation by targeting ubiquitin ligase mind bomb-1, Stem Cells source

      Not checked in the free run.

    • cites [13] BW Luikart, 2011, miR-132 Mediates the Integration of Newborn Neurons into the Adult Dentate Gyrus, PLoS ONE source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Failure of neurons to maintain the G0 phase is associated with neurodegenerative diseases such as Alzheimer's and Parkinson's disease.

    “Failure of neurons to maintain the G0 phase is associated with numerous neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease [8–10].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [8] AB West, 2005, To die or grow: Parkinson&apos;s disease and cancer, Trends Neurosci source
    • cites [9] A Johansson, 2003, Increased frequency of a new polymorphism in the cell division cycle 2 (cdc2) gene in patients with Alzheimer&apos;s disease and frontotemporal dementia, Neurosci Lett source
    • cites [10] K Herrup, 2007, Cell cycle regulation in the postmitotic neuron: oxymoron or new biology?, Nat Rev Neuro source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-338-3p induces maturation and differentiation of spinal cord oligodendrocyte precursors by repressing Sox5 and Hes6.

    “MiR-338-3p is also expressed in spinal cord oligodendrocytes, where it exerts a related effect, inducing maturation and differentiation of precursors into mature oligodendrocytes by repressing Sox5 and Hes6, two maturation-inhibiting transcription factors [31, 32].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [31] JC Dugas, 2010, Dicer1 and miR-219 Are required for normal oligodendrocyte differentiation and myelination, Neuron source

      Not checked in the free run.

    • cites [32] X Zhao, 2010, MicroRNA-mediated control of oligodendrocyte differentiation, Neuron source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    U251 and SF295 GBM cell lines are miR-338-3p deficient.

    “The U251 and SF295 GBM cell lines have previously been shown to be miR-338-3p deficient [24].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [24] A Gaur, 2007, Characterization of microRNA expression levels and their biological correlates in human cancer cell lines, Cancer Res source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-338-3p regulates axonal outgrowth in superior cervical ganglion neurons through COXIV and ATP5G1.

    “It also regulates axonal outgrowth in superior cervical ganglion neurons through COXIV and ATP5G1, two mitochondrial mRNAs active in oxidative phosphorylation [30].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] A Aschrafi, 2012, MicroRNA-338 regulates the axonal expression of multiple nuclear-encoded mitochondrial mRNAs encoding subunits of the oxidative phosphorylation machinery, Cell Mol Life Sci source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    If newborn neurons do not receive sufficient stimulation, they become much less likely to survive to maturity.

    “If these newborn neurons do not receive sufficient stimulation, they become much less likely to survive to maturity [17].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] N Kaneko, 2006, Role of the cholinergic system in regulating survival of newborn neurons in the adult mouse dentate gyrus and olfactory bulb, Genes Cells source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-338 is downregulated in the brains of individuals with schizophrenia.

    “Additionally, miR-338 is downregulated in the brains of individuals suffering from schizophrenia [37].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] MP Moreau, 2011, Altered microRNA expression profiles in post-mortem brain samples from individuals with schizophrenia and bipolar disorder, Biol Psychiatry source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Aberrant dendrite orientation and multiple primary dendrites have been previously described in studies of hippocampal neuron phenotypes in schizophrenia.

    “Surprisingly, both aberrant dendrite orientation and the presence of multiple primary dendrites have been previously described in studies of hippocampal neuron phenotypes in schizophrenia [33–36].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [33] M Kvajo, 2008, A mutation in mouse Disc1 that models a schizophrenia risk allele leads to specific alterations in neuronal architecture and cognition, Proc Natl Acad Sci USA source
    • cites [34] M Lauer, 2003, Increased frequency of dentate granule cells with basal dendrites in the hippocampal formation of schizophrenics, Psychiat Res Neuroimaging source
    • cites [35] X Duan, 2007, Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain, Cell source
    • cites [36] D Senitz, 2003, Granule cells of the dentate gyrus with basal and recurrent dendrites in schizophrenic patients and controls. A comparative Golgi study, J Neural Transm (Vienna) source

  • May combine two claims.

    Interpretationown data

    miR-338-3p knockdown only induces significant changes in the number of dendrites and their orientation, but not to the soma itself or to dendritic arborization and spine density.

    “Thus, we can infer that miR-338-3p knockdown only induces significant changes in the number of dendrites and their orientation, but not to the soma itself or to dendritic arborization and spine density.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MiR-338-3p has previously been implicated in tumor suppression in hepatocarcinoma, gastric cancer, and brain cancer.

