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Cited by 117MicroRNA in disease regulationCancer-related molecular mechanisms researchGlioma Diagnosis and Treatment

Stress Response of Glioblastoma Cells Mediated by miR-17-5p Targeting PTEN and the Passenger Strand miR-17-3p Targeting MDM2

Open access
Li, Haoran, Yang, Burton B·2012·Oncotarget
DOI 10.18632/oncotarget.810PMC PMC3681502
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Abstract

// Haoran Li 1,2 , and Burton B Yang 1,2 1. Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto 2. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto Correspondence: Burton B Yang, email: // Keywords : microRNA, stem cell, miR-17, stress response, drug resistance Received : December 28, 2012, Accepted : December 31, 2012, Published : December 31, 2012 Abstract Tumor development not only destroys the homeostasis of local tissues but also the whole body, and thus the tumor cells have to face the body’s defense system, a shortage of nutrition and oxygen, and chemotherapeutic drug treatment. In response to these stresses, tumor cells often alter gene expression and microRNA levels to facilitate survival. We have demonstrated that glioblastoma cells deprived of nutrition or treated with chemotherapeutics drugs expressed increased levels of miR-17. Ectopic transfection of miR-17 prolonged glioblastoma cell survival when the cells were deprived with nutrition or treated with chemotherapeutic drugs. Expression of miR-17 also promoted cell motility, invasion, and tube-like structure formation. We found that these phenotypes were the results of miR-17 targeting PTEN. As a consequence, HIF1α and VEGF were up-regulated. Ectopic expression of miR-17 was found to facilitate enrichment of stem-like tumor cells, since the cells became drug-resistant, showed increased capacity to form colonies and neurospheres, and expressed higher levels of CD133, a phenotype similar to ectopic expression of HIF1α. To further confirm the phenotypic property of stem cells, we demonstrated that glioblastoma cells transfected with miR-17 proliferated slower in different nutritional conditions by facilitating more cells staying in the G1 phase than the control cells. Finally, we demonstrated that miR-17 could repress MDM2 levels, resulting in decreased cell proliferation and drug-resistance. Our results added a new layer of functional mechanism for the well-studied miRNA miR-17.

AI snapshot

Written from the paper’s full text.

TL;DR

In glioblastoma cells, stress from serum deprivation or chemotherapy increases miR-17, which promotes survival, motility, invasion, tube formation, and stem-like features by targeting PTEN and activating HIF-1α/VEGF. The passenger strand miR-17-3p targets MDM2, reducing proliferation under normal conditions and contributing to drug resistance.

Key findings

  • Nutrition deprivation and Temozolomide treatment increased miR-17-5p levels in U87 and U343 glioblastoma cells; miR-17 overexpression prolonged survival under serum-free conditions (p<0.001, n=3) and increased migration (p<0.01, n=10) and invasion (p<0.01, n=6).
  • miR-17 targeted PTEN at two 3′UTR sites, reducing PTEN and increasing HIF-1α and VEGF under serum deprivation; PTEN siRNA increased HIF-1α and prolonged survival, while ectopic PTEN reversed miR-17’s survival effect.
  • miR-17 promoted stem-like phenotypes: larger neurospheres, more secondary spheres, more colonies in soft agar, and drug resistance to Docetaxel, Carmustine, and Temozolomide. CD133+ cells were higher in miR-17-transfected cells in serum-containing medium (0.31% vs. 0.14%) and serum-free medium (30.6% vs. 0.26%); HIF-1α transfection also increased CD133 (2.8% vs. 0.68%).
  • miR-17 reduced proliferation and increased G1-phase cells; miR-17-3p targeted MDM2 at three 3′UTR sites, and MDM2 knockdown reduced proliferation while MDM2 overexpression enhanced growth of miR-17-transfected cells.

Methods

  • U87 and U343 human glioblastoma cell lines were cultured in DMEM with 10% FBS or serum-free medium; stable miR-17-expressing and control lines were generated, and cells were treated with Temozolomide, Docetaxel, or Carmustine.
  • Functional assays included survival/proliferation counts, wound scratch migration, transwell invasion, tube formation with YPEN endothelial cells, neurosphere/self-renewal and soft agar colony formation, flow cytometry for cell cycle and CD133, Western blot, MTT, and luciferase reporter assays for PTEN and MDM2 3′UTRs.
  • Experiments were performed in triplicate; numerical data were analyzed by independent-sample t test with significance at *p<0.05 and p<0.01 unless otherwise specified.

