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Cited by 626MicroRNA in disease regulationCancer-related molecular mechanisms researchCancer-related Molecular Pathways

Modulation of K-Ras-Dependent Lung Tumorigenesis by MicroRNA-21

Open access
Hatley, Mark E., Patrick, David M., Garcia, Matthew R., Richardson, James A., Bassel-Duby, Rhonda, van Rooij, Eva, Olson, Eric N.·2010·Cancer Cell
DOI 10.1016/j.ccr.2010.08.013PMC PMC2971666
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Abstract

Lung cancer is the leading cause of cancer-related deaths in the world, and non-small-cell lung cancer (NSCLC) accounts for 80% of cases. MicroRNA-21 (miR-21) expression is increased and predicts poor survival in NSCLC. Although miR-21 function has been studied in vitro with cancer cell lines, the role of miR-21 in tumor development in vivo is unknown. We utilize transgenic mice with loss-of-function and gain-of-function miR-21 alleles combined with a model of NSCLC to determine the role of miR-21 in lung cancer. We show that overexpression of miR-21 enhances tumorigenesis and that genetic deletion of miR-21 partially protects against tumor formation. MiR-21 drives tumorigenesis through inhibition of negative regulators of the Ras/MEK/ERK pathway and inhibition of apoptosis.

AI snapshot

Written from the paper’s full text.

TL;DR

In a K-rasLA2 mouse model of NSCLC, miR-21 overexpression increased lung tumor number/area and proliferation, while miR-21 deletion reduced tumor burden and sensitized transformed MEFs to doxorubicin-induced apoptosis. miR-21 acts as a tumor promoter by repressing Ras/MEK/ERK negative regulators (Spry1, Spry2, Btg2, Pdcd4) and pro-apoptotic genes (Apaf1, Faslg, Pdcd4, RhoB).

Key findings

  • CAG-miR-21;K-rasLA2 mice overexpressing miR-21 4–6 fold had significantly more gross lung tumors at 18 weeks and increased microscopic tumor number and total tumor area versus K-rasLA2 littermates; tumors showed increased proliferation, but survival did not differ, possibly due to decreased thymic tumor incidence.
  • miR-21−/−;K-rasLA2 mice had significantly fewer surface lung tumors at 20 weeks and decreased total tumor area; no adenocarcinomas were noted in any of 14 miR-21−/−/K-rasLA2 animals evaluated, and miR-21 deletion did not alter Ki67 proliferation.
  • miR-21 overexpression alone was not sufficient for tumorigenesis: CAG-miR-21 mice without K-rasLA2 (n = 39) followed up to 555 days showed no tumors.
  • Reporter assays showed 13 of 24 putative mouse miR-21 target 3′UTRs were repressed by miR-21, with mutation of the miR-21 site abrogating repression in 11; in tumors, miR-21 overexpression decreased Spry1, Spry2, Btg2, Pdcd4, Apaf1, Faslg, and RhoB and increased ERK phosphorylation. K-ras activation increased miR-21 expression 4-fold; miR-21−/− MEFs formed fewer soft agar colonies and smaller xenograft tumors and showed increased doxorubicin-induced apoptosis.

Methods

  • Generated conditional CAG-miR-21 overexpressing and miR-21−/− mice, crossed to the K-rasLA2 NSCLC model; comparisons were to littermate controls, with tumor burden assessed by H&E/ImageJ, proliferation by Ki67, and apoptosis by TUNEL.
  • MEFs from wild-type and miR-21−/− mice were immortalized with SV40 large T/small t and transformed with H-rasG12V; assays included soft agar, xenografts, doxorubicin treatment (100 nM–10 μM for 12 h), and TUNEL (1 μM for 12 h).
  • Luciferase reporter assays tested 24 putative miR-21 target 3′UTRs; Western blots were performed on micro-dissected tumors; statistics used two-tailed unpaired Student’s t-test with p < 0.05 considered significant.