    “MiR-338-3p has previously been implicated in tumor suppression in hepatocarcinoma, gastric cancer, and brain cancer [40–44].”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [40] C Shang, 2016, Mir-338-3p Inhibits Malignant Biological Behaviors of Glioma Cells by Targeting MACC1 Gene, Med Sci Monit source
    • cites [41] XH Huang, 2011, miR-338-3p suppresses invasion of liver cancer cell by targeting smoothened, J Pathol source
    • cites [42] P Li, 2013, Epigenetic silencing of miR-338-3p contributes to tumorigenicity in gastric cancer by targeting SSX2IP, PLoS ONE source
    • cites [43] X Chen, 2013, miR-338-3p suppresses neuroblastoma proliferation, invasion and migration through targeting PREX2a, FEBS Lett source
    • cites [44] K Sun, 2013, miRNA-338-3p suppresses cell growth of human colorectal carcinoma by targeting smoothened, World J Gastroenterol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    One prior study in pituitary adenoma describes a mechanism of tumor suppression via regulation of Pttg, a paracrine growth factor, in pituitary adenoma.

    “One prior study in pituitary adenoma describes a mechanism of tumor suppression via regulation of Pttg, a paracrine growth factor, in pituitary adenoma.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Y Lee, 2016, MiR-338-3p As a Tumor Suppressor through Pttg Modulation in GH3 Cell Lines, Neuro Oncol

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    Such a pattern of proliferation suggests a potential cell-nonautonomous regulatory mechanism.

    “Such a pattern of proliferation suggests a potential cell-nonautonomous regulatory mechanism, which has not described previously.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    We accounted for individual differences in counts and measurements of neurons between mice using previously described models.

    “We accounted for individual differences in counts and measurements of neurons between mice using previously described models [41].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] XH Huang, 2011, miR-338-3p suppresses invasion of liver cancer cell by targeting smoothened, J Pathol source

  • May combine two claims.

    Premisecitation

    Overexpression of miR-338-3p reduces cell viability and increases apoptosis in certain glioma cell lines.

    “Another study shows reduced cell viability and increased apoptosis in certain glioma cell lines following overexpression of miR-338-3p”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] C Shang, 2016, Mir-338-3p Inhibits Malignant Biological Behaviors of Glioma Cells by Targeting MACC1 Gene, Med Sci Monit source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The mechanisms underlying initiation and termination of both neurite formation and neuronal polarization are not particularly well characterized.

    “The mechanisms underlying initiation and termination of both neurite formation and neuronal polarization are not particularly well characterized, and verification of these hypotheses will require further study [28].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] R Sainath, 2015, Cytoskeletal and signaling mechanisms of neurite formation, Cell Tissue Res source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CyclinD1 and associated CDKs may act as a 'switch' in the cell cycle, and their activity is inhibited in terminally differentiated neurons but high in the embryonic nervous system.

    “CyclinD1 and associated CDKs may act as a “switch” in the cell cycle, and their activity is inhibited in terminally differentiated neurons but high in the embryonic nervous system [48–51].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [48] X Fu, 2012, miR-338-3p is down-regulated by hepatitis B virus X and inhibits cell proliferation by targeting the 3&apos;-UTR region of CyclinD1, Int J Mol Sci source
    • cites [49] M De Falco, 2004, Evaluation of cyclin D1 expression and its subcellular distribution in mouse tissues, J Anat source
    • cites [50] P Sumrejkanchanakij, 2003, Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons, Oncogene source
    • cites [51] S Jessberger, 2008, Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus, PLoS Biol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    To correct for variability in overall staining intensity, the background fluorescence intensity of the entire image was subtracted from the intensity of the individual cell of interest.

    “To correct for variability in overall staining intensity, the background fluorescence intensity of the entire image was subtracted from the intensity of the individual cell of interest.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Experiments used mixed-sex groups of mice with equivalent numbers of each sex.

    “Experiments used mixed-sex groups of mice with equivalent numbers of each sex.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Growth curves and proliferation rates could be calculated from the data at significance for both cell lines despite SF295 cells becoming overconfluent at 96 hours.

    “We were able to perform a cell count for the U251 line at all time points, but SF295 cells became overconfluent at 96 hours. Despite this, growth curves and proliferation rates could be calculated from the data at significance for both cell lines.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A similar analysis on the role of miR-338-3p in colorectal carcinoma implicates loss of SMO inhibition.

    “mirroring the results of a similar analysis done on the role of miR-338-3p in colorectal carcinoma implicating loss of SMO inhibition [44].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] K Sun, 2013, miRNA-338-3p suppresses cell growth of human colorectal carcinoma by targeting smoothened, World J Gastroenterol source

  • May combine two claims.

    Premisecitation

    A phenotype with multiple primary dendrites is associated with loss of Pten, which inhibits the PI3K/AKT pathway.

