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Cited by 123Extracellular vesicles in diseaseMicroRNA in disease regulationRNA Interference and Gene Delivery

Potential Therapeutic Effects of Exosomes Packed With a miR-21-Sponge Construct in a Rat Model of Glioblastoma

Open access
Monfared, Hamideh, Jahangard, Yavar, Nikkhah, Maryam, Mirnajafi-Zadeh, Javad, Mowla, Seyed Javad·2019·Frontiers in Oncology
DOI 10.3389/fonc.2019.00782PMC PMC6710330
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Abstract

Glioblastoma multiforme (GBM) is the grade 4 and the most aggressive form of glioma, with a poor response to current treatments. The expression of microRNAs (miRNAs) is widely dysregulated in various cancers, including GBM. One of the overexpressed miRNAs in GBM is miR-21 which promotes proliferation, invasion and metastatic behaviors of tumor cells. With a size of 30-100 nm, the extracellular vesicles “exosomes” have emerged as a novel and powerful drug delivering systems. Recently, exosomal transfer of miRNAs or anti-miRNAs to tumor cells has introduced a new approach for therapeutic application of miRNAs to combat cancer. Here, we have tried to down-regulate miR-21 expression in glioma cell lines, U87-MG and C6, by using engineered exosomes, packed with a miR-21-sponge construct. Our data revealed that the engineered exosomes have the potential to suppress miR-21 and consequently to upregulate miR-21 target genes, PDCD4 and RECK. Interestingly, in cells treated with miR-21-sponge exosomes we observed a decline in proliferation and also an elevation in apoptotic rates. Finally, in a rat model of glioblastoma, administrating exosomes loaded with a miR-21-sponge construct leads to a significant reduction in the volume of the tumors. In brief, our findings suggest a new therapeutic strategy to use engineered exosomes to deliver a miR-21-sponge construct to GBM cells, in order to block its malignant behavior.

AI snapshot

Written from the paper’s full text.

TL;DR

Engineered exosomes carrying a miR-21-sponge construct reduced miR-21, upregulated PDCD4 and RECK, decreased viability, and increased apoptosis in glioblastoma cell lines. In a rat C6 glioblastoma model, administration of miR-21-sponge exosomes significantly retarded tumor growth, with the best effect from freshly prepared exosomes.

Key findings

  • In stably transfected HEK-293T cells, miR-21-sponge reduced miR-21 (P < 0.05), while pri-miR-21 increased miR-21 about 1,000-fold versus untransfected cells (P < 0.0001).
  • Exosomes from pri-miR-21 cells showed ~10-fold higher miR-21 than mock controls (P < 0.0001); exosomes from miR-21-sponge cells showed a significant decline in endogenous miR-21 (P < 0.0001).
  • In U87-MG cells, miR-21-sponge exosomes reduced miR-21 significantly at 48 h (P < 0.001) and upregulated PDCD4 and RECK at 48 h; MTT showed decreased viability at 24 h (P < 0.001) and 48 h (P < 0.05).
  • Annexin V/PI flow cytometry at 24 h showed late apoptosis increased to 30.9% versus 17% in untreated cells, and alive cells decreased from 80% to 64%; in vivo, miR-21-sponge exosomes significantly retarded tumor growth, and fresh exosomes reduced tumor volume by 50% between day 11 and day 18.

Methods

  • A miR-21-sponge with three miR-21 complementary sequences was cloned into Tracer vector; pri-miR-21 was cloned into pLentiIII. Stable HEK-293T lines were generated and co-cultured with U87-MG cells.
  • Exosomes were purified by differential centrifugation, characterized by DLS (66.65 nm peak), SEM (<100 nm), Western blot for CD63/CD81, and PKH-26 uptake. U87-MG and C6 cells were treated with 50 μg/ml exosomes; miR-21 and target genes were measured by RT-PCR, viability by MTT, and apoptosis by Annexin V-FITC/PI flow cytometry.
  • In vivo, 1 million C6 cells were stereotaxically injected into the CPu of male Wistar rats (250–300 g); tumor volume was measured by T2 MRI and FSL on day 11 and day 18 after injection of 15–20 μg miR-21-sponge exosomes. Statistics used ΔΔCT, GraphPad Prism 6, ordinary ANOVA, mean ± SD, and P < 0.05.