Limitations

  • No formal limitations section is present; the authors state miR-21 overexpression alone was not sufficient for tumorigenesis and that tumors arising after miR-21 deletion may have developed escape mechanisms, possibly explaining latency.
  • The text notes miR-21 deletion did not alter apoptosis in lung lesions, suggesting those tumors escaped miR-21 regulatory effects; no adenocarcinomas were observed in 14 miR-21−/−;K-rasLA2 animals, a limited sample for that endpoint.
  • Evident from the text: all mice used were of mixed genetic backgrounds, and findings are from a mouse K-rasLA2 model and transformed MEFs, so human NSCLC causality and therapeutic implications remain to be tested.

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

Integrity signals

Registry

Crossref · 10.1016/j.ccr.2010.08.013

Cites

70 of 70 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

14 of 43 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Four of the validated miR-21 target genes are known negative regulators of the Ras/MEK/ERK pathway: Spry1, Spry2, Btg2, and Pdcd4.

    “Four of the validated miR-21 target genes are known negative regulators of the Ras/MEK/ERK pathway: sprouty 1 (Spry1), sprouty 2 (Spry2), B-cell translocation gene 2 (Btg2), and programmed cell death 4 (Pdcd4).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Casci et al., 1999 source

      The Discussion identifies Drosophila Sprouty as an inhibitor of Ras/MAP kinase signaling. The paper predicts that mammalian Sprouty homologs may have the same function, but does not establish it for Spry1 or Spry2 specifically. It does not address Btg2, Pdcd4, or whether these genes are validated miR-21 targets. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Hanafusa et al., 2002 source
    • cites Lo et al., 2006

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

  • Premisecitation

    Oncogenic Ras activation has been shown to increase the expression of Spry2, providing feedback inhibition of Ras activation.

    “Oncogenic Ras activation has been shown to increase the expression of Spry2, providing feedback inhibition of Ras activation (Shaw et al., 2007).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Shaw et al., 2007 source

      In “Negative feedback signaling in development” (page not available), the paper reports that oncogenic K-ras up-regulates Spry-2 and that Spry-2 inhibits MAPK activation. It does not establish that Spry-2 inhibits activation of Ras itself; it describes possible effects at different points in the Ras/MAPK pathway, including downstream of activated K-ras. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Findingown data

    Over-expression of miR-21 enhances tumorigenesis.

    “We show that over-expression of miR-21 enhances tumorigenesis and genetic deletion of miR-21 partially protects against tumor formation.”
    Quote in the paperSubstantial claim
  • Findingown data

    Genetic deletion of miR-21 partially protects against tumor formation.

    “We show that over-expression of miR-21 enhances tumorigenesis and genetic deletion of miR-21 partially protects against tumor formation.”
    Quote in the paperSubstantial claim
  • Findingown data

    K-ras activation in the tumors of K-rasLA2 mice increased miR-21 expression 4-fold similar to the expression level in the CAG-miR-21 transgenic mouse lung.

    “K-ras activation in the tumors of K-rasLA2 mice increased miR-21 expression 4-fold similar to the expression level in the CAG-miR-21 transgenic mouse lung.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 25
  • May combine two claims. 35

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

    Premisecitation

    miR-21 expression levels are useful prognostic markers in non-small cell lung cancer.

    “Recently, miR-21 expression levels have proven useful prognostic markers in non-small cell lung cancer (Markou et al., 2008; Yanaihara et al., 2006).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Markou et al., 2008 source
    • cites Yanaihara et al., 2006 source

      The Results report that high hsa-mir-21 expression was associated with significantly worse survival in lung adenocarcinoma patients. They do not establish miR-21 as a validated prognostic marker across non-small cell lung cancer: the reported prognostic validation focused on other miRNAs. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The level of miR-21 expression in sputum distinguishes NSCLC patients from cancer-free controls with a greater sensitivity than conventional cytology.

    “The level of miR-21 expression in sputum distinguishes NSCLC patients from cancer-free controls with a greater sensitivity than conventional cytology (Xie et al., 2009).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Xie et al., 2009 source

  • May combine two claims.

    Findingown data

    Several components of the PI3K pathway, including PTEN and PIK3R1, have been suggested as miR-21 target genes; however, miR-21 over-expression did not alter PTEN and PIK3R1 protein expression or Akt phosphorylation.