    “A phenotype with multiple primary dendrites is associated with loss of Pten, which inhibits the PI3K/AKT pathway [52].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] MR Williams, 2015, Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive, J Neurosci source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-338-3p regulates morphological polarity in other cell types by facilitating the activity of β-integrin.

    “Others have shown miR-338-3p regulates morphological polarity in other cell types by facilitating the activity of β-integrin [26].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] S Tsuchiya, 2009, MicroRNA-338-3p and microRNA-451 contribute to the formation of basolateral polarity in epithelial cells, Nucleic Acids Res source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Induction of miR-338-3p expression has no effect on U251 proliferation.

    “a recent study expressing miR-338-3p in U251 cells describes a different phenomenon, where induction of miR-338-3p expression has no effect on U251 proliferation [45].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] L Wang, 2012, MiR-483-5p suppresses the proliferation of glioma cells via directly targeting ERK1, FEBS Lett source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The prior pituitary adenoma study did not examine in vivo proliferation, and Pttg expression has not been described in the hippocampus.

    “However, this study did not examine in vivo proliferation, and the gene’s expression has not been described in the hippocampus thus far [47].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Y Lee, 2016, MiR-338-3p As a Tumor Suppressor through Pttg Modulation in GH3 Cell Lines, Neuro Oncol

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CyclinD1 and these CDKs may control angles of neurite formation in hippocampal granule cells.

    “Furthermore, CyclinD1 and these CDKs may control angles of neurite formation in hippocampal granule cells [51].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] S Jessberger, 2008, Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus, PLoS Biol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cells were grown in triplicate.

    “Cells were grown in triplicate in 10 cm plates (Corning Incorporated) in Dulbecco’s Modified Eagle Medium (Life Technologies) containing 10% fetal bovine serum and 1% penicillin/streptomycin.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-338-3p's inhibition of PREX2a is crucial in suppression of neuroblastoma proliferation.

    “Prior studies have already shown miR-338-3p’s inhibition of PREX2a is crucial in suppression of neuroblastoma proliferation [43].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] X Chen, 2013, miR-338-3p suppresses neuroblastoma proliferation, invasion and migration through targeting PREX2a, FEBS Lett source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    General loss of miRNA function through Dicer1 ablation produces a wide range of changes in hippocampal dendrites, including increased dendritic spine length, reduced dendritic arborization, and a bias toward arborization more distal to the soma.

    “General loss of miRNA function through Dicer1 ablation produces a wide range of changes in hippocampal dendrites, including but not limited to increased dendritic spine length, reduced dendritic arborization, and a bias toward arborization more distal to the soma [38, 39].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [38] W Konopka, 2010, MicroRNA loss enhances learning and memory in mice, J Neurosci source
    • cites [39] TH Davis, 2008, Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus, J Neurosci source

  • May combine two claims.

    Interpretationown data

    miR-338-3p has a regulatory role in neuronal proliferation, maturation, and neurite outgrowth and organization in the dentate gyrus, and acts as a tumor suppressor in vivo.

    “In this study, we identified a regulatory role for miR-338-3p in neuronal proliferation, maturation, and neurite outgrowth and organization in the dentate gyrus, while also acting as a tumor suppressor in vivo.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The miR-338-3p sponge specifically reduces miR-338-3p activity without affecting other miRNAs.
    • Rests on an assumption: The neoplastic growths observed in miR-338-3p knockdown mice are a valid model of human GBM.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All procedures were approved by the Institutional Animal Care and Use Committee at the Geisel School of Medicine at Dartmouth College and conformed to federal, state, local, and Association for Assessment and Accreditation of Laboratory Animal Care standards, under Protocol Numbers 11-01-01 and 00002030(a).

    “All procedures were approved by the Institutional Animal Care and Use Committee at the Geisel School of Medicine at Dartmouth College and conformed to federal, state, local, and Association for Assessment and Accreditation of Laboratory Animal Care standards, under Protocol Numbers 11-01-01 and 00002030(a).”
    Quote in the paperNot a substantial claim
55 of 55

  • The paper relies on this without support.

    The level of mCherry fluorescence from the lentiviral sensor quantitatively reflects the endogenous level or activity of the target miRNA.

    “Thus, endogenous miRNAs will bind the mCherry transcript’s target sequences, blocking its translation and reducing the level of mCherry fluorescence in cells expressing the miRNA of interest.”

  • The paper relies on this without support.

    The miR-338-3p sponge specifically knocks down miR-338-3p without affecting other miRNAs or cellular processes.

    “This increase in the number of labeled cells infected with the miR-338-3p sponge shows the sponge effectively knocks down endogenous miR-338-3p.”

  • The paper relies on this without support.

    Retroviral infection specifically labels actively proliferating cells that become dentate gyrus granule neurons.