Limitations

  • The paper states challenges for exosome therapeutics, including low exosome productivity, need for large-scale production, isolation methods, uniform quality, storage conditions, and delivery; it also notes the blood-brain-barrier as a challenge for brain tumor therapy.
  • The paper reports that freshly prepared exosomes worked better than freeze-thawed exosomes, but does not provide a systematic comparison of storage or freeze-thaw conditions.
  • Evident from the text: in vivo results are described as significant but without exact P values or group sizes; flow cytometry used two independent replicates, and MTT used three, while PDCD4/RECK and apoptosis assays were mainly in U87-MG cells, with C6 used for miR-21 and in vivo tumor growth.

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

Integrity signals

Registry

Crossref · 10.3389/fonc.2019.00782

Cites

70 of 70 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

76 of 97 cited sources checked · 45 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    In GBM, communication between tumor cells and the microenvironment has a big impact on tumor progression.

    “In GBM, similar to other cancer types, the communication between tumor cells and its microenvironment has a big impact on tumor progression; and hence it provided a new approach for cancer treatment (3, 4).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [3] Skog, 2008, Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers, Nat Cell Biol. source
    • cites [4] Setti, 2015, Extracellular vesicle-mediated transfer of CLIC1 protein is a novel mechanism for the regulation of glioblastoma growth, Oncotarget. source
  • Premisecitation

    miR-21 targets several genes such as PDCD4, TIMP3, and RECK, which are key regulators of apoptotic and metastatic pathways.

    “miR-21 targets several genes such as PDCD4, TIMP3, and RECK, which are key regulators of apoptotic and metastatic pathways (17–23).”
    Quote in the paperSubstantial claim5 of 7 sources checked
    • cites [17] Gabriely, 2008, MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators, Mol Cell Biol. source
    • cites [18] Gaur, 2011, Downregulation of Pdcd4 by mir-21 faciliates glioblastoma proliferation in vivo, Neuro Oncol source
    • cites [19] Papagiannakopoulos, 2008, MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells, Cancer Res. source

      The Introduction identifies PDCD4 as a miR-21 target, and the target-validation section calls it a known target; the results also link PDCD4 to TGF-β-induced apoptosis. Our copy does not identify TIMP3 or RECK as miR-21 targets or establish their roles in metastatic pathways. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [20] Sekar, 2015, Role of microRNA 21 in mesenchymal stem cell (MSC) differentiation: a powerful biomarker in MSCs derived cells, Curr Pharm Biotechnol. source
    • cites [21] Gao, 2007, Frequent loss of PDCD4 expression in human glioma: possible role in the tumorigenesis of glioma, Oncol Rep. source

      The Introduction describes PDCD4 as a pre-apoptotic tumor suppressor, and the Discussion suggests that its loss in glioma may act through reduced apoptosis. Neither section identifies miR-21 as targeting PDCD4, and our copy does not address TIMP3 or RECK. The central targeting claim is therefore unsupported by this source. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [22] Chai, 2018, MicroRNA-21 promotes glioma cell proliferation and inhibits senescence and apoptosis by targeting SPRY1 via the PTEN/PI3K/AKT signaling pathway, CNS Neurosci Ther. source
    • cites [23] Malhotra, 2018, Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastomacells to lower effective doses of temozolomide in vitro and in a mouse model, Oncotarget. source

      The Introduction (page unavailable) lists PDCD4, TIMP3, and RECK as associated targets of miR-21. The Discussion (page unavailable) links PDCD4 to increased apoptosis, but the paper does not identify TIMP3 or RECK as key regulators of apoptotic or metastatic pathways. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    miRNA inhibition using decoy or sponge-like constructs has potential therapeutic benefit.

    “It has already been shown that miRNA inhibition using decoy or sponge-like constructs have a potential therapeutic benefit.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [29] Bak, 2013, Managing microRNAs with vector-encoded decoy-type inhibitors, Mol Ther source
    • cites [30] Mullokandov, 2012, High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries, Nat Methods. source
    • cites [31] Haraguch, 2009, Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells, Nucleic Acids Res. source
  • Premisecitation

    miR-21 has been demonstrated to be packaged within exosomes and released from different cancer types.