    “Several components of the PI3K pathway, including PTEN and PIK3R1, have been suggested as miR-21 target genes (Meng et al., 2007); however, miR-21 over-expression did not alter PTEN and PIK3R1 protein expression or Akt phosphorylation (Figure S3B).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Meng et al., 2007 source

      The Results identify PTEN as a miR-21 target, and the Discussion places PTEN in the PI3K pathway. The passages do not identify PIK3R1 as a miR-21 target. The statement that over-expression did not alter protein expression or Akt phosphorylation is attributed to the citing paper’s Figure S3B, not to Meng et al. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • Rests on an assumption: The antibodies used in Western blots specifically detect the intended target proteins.

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

    Premisecitation

    Targeted deletion of miR-21 in RKO and DLD1 colon cancer cells revealed miR-21 contributes to tumorigenesis through compromising cell cycle progression and DNA damage-induced checkpoint function through the Cdc25a target gene.

    “Targeted deletion of miR-21 in RKO and DLD1 colon cancer cells revealed miR-21 contributes to tumorigenesis through compromising cell cycle progression and DNA damage-induced checkpoint function through the Cdc25a target gene (Wang et al., 2009).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Wang et al., 2009 source

      The Results report targeted miR-21 deletion in RKO and DLD1 cells, and the Discussion links miR-21, Cdc25A, cell-cycle progression, and checkpoint activation. However, the Discussion describes a contribution to tumorigenesis as a possibility, not a demonstrated outcome; the claim's “revealed” overstates that conclusion. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    EGFR and HER2/neu signaling positively regulates miR-21 expression in human lung and breast carcinoma cell lines, respectively.

    “As well, epidermal growth factor receptor (EGFR) and HER2/neu signaling positively regulates miR-21 expression in human lung and breast carcinoma cell lines, respectively (Huang et al., 2009; Seike et al., 2009).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Huang et al., 2009 source
    • cites Seike et al., 2009 source

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

    Premisecitation

    Forced over-expression of miR-21 in a human gastric cancer cell line enhanced tumor proliferation and invasion.

    “Zhang et al. showed in a human gastric cancer cell line that forced over-expression of miR-21 enhanced tumor proliferation and invasion”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Zhang et al., 2008 source

  • May combine two claims.

    Premisecitation

    SPRY2 negatively regulates Ras/MEK/ERK signaling and is a tumor suppressor in NSCLC in both mice and humans.

    “SPRY2, in turn, negatively regulates Ras/MEK/ERK signaling and is a tumor suppressor in NSCLC in both mice (Minowada and Miller, 2004; Minowada and Miller, 2009; Shaw et al., 2007) and humans (Sutterluty et al., 2007).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Minowada and Miller, 2004 source
    • cites Minowada and Miller, 2009 source

      The study summary and Discussion (page unavailable) report fewer and smaller urethane-induced lung tumors in mice overexpressing Sprouty 2, supporting a tumor-suppressive effect in that mouse model. The Discussion says the findings are consistent with antagonism of Ras signaling, but the mechanism remains unclear; this paper does not demonstrate inhibition of MEK/ERK signaling. The citing sentence attributes the human NSCLC portion to a separate citation, not to this paper. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites Shaw et al., 2007 source

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

    Study design and conductcitation

    All tumors from the survival cohort were analyzed on H&E sections and categorized according to the Mouse Models of Human Cancer Consortium recommendations.

    “All tumors from the survival cohort were analyzed on H&E sections and categorized as hyperplasia, atypical adenomatous hyperplasia, adenoma, adenoma with typia, and adenocarcinoma according to the Mouse Models of Human Cancer Consortium recommendations.(Nikitin et al., 2004)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Nikitin et al., 2004 source

      The Development of Recommendations section says the consortium prepared and reviewed H&E-stained sections, and the classification defines hyperplasia, atypical adenomatous hyperplasia, adenoma, and adenocarcinoma. The cited paper does not identify “adenoma with typia” as a recommended category. The survival-cohort analysis describes the citing authors’ own work, not an analysis reported by Nikitin et al. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Knock-down of miR-21 in a human gastric cancer cell line resulted in a marked reduction in proliferation and increase in apoptosis.