Limitations

  • The paper states that further studies are needed on the prognostic value of miR-17 and notes ongoing controversy/no general agreement on definitions of tumor stem-like cells in vitro.
  • Evident from the text: all experiments use U87 and U343 cell lines and in vitro assays; no in vivo tumor models, patient samples, or clinical outcome analyses are reported.

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

Integrity signals

Registry

Crossref · 10.18632/oncotarget.810

Cites

75 of 75 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 18 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    miR-17 induces the generation of glioblastoma stem-like cells.

    “The ability of miR-17 to induce generation of glioblastoma stem-like cells is another interesting finding of our work.”
    Quote in the paperSubstantial claim
  • Findingown data

    PTEN is down-regulated in miR-17-overexpressing U87 cells compared to control cells.

    “Compared with the control cells, PTEN was down-regulated in cells over-expressing miR-17 (Fig 3b).”
    Quote in the paperSubstantial claim
  • Findingown data

    HIF-1α overexpression increases survival of U87 cells in serum-free medium compared to control.

    “HIF-1α over-expression increased the number of survival cells compared with the control (Fig 5b).”
    Quote in the paperSubstantial claim
  • Findingown data

    HIF-1α-transfected glioblastoma cells have an increased number of GSCs compared with control cells.

    “there was increased number of GSCs in HIF-1α-transfected cells compared with that of the control cells.”
    Quote in the paperSubstantial claim
  • Findingreasoning

    miR-17-transfected glioblastoma cells have the potential to develop tube-like structures of endothelial cells.

    “with the potential to develop the tube-like structures of endothelial cells”
    Quote in the paperSubstantial claim
  • May combine two claims. 10
  • Classified as probably not a substantial claim. 11

  • May combine two claims.

    Findingown data

    miR-17 represses luciferase activity of constructs containing PTEN 3'UTR binding sites, and mutation of the binding sites abolishes this repression.

    “The experiments showed that luciferase activities were repressed when the luciferase constructs were co-transfected with miR-17 plasmid, and the inhibitory effect of miR-17 was abolished when the binding sites were mutated (Fig 3d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HIF-1α is suppressed in cells cultured in 10% FBS, but highly expressed in miR-17-transfected cells cultured in serum-free medium.

    “Interestingly, HIF-1α was suppressed in cells cultured in medium containing 10% FBS, but was highly expressed in the miR-17-transfected cells cultured in serum-free medium (Fig 2b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miR-17-3p represses luciferase activity of constructs containing MDM2 3'UTR binding sites, and mutation of the binding sites abolishes this repression.

    “There was a decrease in luciferase activities in the cells transfected with the MDM2 3'UTR construct, but the inhibitory effect was abolished when the miR-17-3p binding sites were mutated (Fig 7d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miR-17 overexpression increases tube-like structure formation when co-cultured with endothelial YPEN cells in low serum medium, but not in 10% FBS.

    “When co-cultured with endothelial cell line, YPEN, in low serum medium, the miR-17-transfected cells induced formation of more tube-like structures than the control cells, but there was no significant difference when the cells were cultured in medium containing 10% FBS (Fig 2a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    miR-17 has a dual function in glioblastoma: it suppresses tumor cell growth in normal conditions and promotes tumor cell survival in unfavorable conditions.

    “Hereby we show miR-17 has a dual function in glioblastoma: it suppresses tumor cell growth in normal conditions, and it also promotes tumor cell survival in unfavorable conditions.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The passenger strand of a miRNA is believed to be degraded and inactivated.

    “while the passenger strand is believed to be degraded and inactivated [5, 6].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Lu, 2010, The fate of miRNA* strand through evolutionary analysis: implication for degradation as merely carrier strand or potential regulatory molecule?, PLoS One source
    • cites [6] Zamore, 2005, Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes, Cell source

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

    Study design and conductown data

    β-actin staining from the same membrane confirmed equal loading in Western blot analyses.