    “Moreover, miR-21 has been demonstrated to be packaged within exosomes and released from different cancer types (51–54).”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [51] Tanaka, 2013, Clinical impact of serum exosomal microRNA-21 as a clinical biomarker in human esophageal squamous cell carcinoma, Cancer. source
    • cites [52] Tian, 2014, Exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating miR-21 delivery, J Biol Chem. source

      The Discussion states that PC12 cell-derived exosomes enclosed abundant miR-21, and the section on delivery into BMSCs reports miR-21 in those exosomes. This demonstrates packaging by one tumor-cell type, but the paper does not demonstrate miR-21 release in exosomes from different cancer types. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [53] Tsukamoto, 2017, Circulating exosomal microRNA-21 as a biomarker in each tumor stage of colorectal cancer, Oncology. source

      The Results in the indexed Japanese thesis summary (page unavailable) report miR-21 in plasma exosomes from colorectal cancer patients. The discussion says its correlation with tumor-tissue miR-21 suggests a possible tumor origin, but does not demonstrate release from the tumors. The copy addresses colorectal cancer, not different cancer types. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [54] Wang, 2015, Macrophage-secreted exosomes delivering miRNA-21 inhibitor can regulate BGC-823 cell proliferation, Asian Pac J Cancer Prev. source
  • Premisecitation

    Suppressing miR-21 enhanced the therapeutic effects of antiangiogenic drug sunitinib in glioblastoma.

    “Suppressing miR-21 also enhanced the therapeutic effects of antiangiogenic drug, sunitinib, in glioblastoma (55).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [55] Costa, 2013, MicroRNA-21 silencing enhances the cytotoxic effect of the antiangiogenic drug sunitinib in glioblastoma, Hum Mol Genet. source
  • Classified as probably not a substantial claim. 26
  • May combine two claims. 24
  • The quote only partly states this claim. 1

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

    Premisecitation

    Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors/gliomas.

    “Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors/gliomas.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [1] Van Meir, 2010, Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma, CA Cancer J Clin source

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

    Premisecitation

    Extracellular vesicles are membrane fragments shed from cell surfaces to transfer cytoplasmic or membrane contents to neighboring cells or body fluids.

    “Extracellular vesicles (Evs) are membrane fragments shed from cell surfaces to transfer cytoplasmic or membrane contents to neighboring cells or body fluids.”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [5] Ratajczak, 2006, Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication, Leukemia. source
    • cites [6] Yuana, 2013, Extracellular vesicles in physiological and pathological conditions, Blood Rev. source

      The Introduction and Practice points (pages not provided) describe membrane-enclosed EVs that carry biomolecules to recipient cells, and the Introduction reports EVs in body fluids. But the Formation and shedding section distinguishes microvesicles shed directly from the cell surface from exosomes released through multivesicular bodies; it does not characterize all EVs as surface-shed fragments. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [7] Tkach, 2016, Communication by extracellular vesicles: where we are and where we need to go, Cell. source

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

    Premisecitation

    GBM is grade 4 according to the World Health Organization (WHO) classification.

    “Glioma is divided into 4 grades and GBM is the grade 4, according to the World Health Organization (WHO) classifications (1).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [1] Van Meir, 2010, Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma, CA Cancer J Clin source

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

    Premisecitation

    miRNAs are small (~22 nt) non-coding RNAs involved in diverse physiological and developmental processes, and their dysregulation leads to several diseases including cancer.

    “miRNAs are small (~22 nt) non-coding RNAs involved in diverse physiological and developmental processes, and their dysregulation lead to several diseases including cancer (14–16).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [14] Hayes, 2014, MicroRNA in cancer: biomarkers, functions and therapy, Trends Mol Med. source
    • cites [15] Paul, 2018, Interplay between miRNAs and human diseases, J Cell Physiol source
    • cites [16] Lyudmila, 2016, Regulatory mechanisms of microRNA expression, J Transl Med source

      The Background describes miRNAs as 18–22-nucleotide regulators involved in many biological processes and reports altered expression in various diseases, especially cancer. The DNA methylation section says deregulated miRNA expression may contribute to cancer development, but the paper does not establish the broader causal claim that miRNA dysregulation leads to several diseases. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The life expectancy of GBM patients is approximately 18 months after diagnosis.