    “knock-down of miR-21 resulted in a marked reduction in proliferation and increase in apoptosis (Zhang et al., 2008).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Zhang et al., 2008 source

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

    Premisecitation

    Methods have been developed to manipulate miRNA function pharmacologically, facilitating the development of new cancer therapeutic strategies.

    “Methods have been developed to manipulate miRNA function pharmacologically, facilitating the development of new cancer therapeutic strategies (Hutvagner et al., 2004; Krutzfeldt et al., 2005).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Hutvagner et al., 2004 source

      The Results and Discussion report that 2′-O-methyl oligonucleotides inhibit specific miRNAs and can be used to study their function in C. elegans. The paper presents them as a research tool; it does not establish that they facilitate new cancer therapeutic strategies. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Krutzfeldt et al., 2005 source

  • May combine two claims.

    Findingown data

    RECK, Cdc25a, Map2k3, NfiB, Trp63 and Ski have been shown to be miR-21 target genes in vitro, but only RECK showed decreased protein levels in the CAG-miR-21;K-rasLA2 tumors.

    “RECK, Cdc25a, Map2k3, NfiB, Trp63 and Ski have been shown to be miR-21 target genes in vitro, but only RECK showed decreased protein levels in the CAG-miR-21;K-rasLA2 tumors (Figure S3A).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The antibodies used in Western blots specifically detect the intended target proteins.

  • May combine two claims.

    Findingown data

    MiR21−/−;K-rasLA2 mice all develop lung tumors, but show an increased incidence of thymic lymphoma.

    “MiR21−/−;K-rasLA2 mice all develop lung tumors, but show an increased incidence of thymic lymphoma (Figure 2F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Caspase 3 and K-ras do not contain miR-21 sites in their 3′ UTR and the 3′ UTR luciferase reporter activity is not suppressed by miR-21.

    “Caspase 3 and K-ras do not contain miR-21 sites in their 3′ UTR and the 3′ UTR luciferase reporter activity is not suppressed by miR-21 (Figure 3B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The decrease in Spry1, Spry2, and Btg2 in CAG-miR-21;K-rasLA2 tumors correlates with increased Ras pathway activity, shown by increased ERK phosphorylation.

    “The decrease in Spry1, Spry2, and Btg2 in CAG-miR-21;K-rasLA2 tumors correlates with increased Ras pathway activity, shown by increased ERK phosphorylation (Figure 4C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The antibodies used in Western blots specifically detect the intended target proteins.

  • May combine two claims.

    Findingown data

    miR-21 dependent repression of the mouse Apaf1 3′ UTR reporter was shown; however, the miR-21 site in the human APAF1 3′ UTR is conserved in primates but not conserved in the mouse.

    “We show miR-21 dependent repression of the mouse Apaf1 3′ UTR reporter; however, the miR-21 site in the human APAF1 3′ UTR is conserved in primates but not conserved in the mouse.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Adult male CAG-miR-21 mice showed no signs of heart failure compared to littermate controls.

    “adult male CAG-miR-21 mice showed no signs of heart failure compared to littermate controls (Figure S1E)(van Rooij et al., 2006; van Rooij et al., 2008).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Apaf1, Pdcd4, Faslg, and RhoB were validated as direct miR-21 target genes by luciferase reporter assays; however, caspase 3 was not directly regulated by miR-21.

    “Apaf1, Pdcd4, Faslg, and RhoB were validated as direct miR-21 target genes by luciferase reporter assays; however, caspase 3 was not directly regulated by miR-21 (Figure 3B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The luciferase reporter assays in COS cells reflect endogenous miR-21 target regulation in mouse lung tumors.

  • May combine two claims.

    Findingown data

    MiR-21 over-expression increased the incidence of all tumor grades without increasing the rate of conversion to adenocarcinoma.

    “MiR-21 over-expression increased the incidence of all tumor grades without increasing the rate of conversion to adenocarcinoma, suggesting that miR-21 participates in tumor promotion rather than progression and metastasis in this model (Figure 1J and 1K).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    No increased tumor spectrum or increased metastasis was observed in CAG-miR-21;K-rasLA2 mutant mice.