    “Staining of β-actin from the same membrane confirmed equal loading.”
    Quote in the paperNot a substantial claim

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

    Interpretationcitation

    miR-17 mediates cellular response to diverse stimuli by targeting closely related signaling pathways.

    “which raises the possibility that miR-17 mediates cellular response to diverse stimuli by targeting closely related signaling pathways [56].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Bedi, 2000, Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha, Genes & development source

      Not checked in the free run.

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

    Premisecitation

    A sub-population of tumor cells is not sensitive to treatment and might be the cause of tumor relapse.

    “It is believed that a sub-population of tumor cells is not sensitive to treatment, and might be the cause of tumor relapse [2, 3].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [2] Bao, 2010, Cancer stem cells in glioblastoma--molecular signaling and therapeutic targeting, Protein & cell source
    • cites [3] Parada, 2012, A restricted cell population propagates glioblastoma growth after chemotherapy, Nature source

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

    Premisecitation

    miR-141 and miR-200a promoted tumor growth but sensitized tumors to chemotherapy.

    “It was concluded that miR-141 and miR-200a promoted tumor growth but sensitized tumors to chemotherapy”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] Mechta-Grigoriou, 2011, miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response, Nature medicine source

  • Classified as probably not a substantial claim.

    Premisecitation

    HIF-1α is involved in response to drug treatment in glioblastoma.

    “Since HIF-1α is involved in response to drug treatment in glioblastoma [44]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [44] Su, 2012, Serum levels of microRNAs can specifically predict liver injury of chronic hepatitis B, World J Gastroenterol

      Not checked in the free run.

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

    Premisecitation

    MDM2 can regulate tumor cell proliferation independently of p53.

    “It is thought to inhibit the activation of p53, but it can also regulate tumor cell proliferation independently [48].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Bargonetti, 2011, A p53-independent role of Mdm2 in estrogen-mediated activation of breast cancer cell proliferation, Breast cancer research : BCR source

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

    Premisecitation

    HIF-1α is often overexpressed in glioblastoma.

    “Notably, HIF-1α is often found overexpressed in glioblastoma [44].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Su, 2012, Serum levels of microRNAs can specifically predict liver injury of chronic hepatitis B, World J Gastroenterol

      Not checked in the free run.

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

    Premisecitation

    MDM2 can ubiquitinate and degrade HIF-1α through the proteasome pathway.

    “It has been proposed that MDM2 can also ubiquitinate and degrade HIF-1α through the proteasome pathway”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Bedi, 2000, Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha, Genes & development source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    miR-17 represses MDM2 levels, resulting in decreased cell proliferation and drug resistance.

    “Finally, we demonstrated that miR-17 could repress MDM2 levels, resulting in decreased cell proliferation and drug-resistance.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All experiments were performed in triplicate.

    “All experiments were performed in triplicate”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Numerical data were analyzed by independent sample t test with significance set at p<0.05 and p<0.01.

    “numerical data were subjected to independent sample t test (unless otherwise specified). The levels of significance were set at *p<0.05 and **p<0.01.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    miR-17-transfected glioblastoma cells may facilitate angiogenesis and increase the number of tumor stem cells (TSCs).

    “These may facilitate angiogenesis and increase the number of TSCs.”
    • Also stated in: § DISCUSSION · plain
      “induced angiogenesis”
    Quote in the paperSubstantial claim
18 of 18

  • The paper relies on this without support.

    The characteristics of treatment-insensitive tumor cells that may cause relapse remain largely unknown.

    “The characteristics of these cells, however, remain largely unknown.”

  • The paper relies on this without support.

    TSCs play a major role in tumor re-vascularization and re-aggregation, eventually leading to tumor relapse.

    “It is generally thought that TSCs play a major role in tumor re-vascularization and re-aggregation, eventually leading to tumor relapse.”

  • The paper relies on this without support.

    CD133 is a valid marker of glioblastoma stem-like cells.

    “CD133, a cell surface glycoprotein, has been used extensively as a marker of glioblastoma stem-like cells (GSC).”

  • The paper relies on this without support.

    GSCs play an important role in drug resistance.

    “GSCs are thought to play an important role in drug resistance.”