    “The life expectancy of the GBM patients is ~18 months after diagnosis (2).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [2] Paolillo, 2018, Glioblastoma under siege: an overview of current therapeutic strategies, Brain Sci. source

  • May combine two claims.

    Premisecitation

    miR-21 is overexpressed in almost all cancer types, and its upregulation promotes cell proliferation, invasion, and metastatic behavior.

    “miR-21 is a well-known miRNA, found to be overexpressed in almost all cancer types, where its upregulation promotes cell proliferation, invasion, and metastatic behavior.”
    Quote in the paperSubstantial claim5 of 7 sources checked
    • cites [17] Gabriely, 2008, MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators, Mol Cell Biol. source

      The Discussion reports miR-21 upregulation in a variety of cancers and links elevated expression to proliferation and migration; the Results show that inhibiting miR-21 reduces glioma-cell invasion. It does not establish overexpression in almost all cancer types or show metastatic behavior, which goes beyond the invasion and migration measured here. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [18] Gaur, 2011, Downregulation of Pdcd4 by mir-21 faciliates glioblastoma proliferation in vivo, Neuro Oncol source

      The Discussion (page unavailable) reports elevated miR-21 in several cancer types, not almost all cancer types. The Abstract (page unavailable) reports that reducing miR-21 decreases proliferation in glioblastoma-derived cells, but this study does not establish that miR-21 upregulation promotes invasion or metastasis; those topics appear in titles of cited works in the References. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [19] Papagiannakopoulos, 2008, MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells, Cancer Res. source

      The Introduction (page unavailable) reports increased miR-21 in most human cancers, while the Abstract and miR-21 target validation section (pages unavailable) support a role in glioblastoma cell growth. The claim's stronger “almost all cancer types” wording is not established, and this paper does not report that miR-21 promotes invasion or metastatic behavior. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [20] Sekar, 2015, Role of microRNA 21 in mesenchymal stem cell (MSC) differentiation: a powerful biomarker in MSCs derived cells, Curr Pharm Biotechnol. source
    • cites [21] Gao, 2007, Frequent loss of PDCD4 expression in human glioma: possible role in the tumorigenesis of glioma, Oncol Rep. source

      The Introduction and Discussion report loss of PDCD4 expression in several cancers and in glioma samples; they do not address miR-21. Searches for miR-21 overexpression and for microRNA-related proliferation, invasion, and metastasis found no supporting passage in our copy. Page numbers were not provided. (LLM, conf=0.77)

      Experimental — accuracy not yet measured
    • cites [22] Chai, 2018, MicroRNA-21 promotes glioma cell proliferation and inhibits senescence and apoptosis by targeting SPRY1 via the PTEN/PI3K/AKT signaling pathway, CNS Neurosci Ther. source
    • cites [23] Malhotra, 2018, Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastomacells to lower effective doses of temozolomide in vitro and in a mouse model, Oncotarget. source

      The Introduction identifies miR-21 as an oncomiR overexpressed in the majority of glioblastomas, and the Discussion links miR-21 downregulation to decreased proliferation. The source does not establish overexpression in almost all cancer types; its explicit account of increased invasion concerns miR-10b, while its mention of metastasis concerns miRNAs generally, not miR-21 specifically. Page numbers are not available in our copy. (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

    Exosomes are a subclass of extracellular vesicles with a size of 30-100 nm and act as important mediators of signaling between tumor cells and other cells in the microenvironment.

    “Exosomes are a subclass of EVs with a size of 30–100 nm, with a recognized role as an important mediator of signaling event between tumor cells and other cells in its microenvironment (8, 9).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [8] Arscott, 2013, Ionizing radiation and glioblastoma exosomes: implications in tumor biology and cell migration, Transl Oncol. source

      The Introduction (page not specified) describes exosomes among extracellular vesicles and says tumor exosomes mediate signaling between tumor and surrounding stromal cells. It gives an average size of 100 nm, however, not the claimed 30–100 nm range. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [9] Kucharzewska, 2013, Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development, Proc Natl Acad Sci USA source

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

    Premisecitation

    Despite current standard of care combining surgery, radiotherapy and chemotherapy, GBM patients have frequent recurrence and very short life expectancy.