    “Unlike K-rasLA1;p53−/− compound mutant mice (Johnson et al., 2001), we did not observe an increased tumor spectrum or increased metastasis in the CAG-miR-21;K-rasLA2 mutant mice.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisereasoning

    The role of miR-21 in tumor development in vivo is unknown.

    “Although miR-21 function has been studied in vitro using cancer cell lines, the role of miR-21 in tumor development in vivo is unknown.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    MiR-21 drives tumorigenesis through inhibition of negative regulators of the Ras/MEK/ERK pathway and inhibition of apoptosis.

    “MiR-21 drives tumorigenesis through inhibition of negative regulators of the Ras/MEK/ERK pathway and inhibition of apoptosis.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    A large scale survey of 540 tumor samples revealed miR-21 was the only miRNA up-regulated in all these tumors.

    “A large scale survey to determine the miRNA signature of 540 tumor samples including lung, breast, stomach, prostate, colon, and pancreatic tumors and their respective normal adjacent tissue revealed miR-21 was the only miRNA up-regulated in all these tumors (Volinia et al., 2006).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Volinia et al., 2006 source

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

    Premisecitation

    miR-21 expression is increased in glioblastoma, head and neck carcinomas, ovarian cancer, B-cell lymphoma, hepatocellular, and cervical carcinoma.

    “Further miRNA profiling in tumor samples and cancer cell lines show increased miR-21 expression in glioblastoma (Chan et al., 2005; Ciafre et al., 2005), head and neck carcinomas (Tran et al., 2007), ovarian cancer (Iorio et al., 2007), B-cell lymphoma (Lawrie et al., 2007), hepatocellular (Meng et al., 2007), and cervical carcinoma (Lui et al., 2007).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Chan et al., 2005 source
    • cites Ciafre et al., 2005 source
    • cites Tran et al., 2007 source

  • May combine two claims.

    Study design and conductown data

    CAG-miR-21 transgenic mice were viable, fertile and born in expected Mendelian ratios.

    “CAG-miR-21 transgenic mice were viable, fertile and born in expected Mendelian ratios.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Knock-down of miR-21 in the metastatic breast cancer MD-MBA-231 cells reduced invasion and metastasis.

    “Knock-down of miR-21 in the metastatic breast cancer MD-MBA-231 cells reduced invasion and metastasis (Zhu et al., 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Zhu et al., 2008 source

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

    Study design and conductown data

    Homozygous miR-21 knock-out mice are viable, fertile, and born in expected Mendelian ratios without any gross phenotypic differences compared to wild type or heterozygous littermates, and without a cardiac phenotype.

    “Homozygous miR-21 knock-out mice are viable, fertile, and born in expected Mendelian ratios without any gross phenotypic differences compared to wild type or heterozygous littermates, and without a cardiac phenotype (D.P., E.V.R. and E.N.O., manuscript in preparation).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The miR-21 knockout mice do not have compensatory molecular changes that confound the interpretation of tumorigenesis experiments.

  • May combine two claims.

    Interpretationown data

    MiR-21 over-expression decreases the protein expression of four miR-21 target genes that are involved in apoptosis thus promoting survival.

    “These data illustrate that miR-21 over-expression decreases the protein expression of four miR-21 target genes that are involved in apoptosis thus promoting survival.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The antibodies used in Western blots specifically detect the intended target proteins.
    • Rests on an assumption: The luciferase reporter assays in COS cells reflect endogenous miR-21 target regulation in mouse lung tumors.

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

    Premisecitation

    miR-21 expression was induced by AP-1 in response to Ras activation, and miR-21 mediated Ras-dependent downregulation of Pdcd4.

    “miR-21 expression was induced by AP-1 in response to Ras activation, and miR-21 mediated Ras-dependent downregulation of Pdcd4 (Talotta et al., 2009)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Talotta et al., 2009 source

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

    Premisecitation

    Knockdown of miR-21 in cultured glioblastoma cells activates caspases leading to apoptotic cell death, suggesting miR-21 is an anti-apoptotic factor.

    “Knockdown of miR-21 in cultured glioblastoma cells activates caspases leading to apoptotic cell death, suggesting miR-21 is an anti-apoptotic factor (Chan et al., 2005).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chan et al., 2005 source

  • May combine two claims.