    “Injection of this retrovirus results in specific infection of actively proliferating cells, allowing us to examine the development of newborn granule neurons in vivo.”

  • The paper relies on this without support.

    The U251 and SF295 cell lines are valid models of glioblastoma for studying miR-338-3p effects on proliferation.

    “The U251 and SF295 GBM cell lines have previously been shown to be miR-338-3p deficient [24].”

  • The paper relies on this without support.

    The TCGA GBM patient data are representative of glioblastoma patients generally.

    “Using data from 157 GBM patients with miRNA expression profiling of their tumors, we show that low expression of miR-338-3p corresponded to a decrease in overall and disease-free survival (Fig 8).”

  • The paper relies on this without support.

    The neoplasms observed in mice after miR-338-3p knockdown are a valid model of human glioblastoma.

    “This histological organization is reminiscent of GBM [23].”

  • The paper relies on this without support.

    BrdU incorporation accurately reflects cellular proliferation in this tissue.

    “BrdU (blue), a marker of cellular proliferation, in a dentate gyrus neoplasm following miR-338-3p knockdown.”

  • The paper relies on this without support.

    Normalization to the mCherry control corrects for differential tropism biases in sensor expression across cell types.

    “To illustrate the changes in expression for miR-338-3p and miR-132-3p specific to different points in neuronal maturity while compensating for any possible unforeseen biases toward specific cell types potentially induced by differential tropism, we described these percentages normalized to an mCherry control utilizing the same backbone (Fig 2H–2J).”

  • The paper relies on this without support.

    This study is the first to examine the effects of miR-338-3p on the morphology of dentate gyrus granule neurons.

    “This study is the first to examine the effects of miR-338-3p on the morphology of dentate gyrus granule neurons, but other studies have described other changes to neural morphology induced by miR-338-3p, by a variety of molecular targets.”

  • The paper relies on this without support.

    The miR-338-3p sponge specifically reduces miR-338-3p activity without affecting other miRNAs.

    “The miR-338-3p sponge was constructed by inserting 6 perfect miR-338-3p target sequences downstream of the U6 promotor and 6 perfect miR-338-3p target sequences downstream of the H1 promotor via complimentary overhangs to Bbs1/BglII.”

  • The paper relies on this without support.

    The U251 and SF295 cell lines are valid models of GBM for studying miR-338-3p effects on proliferation.

    “The U251 and SF295 cell lines were generously donated by the Israel laboratory at the Geisel School of Medicine at Dartmouth.”

  • The paper relies on this without support.

    The neoplastic growths observed in miR-338-3p knockdown mice are a valid model of human GBM.

    “we found miR-338-3p knockdown leads to the formation of multiple neoplastic growths resembling GBM”
12 of 12

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • DCX co-labeling is described as decreasing despite higher reported percentages

    Major

    The mCherry control has 14.60% DCX-positive cells, versus 33.56% for the miR-132-3p sensor and 43.97% for the miR-338-3p sensor. Those sensor percentages are higher than control, not the stated 2.30- and 3.01-fold decreases.

1 of 1

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Authors
Howe, J. R., Li, E. S., Streeter, S. E., Rahme, G. J., Chipumuro, E., Russo, G. B., Litzky, J. F., Hills, L. B., Rodgers, K. R., Skelton, P. D., & Luikart, B. W.
Affiliations
Dartmouth College (US)
Corresponding author
Luikart, Bryan W.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2017-05-11
Volume
12
Issue
5
Pages
e0177661
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0177661
PMC
PMC5426787
OpenAlex
W2612054083
Cited by
41
References
54
Citations by year
2026: 1 · 2025: 5 · 2024: 2 · 2023: 1 · 2022: 6 · 2021: 11 · 2020: 6 · 2019: 4 · 2018: 4 · 2017: 1
FWCI
1.38
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Audrey and Theodor Geisel School of Medicine at Dartmouth
  • Norris Cotton Cancer Center (US) (Pilot Grant Funded by Skip Matthews Run)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
Keywords
miR-338-3p, microRNA, neuronal maturation, glioblastoma, tumor suppressor, neurogenesis, dentate gyrus, dendrite formation, neuronal proliferation, miRNA expression profiling, patient mortality
MeSH terms
Animals, Brain Neoplasms, Cell Differentiation, Glial Fibrillary Acidic Protein, Glioblastoma, Humans, Mice, Inbred C57BL, Neurons, Reproducibility of Results, Gene Expression Regulation, Neoplastic, Treatment Outcome, Disease-Free Survival, Dentate Gyrus, MicroRNAs, Cell Line, Tumor, Cell Shape, Cell Proliferation, Gene Knockdown Techniques

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-06.

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