    “Despite the current standard of care that combines surgery, radiotherapy and chemotherapy, GBM patients are nonetheless confronted with frequent recurrence, and hence their life expectancy is very short (1, 2, 34).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [1] Van Meir, 2010, Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma, CA Cancer J Clin source
    • cites [2] Paolillo, 2018, Glioblastoma under siege: an overview of current therapeutic strategies, Brain Sci. source
    • cites [34] Gilbert, 2011, Recurrent glioblastoma: a fresh look at current therapies and emerging novel approaches, Semin Oncol. source

  • Classified as probably not a substantial claim.

    Premisecitation

    Sponge constructs are designed to bind complementary miRNA(s) or their seed sequences, and hence block the binding of the miRNA to its biological targets.

    “The sponge construct are designed to bind its complementary miRNA(s) or their seed sequences, and hence block the binding of the miRNA to its biological targets (29–31).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [29] Bak, 2013, Managing microRNAs with vector-encoded decoy-type inhibitors, Mol Ther source
    • cites [30] Mullokandov, 2012, High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries, Nat Methods. source
    • cites [31] Haraguch, 2009, Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells, Nucleic Acids Res. source

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

    Premisecitation

    Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors, glioma.

    “Glioblastoma multiform (GBM) is the most aggressive form of brain tumors, glioma.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [1] Van Meir, 2010, Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma, CA Cancer J Clin source
    • cites [2] Paolillo, 2018, Glioblastoma under siege: an overview of current therapeutic strategies, Brain Sci. source
    • cites [34] Gilbert, 2011, Recurrent glioblastoma: a fresh look at current therapies and emerging novel approaches, Semin Oncol. source

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

    Premisecitation

    GBM cells employ exosomes for tumor cell/microenvironment communications and to facilitate proliferation, invasion, and metastatic behaviors.

    “Similar to other cancer cell types, GBM cells employ exosomes for tumor cells/microenvironment communications and to facilitate its proliferation, invasion, and metastatic behaviors (8, 9).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [8] Arscott, 2013, Ionizing radiation and glioblastoma exosomes: implications in tumor biology and cell migration, Transl Oncol. source

      The Introduction (page not provided) describes exosome-mediated communication between tumor and stromal cells, and the Discussion reports that exosomes from irradiated GBM cells enhance recipient-cell migration. The paper does not establish that GBM exosomes facilitate proliferation or metastatic behavior; its discussion of a premetastatic niche concerns cancer generally, while it describes GBM invasion as local. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [9] Kucharzewska, 2013, Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development, Proc Natl Acad Sci USA source

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Premisecitation

    Exosomes are currently considered an important tool for extracellular communications and clinical process.

    “The extracellular vesicles, exosomes, are currently considered an important tool for extracellular communications and clinical process (35–37).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [35] Huang, 2019, Current progresses of exosomes as cancer diagnostic and prognostic biomarkers, Int J Biol Sci. source
    • cites [36] Sun, 2018, Effect of exosomal miRNA on cancer biology and clinical applications, Mol Cancer. source
    • cites [37] Tai, 2018, Exosomes in cancer development and clinical applications, Cancer Sci. source

  • May combine two claims.

    Premisecitation

    Inhibition of miR-21 via different strategies elevated apoptotic cell death, sensitivity to chemotherapy/radiotherapy, and diminished tumor progression.

    “Inhibition of miR-21 via different strategies elevated apoptotic cell death, sensitivity to chemotherapy/radiotherapy, and diminished tumor progression (24–28).”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [24] Devulapally, 2015, Polymer nanoparticles mediated codelivery of antimiR-10b and antimiR-21 for achieving triple negative breast cancer therapy, ACS Nano source

      The paper’s Abstract (page unavailable) reports that antisense inhibition of miR-21 induces apoptosis and impairs tumor-cell growth. Its Conclusion (page unavailable) reports reduced tumor growth with combined anti-miR-21/anti-miR-10b nanoparticles. It does not show increased sensitivity to chemotherapy or radiotherapy; the Conclusion proposes combining the approach with chemotherapy as future work. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [25] Sicard, 2013, Targeting miR-21 for the therapy of pancreatic cancer, Mol Ther source