    Premisecitation

    K-rasLA2 mice develop multi-focal lung tumors with 100% penetrance, and less frequently develop thymic lymphomas and skin papillomas.

    “K-rasLA2 mice develop multi-focal lung tumors with 100% penetrance, and less frequently develop thymic lymphomas and skin papillomas (Johnson et al., 2001).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Johnson et al., 2001 source

  • May combine two claims.

    Interpretationown data

    miR-21 over-expression in human tumors does not simply reflect cancer pathology, but instead enhances aspects of the pathology of NSCLC.

    “These data suggest that miR-21 over-expression in human tumors does not simply reflect cancer pathology, but instead enhances aspects of the pathology of NSCLC.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The K-rasLA2 mouse model is a valid model of human NSCLC for studying miR-21.
    • Rests on an assumption: The level of miR-21 over-expression achieved in CAG-miR-21 mice (4–6 fold) is representative of the increased miR-21 expression in human NSCLC.

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

    Premisecitation

    An autoregulatory loop connecting miR-21 and Ras activation through AP-1 was illustrated in a rat thyroid cell system.

    “Previously, an autoregulatory loop connecting miR-21 and Ras activation through AP-1 was illustrated in a rat thyroid cell system (Talotta et al., 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Talotta et al., 2009 source

  • May combine two claims.

    Interpretationown data

    MiR-21 over-expression decreases the protein expression of four miR-21 target genes that are tumor suppressors and negative regulators of the Ras/MEK/ERK pathway resulting in a concomitant increased ERK activity.

    “These data show that miR-21 over-expression decreases the protein expression of four miR-21 target genes that are tumor suppressors and negative regulators of the Ras/MEK/ERK pathway resulting in a concomitant increased ERK activity.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The antibodies used in Western blots specifically detect the intended target proteins.
    • Rests on an assumption: The luciferase reporter assays in COS cells reflect endogenous miR-21 target regulation in mouse lung tumors.

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

    Premisecitation

    In breast cancer MCF-7 cells, miR-21 knock-down results in suppression of cell growth in vitro and tumor growth in xenografts.

    “In breast cancer MCF-7 cells, miR-21 knock-down results in suppression of cell growth in vitro and tumor growth in xenografts (Si et al., 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Si et al., 2007

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

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

    Study design and conductnone given

    All mice used in these studies were of mixed genetic backgrounds and all comparisons were performed on littermate controls.

    “All mice used in these studies were of mixed genetic backgrounds and all comparisons were performed on littermate controls.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    PDCD4 is a pro-apoptotic tumor suppressor gene and has been verified as a miR-21 target in human colorectal and breast cancer cell lines.

    “PDCD4 is a pro-apoptotic tumor suppressor gene and has been verified as a miR-21 target in human colorectal and breast cancer cell lines (Afonja et al., 2004; Asangani et al., 2007; Bitomsky et al., 2008; Frankel et al., 2007; Lu et al., 2008; Zhang et al., 2006).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Afonja et al., 2004 source
    • cites Asangani et al., 2007

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

    • cites Bitomsky et al., 2008 source

  • May combine two claims.

    Premisecitation

    In laryngeal carcinoma, miR-21 targets BTG2, a p53 target gene, recently shown to suppress oncogenic Ras activity by reducing the GTP bound, active state.

    “In laryngeal carcinoma, miR-21 targets BTG2 (Liu et al., 2009), a p53 target gene, recently shown to suppress oncogenic Ras activity by reducing the GTP bound, active state (Buganim et al., 2010).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Liu et al., 2009 source
    • cites Buganim et al., 2010 source

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

    Premisecitation

    Knock-down of miR-21 in glioblastoma cell lines triggers the activation of caspases and increases apoptosis.

    “Knock-down of miR-21 in glioblastoma cell lines triggers the activation of caspases and increases apoptosis (Chan et al., 2005).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chan et al., 2005 source

  • May combine two claims.

    Premisecitation

    RhoB promotes growth inhibition and induces apoptosis in cancer cells.

    “RhoB promotes growth inhibition and induces apoptosis in cancer cells (Kim et al., 2009)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kim et al., 2009 source

  • May combine two claims.