  • The paper relies on this without support.

    During tumor progression to an advanced stage, tumors can be deprived of serum, causing growth factor deficiency stress.

    “During tumor progression to an advanced stage, it could be deprived of serum, under the stress of growth factor deficiency.”

  • The paper relies on this without support.

    A subpopulation of glioblastoma cells is resistant to tumor-starving treatment.

    “It is believed that a subpopulation of cells is resistant to “starving” treatment.”

  • The paper relies on this without support.

    Glioblastoma is characterized by high expression of miR-17 in tumor samples.

    “glioblastoma is characterized by its uncontrolled vascularization and high expression of miR-17 in tumor samples”

  • The paper relies on this without support.

    GSCs are responsible for drug resistance.

    “GSCs are often thought to be responsible for drug resistance”

  • The paper relies on this without support.

    A slower metabolic rate requires limited nutritional supply.

    “a slower metabolic rate requires limited nutritional supply.”
9 of 9

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?

  • Mir-17 can repress MDM2 levels and reduce cell proliferation by protecting p53 from MDM2-mediated degradation.

    “Mir-17 can repress MDM2 levels and reduce cell proliferation by protecting p53 from MDM2-mediated degradation [55].”

    From The p53 Pathway in Glioblastoma (2018) · cites this paper as [55]

    The Results sections report that miR-17 lowers MDM2 levels and reduces glioblastoma cell proliferation. They do not support the proposed p53-protection mechanism: p53 expression did not change, and the authors suggest miR-17 may act through a p53-independent pathway. Page numbers are unavailable in our copy. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

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.

  • Transwell upper-chamber medium differs

    Major

    The Figure 1 caption describes serum-free medium in the insert, while the Methods specify medium containing 1% FBS for the upper chamber.

  • Proposed pathway is assigned conflicting figure panels

    Minor

    The Discussion points to Figure 8d for the proposed pathway, but the Figure 8 caption assigns the proposed pathway to panel 8e and describes panel 8d as a proliferation assay.

  • Colony-formation assay duration differs

    Minor

    The Results say colonies were assessed after three weeks, but the Methods specify four weeks.

  • Figure 7 lists three mutants but calls them four

    Minor

    The caption claims four mutant constructs resulted, but names only three.

4 of 4

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Related

Authors
Li, H., & Yang, B. B.
Affiliations
Sunnybrook Health Science Centre (CA)
Corresponding author
Li, Haoran
ORCID
Venue
Oncotarget
Publisher
Impact Journals, LLC
Published
2012-12-31
Volume
3
Issue
12
Pages
1653-1668
ISSN
1949-2553 (ISSN-L)
DOI
10.18632/oncotarget.810
PMC
PMC3681502
OpenAlex
W1842367235
Cited by
117
References
75
Citations by year
2026: 1 · 2025: 2 · 2024: 1 · 2023: 5 · 2022: 7 · 2021: 6 · 2020: 7 · 2019: 10 · 2018: 9 · 2017: 7 · 2016: 14 · 2015: 21 · 2014: 17 · 2013: 10
FWCI
4.95
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Oncotarget (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Heart and Stroke Foundation of Canada
  • Canadian Institutes of Health Research (MOP111171, 102635_1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
  • Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
Keywords
miR-17, glioblastoma, PTEN, MDM2, microRNA, drug resistance, hypoxia, HIF-1α, vascular endothelial growth factor, cancer stem cells, CD133, cell proliferation, cell motility and invasion, neurospheres, G1 phase
MeSH terms
AC133 Antigen, Antineoplastic Agents, Cell Movement, Cell Survival, Dose-Response Relationship, Drug, Genotype, Glioblastoma, Glycoproteins, Humans, Neoplasm Invasiveness, Neovascularization, Pathologic, Peptides, Phenotype, Stress, Physiological, Time Factors, Transfection, Neoplastic Stem Cells, Antigens, CD, Up-Regulation, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Neoplastic, Drug Resistance, Neoplasm, MicroRNAs, Vascular Endothelial Growth Factor A, Cell Line, Tumor, PTEN Phosphohydrolase, Proto-Oncogene Proteins c-mdm2, Hypoxia-Inducible Factor 1, alpha Subunit

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