      The Discussion and Results (page unavailable in our copy) report increased apoptosis and reduced pancreatic tumor progression after miR-21 depletion. The Results also report tumor regression when miR-21 inhibition was combined with gemcitabine. The paper does not establish increased radiotherapy sensitivity, and its own findings do not demonstrate these effects across different inhibition strategies. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [26] Belter, 2016, Inhibition of miR-21 in glioma cells using catalytic nucleic acids, Sci Rep source
    • cites [27] Yang, 2014, MicroRNA-21 promotes glioblastoma tumorigenesis by down-regulating insulin-like growth factor-binding protein-3 (IGFBP3), J Biol Chem. source

      The Discussion (page unavailable) reports that miR-21 knockdown sensitized cells to the apoptotic effects of chemotherapeutic agents, and the paper reports that knockdown markedly inhibited tumor formation in vivo. It describes miR-21 as involved in resistance to radiation, but does not show that inhibition increased radiotherapy sensitivity; this study also uses an antagomiR-21 knockdown strategy rather than demonstrating different inhibition strategies. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [28] Lu, 2008, MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene, Oncogene. source

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

    Premisecitation

    Exosome stability under different pH, temperature and other conditions has been studied and reported.

    “Exosomes stability in different PH, temperature and other conditions has been already studied and reported.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [61] Cheng, 2019, Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes, Protein Cell. source
    • cites [62] Maroto, 2017, Effect of storage temperature on airway exosomes for diagnostic and analyses, J Extracell Vesicles. source

      The Introduction and Results report that storage temperature affects airway exosome morphology and protein content. The Materials and methods section says the solution’s pH was not adjusted; this paper does not report testing exosome stability across different pH conditions. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    C6 cell line has a close gene expression pattern to human brain tumors and is widely used in neuro-oncological studies.

    “To confirm our results, we also employed C6 cell line for generating xenograft rat models, because of a close gene expression pattern to human brain tumors and they are also widely used in neuro-oncological studies (58–60).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [58] Zheng, 2007, Most C6 cells are cancer stem cells: evidence from clonal and population analyses, Cancer Res. source
    • cites [59] Barth, 2009, Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9, RG2, F98, BT4C, RT-2 and CNS-1 gliomas, J Neurooncol. source
    • cites [60] Shi, 2015, Establishment of C6 brain glioma models through stereotactic technique for laser interstitial thermotherapy research, Surg Neurol Int. source

      The Discussion (page not provided) describes stereotactically implanted C6 glioma models as among the most widely used, supporting the claim about their use in neuro-oncological research. The Results report resemblance to human glioma in histopathological features, not a close gene-expression pattern; our copy does not establish that genetic claim. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Watson et al. showed that yield of exosomes can be increased by 5-10 fold using a hollow fiber bioreactor.

    “Watson et al. (38) showed that yield of exosomes can be increased by 5–10 fold using a hollow fiber bioreactor.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] Watson, 2016, Efficient production and enhanced tumor delivery of engineered extracellular vesicles, Biomaterials. source

      Results §3.1 reports approximately fivefold more extracellular vesicles (EV) from bioreactor-conditioned medium than from conventional flasks. The tenfold total improvement also required an optimized purification step. The paper measures EV, a category that includes exosomes, rather than establishing a five- to tenfold increase specifically in exosome yield from the bioreactor alone. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    The existence of the blood-brain-barrier introduces a unique challenge for applying cell-based gene therapy of brain tumors.

    “The existence of the blood-brain-barrier introduces a unique challenge for applying cell-based gene therapy of brain tumors (63, 64).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [63] Van Tellingen, 2015, Overcoming the blood-brain tumor barrier for effective glioblastoma treatment, Drug Resist Updat source
    • cites [64] Sarkaria, 2018, Is the blood-brain barrier really disrupted in all glioblastomas?, Neuro Oncol source

      The paper’s Abstract says that an intact blood–brain barrier limits delivery of many cancer therapeutics to glioblastoma regions. It does not address cell-based gene therapy specifically, so it supports the general treatment challenge but not that application. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    microRNA's presence in exosomes has been reported in different cells and cancer types.