    Interpretationreasoning

    Although the reductions in protein levels of individual miR-21 targets appear modest with miR-21 over-expression, collectively this diminution of tumor suppressor activity allows for more robust tumor formation through relieving multiple nodes of inhibition of the Ras/MEK/ERK pathway, thus facilitating tumor proliferation.

    “Although the reductions in protein levels of individual miR-21 targets appear modest with miR-21 over-expression, collectively this diminution of tumor suppressor activity allows for more robust tumor formation through relieving multiple nodes of inhibition of the Ras/MEK/ERK pathway, thus facilitating tumor proliferation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The K-rasLA2 mouse model is a valid model of human NSCLC for studying miR-21.
    • Rests on an assumption: The antibodies used in Western blots specifically detect the intended target proteins.

  • May combine two claims.

    Interpretationown data

    Ras activation increases miR-21 expression and in turn decreasing the expression of Spry2.

    “We show that Ras activation increases miR-21 expression and in turn decreasing the expression of Spry2.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The K-rasLA2 mouse model is a valid model of human NSCLC for studying miR-21.

  • May combine two claims.

    Interpretationown data

    miR-21 modulates several components critical to NSCLC pathogenesis through both relieving antagonism of the Ras pathway and reducing apoptotic effectors.

    “miR-21 modulates several components critical to NSCLC pathogenesis through both relieving antagonism of the Ras pathway and reducing apoptotic effectors (Figure 7).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The K-rasLA2 mouse model is a valid model of human NSCLC for studying miR-21.

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

    Premisecitation

    RhoB down-regulation in human NSCLC suggests a role as a tumor suppressor gene.

    “RhoB expression is down-regulated in human NSCLC suggesting a role as a tumor suppressor gene (Sato et al., 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sato et al., 2007 source

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Relieving miR-21 inhibition of pro-apoptotic genes could provide a means to augment the effect of current chemotherapy.

    “Relieving miR-21 inhibition of pro-apoptotic genes could provide a means to augment the effect of current chemotherapy.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Apaf1, Faslg, Pdcd4 and RhoB promote apoptosis.

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

    Premisecitation

    MiR-21 targets SPRY2 in cardiomyocytes and SW480 colon cancer cells, enhancing cell migration.

    “MiR-21 targets SPRY2 in cardiomyocytes and SW480 colon cancer cells, enhancing cell migration (Sayed et al., 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sayed et al., 2008 source

  • May combine two claims.

    Interpretationown data

    miR-21 expression modulates tumor number, incidence and size in a mouse lung cancer model initiated by oncogenic K-rasG12D, consistent with miR-21 functioning as a tumor promoter.

    “Our results show that miR-21 expression modulates tumor number, incidence and size in a mouse lung cancer model initiated by oncogenic K-rasG12D, consistent with miR-21 functioning as a tumor promoter.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The K-rasLA2 mouse model is a valid model of human NSCLC for studying miR-21.
    • Rests on an assumption: The level of miR-21 over-expression achieved in CAG-miR-21 mice (4–6 fold) is representative of the increased miR-21 expression in human NSCLC.

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

    Premisecitation

    Pdcd4 functions both as an inducer of apoptosis and a suppressor of AP-1 activity.

    “Pdcd4 functions both as an inducer of apoptosis and a suppressor of AP-1 activity (Hwang et al., 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hwang et al., 2007 source

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

    Premisecitation

    Loss of PDCD4 expression in human lung cancer correlates with higher grade and disease stage.

    “Loss of PDCD4 expression in human lung cancer correlates with higher grade and disease stage (Chen et al., 2003).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chen et al., 2003 source
48 of 48

  • The paper relies on this without support.

    The K-rasLA2 mouse model is a valid model of human NSCLC for studying miR-21.

    “To explore the role of miR-21 in the pathogenesis of NSCLC, we utilized the K-rasLA2 murine lung cancer model, which harbors a targeted, latent K-ras G12D allele that is activated by two distinct recombination events (Johnson et al., 2001).”

  • The paper relies on this without support.

    The level of miR-21 over-expression achieved in CAG-miR-21 mice (4–6 fold) is representative of the increased miR-21 expression in human NSCLC.

    “which globally over-expressed miR-21 4–6 fold over normal levels of expression (Figure S1B–D).”

  • The paper relies on this without support.

    The miR-21 knockout mice do not have compensatory molecular changes that confound the interpretation of tumorigenesis experiments.

    “Homozygous miR-21 knock-out mice are viable, fertile, and born in expected Mendelian ratios without any gross phenotypic differences compared to wild type or heterozygous littermates, and without a cardiac phenotype (D.P., E.V.R. and E.N.O., manuscript in preparation).”

  • The paper relies on this without support.

    The antibodies used in Western blots specifically detect the intended target proteins.

    “We isolated individual tumors from mice, made whole tumor lysates, and performed Western blots for putative miR-21 targets to address the effect of miR-21 expression on the protein levels of the miR-21 target genes in vivo within lung tumors.”

  • The paper relies on this without support.

    The luciferase reporter assays in COS cells reflect endogenous miR-21 target regulation in mouse lung tumors.

    “Reporter assays in COS cells revealed miR-21 dependent repression of 13 of the 24 3′UTRs”

  • The paper relies on this without support.

    MicroRNAs are small non-coding RNAs that are commonly dysregulated in human malignancies and play a substantial role in the pathogenesis and survival of many cancers.

    “MicroRNAs are small non-coding RNAs that are commonly dysregulated in human malignancies and play a substantial role in the pathogenesis and survival of many cancers.”

  • The paper relies on this without support.

    MiR-21 is over-expressed in the majority of human malignancies including non-small cell lung cancer (NSCLC).

    “MiR-21 is over-expressed in the majority of human malignancies including non-small cell lung cancer (NSCLC).”

  • The paper relies on this without support.

    It is not clear whether miR-21 determines important aspects of lung cancer pathogenesis or is instead simply a marker for advanced disease.

    “However, it is not clear whether miR-21 determines important aspects of lung cancer pathogenesis or is instead simply a marker for advanced disease.”

  • The paper relies on this without support.

    Spry1, Spry2, Btg2, and Pdcd4 act as negative regulators of the Ras/MEK/ERK pathway.

    “which act as negative regulators of the Ras/MEK/ERK pathway”

  • The paper relies on this without support.

    Apaf1, Faslg, Pdcd4 and RhoB promote apoptosis.

    “which promote apoptosis”
10 of 10

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Related

Authors
Hatley, M. E., Patrick, D. M., Garcia, M. R., Richardson, J. A., Bassel-Duby, R., van Rooij, E., & Olson, E. N.
Affiliations
The University of Texas Southwestern Medical Center (US)
Corresponding author
Olson, Eric N.
ORCID
Venue
Cancer Cell
Publisher
Elsevier BV
Published
2010-09
Volume
18
Issue
3
Pages
282-293
ISSN
1535-6108 (ISSN-L)
DOI
10.1016/j.ccr.2010.08.013
PMC
PMC2971666
OpenAlex
W2169354377
Semantic Scholar
ea1aaa83b1883a1290ce31db3401b12568b668c7
Cited by
626
References
74
Citations by year
2026: 12 · 2025: 15 · 2024: 26 · 2023: 22 · 2022: 38 · 2021: 31 · 2020: 28 · 2019: 29 · 2018: 42 · 2017: 31 · 2016: 40 · 2015: 72 · 2014: 57 · 2013: 62 · 2012: 63
FWCI
16.52
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancer Cell (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Heart, Lung, and Blood Institute (R01HL093039)
  • National Cancer Institute
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (K12HD000850)
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 › Oncology › Cancer-related Molecular Pathways
Keywords
miR-21, KRAS, lung carcinogenesis, non-small cell lung cancer, Ras/MEK/ERK pathway, apoptosis inhibition, transgenic mouse model, tumor suppressor microRNA
MeSH terms
Animals, Carcinoma, Non-Small-Cell Lung, Cell Transformation, Neoplastic, Humans, Lung Neoplasms, Mice, Nude, Mice, Transgenic, Genes, ras, Signal Transduction, MicroRNAs, Cell Line, Tumor, Mice

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