    “microRNA's presence in exosomes have been already reported in different cells and cancer types (44–50).”
    Quote in the paperNot a substantial claim7 of 7 sources checked
    • cites [44] Collino, 2010, Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs, PLoS ONE. source

      The Introduction (page not available) reports miRNAs in mast-cell exosomes and in microvesicles from several other cell sources, including the blood of patients with ovarian tumors. It does not establish their presence in exosomes across different cancer types; the tumor-related example concerns blood microvesicles, not identified cancer-cell exosomes. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [45] Gonzalez-Villasana, 2019, Presence of circulating miR-145, miR-155, and miR-382 in exosomes isolated from serum of breast cancer patients and healthy donors, Dis Markers source
    • cites [46] Huang, 2017, A novel serum microRNA signature to screen esophageal squamous cell carcinoma, Cancer Med. source
    • cites [47] Li, 2018, Exosomal miR-21-5p derived from gastric cancer promotes peritoneal metastasis via mesothelial-to-mesenchymal transition, Cell Death Dis. source
    • cites [48] Ogata-Kawata, 2014, Circulating exosomal microRNAs as biomarkers of colon cancer, PLoS ONE. source
    • cites [49] Yuan, 2009, Transfer of microRNAs by embryonic stem cell microvesicles, PLoS ONE. source

      The Results section reports miRNAs in embryonic stem-cell microvesicles, and the Introduction reports prior findings in peripheral-blood and ovarian-tumor microvesicles. The paper does not identify these vesicles as exosomes or establish miRNA presence across multiple cancer types. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [50] Zhu, 2019, Macrophages derived exosomes deliver miR-223 to epithelial ovarian cancer cells to elicit a chemoresistant phenotype, J Exp Clin Cancer Res. source

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

    Premisecitation

    Numerous miRNAs with oncogenic and tumor-suppressive properties have been identified in GBM.

    “Numerous miRNAs with oncogenic and tumor-suppressive properties have been identified in GBM (42).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [42] Haapa-Paananen, 2013, Functional profiling of precursor microRNAs identifies microRNAs essential for glioma proliferation, PLoS ONE. source

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

    Premisecitation

    Exosome modifications under different maintenance conditions and freeze-thawing cycles have been reported.

    “Moreover, exosomes modifications under a different situation of maintenance and with a different cycle of freeze thawing has been already reported (61, 62).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [61] Cheng, 2019, Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes, Protein Cell. source
    • cites [62] Maroto, 2017, Effect of storage temperature on airway exosomes for diagnostic and analyses, J Extracell Vesicles. source

  • May combine two claims.

    Premisecitation

    The engineered exosomes could cross the multiple layers of the blood-brain-barrier and target cancer cells within the brain tissue.

    “Importantly, the engineered exosomes could cross the multiple layers of the blood-brain-barrier (BBB) and target cancer cells within the brain tissue (40, 65–68).”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [40] Ha, 2016, Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges, Acta Pharm Sin B source

      The section on drug delivery vehicles for small molecules (page not available) reports drug distribution in zebrafish brains after exosome delivery across the BBB and improved efficacy in a zebrafish brain-cancer model. It does not establish that the exosomes crossed multiple distinct BBB layers or specifically targeted cancer cells within brain tissue. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [65] Chen, 2016, Elucidation of exosome migration across the blood-brain barrier model in vitro, Cell Mol Bioeng. source

      The Results and Discussion report that engineered exosomes crossed a single brain microvascular endothelial cell monolayer in vitro under TNF-α-induced, stroke-like conditions. The Conclusions say that astrocytes, pericytes, and basement membrane were not included in that model. The paper does not show passage through the BBB’s multiple layers or targeting of cancer cells in brain tissue. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [66] Druzhkova, 2018, Exosome drug delivery through the blood–brain barrier: experimental approaches and potential applications, Neurochem J. source
    • cites [67] Morales-Prieto, 2018, Peripheral blood exosomes pass blood-brain-barrier and induce glial cell activation, bioRxiv [Preprint] source

      The Discussion states that exosomes can cross the blood-brain barrier and reports that injected peripheral-blood exosomes localized in recipient mice’s brains (page unavailable). It does not show that engineered exosomes crossed multiple BBB layers or targeted cancer cells in brain tissue; the study examined peripheral-blood exosomes and glial activation. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [68] Yuan, 2017, Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain, Biomaterials. source

      The Conclusion and Future Directions section says macrophage exosomes can cross the BBB and carry cargo into brain tissue, particularly under inflammation. It does not show that they target cancer cells, and it expressly limits its barrier conclusion to the vascular barrier rather than multiple barriers. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    SEM showed extracted vesicles have <100 nm diameters, corresponding to typical exosomes.

    “Finally, SEM showed a <100 nm diameters of the extracted vesicles, corresponding to the size of the typical exosomes (Supplementary Figure 2C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with freshly prepared exosomes containing miR-21-sponge inhibited tumor growth and eradicated tumor mass.

    “Results from the third repeat treated with freshly prepared exosomes containing miR-21-sponge demonstrated good effects not only in inhibiting tumor growth, but also in eradicating tumor mass.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Secreted miR-21 had a significant effect on U87-MG miR-21 level after 24 h of co-culture, while the effect of miR-21-sponge was more evident after 48 h.

    “Although the secreted miR-21 had a significant effect in U87-MG's miR-21 level after 24 h of co-culture, however, the effect of miR-21-sponge was more evident after 48 h of treatment.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    At 48 h post-treatment, less effects on Q2 and Q4 were seen compared to 24 h, but an increase in early apoptosis (Q3) and necrotic cells (Q1) was observed (5.54% and 4.29% of total cell count).

    “At 48 h post-treatment, less effects on Q2 and Q4 were seen, in comparison with the 24 h treatment group. However, an increase in the percentage of the cells in early apoptosis (Q3) and necrotic cells (Q1) was observed (respectively, 5.54 and 4.29% of total cell count; Figure 6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Secreted miR-21-sponge can be transferred from producing HEK-293T cells to U87-MG cells and reduce miR-21 level in target cells.

    “Our data revealed that secreted miR-21-sponge can be transferred from the producing cells to the U87-MG cells and reduce the level of miR-21 in the target cells (Figure 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After 48 h of treatment, MTT absorbance alteration was still significant (P < 0.05), but lower than at 24 h.

    “After 48 h of treatment, MTT absorbance alteration was still significant (P < 0.05), however, the level of alterations was lower than that of the 24 h time point (Figure 5).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    At 24 h after treatment, miR-21-sponge containing exosomes caused an increase up to 30.9% (compared to 17% of untreated cells) in late apoptosis (Q2) and a decrease in alive cells (Q4) from 80% untreated to 64% treated.

    “At 24 h after treatment, miR-21-sponge containing exosomes caused an increase (up to 30.9%, compared to 17% of untreated cells) in the rate of late apoptosis (Q2) and a decrease in the percentage of the alive cells (Q4), from 80% on untreated samples to 64% in miR-21-sponge treated samples (Figure 6).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Western blot confirmed the presence of specific exosome markers CD81 and CD63 on the surface of extracted exosomes.

    “Moreover, Western blot assay confirmed the presence of the specific exosomes markers on the surface of the extracted exosomes (Supplementary Figure 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Exosomes filled with miR-21-sponge downregulated miR-21 in U87-MG and C6 cells and upregulated PDCD4 and RECK in U87-MG target cells.

    “On the other hand, as expected, exosomes filled with miR-21-sponge downregulated miR-21 in U87-MG and C6 cells and upregulated PDCD4 and RECK in U87-MG target cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    miR-21-sponge containing exosomes have the potential to cause apoptosis and reduce cell viability in U87-MG target cells, through miR-21 inhibition.

    “Altogether, our data revealed that miR-21-sponge containing exosomes have the potential to cause apoptosis and reduce cell viability in U87-MG target cells, through miR-21 inhibition.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Tumor production was verified by MRI with T2 method after 11 days.

    “After 11 days, the tumor production was verified by Magnetic Resonance Imaging (MRI) with T2 method.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All data were repeated at least 3 times and analyzed with ΔΔCT method by GraphPad Prism 6, with ordinary ANOVA and P < 0.05 considered significant.

    “All data repeated at least 3 times and were analyzed with ΔΔCT method by GraphPad Prism 6 software. The statistically significant changes tested with the ordinary ANOVA test. All histograms were presented as mean ±SD, and differences were considered as significant when the P < 0.05.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Three independent replicates were used for each MTT experiment.

    “Three independent replicates were used for each experiment.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Stable cell lines were cultured in media supplemented with exosome-free FBS to avoid exosome contamination.

    “Stable cell lines were cultured in a T75 flask in media supplemented with exosome-free FBS [exosomes depleted by centrifugation at 100,000 g for 2.5 h; (33)].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [33] Huan, 2013, RNA trafficking by acute myelogenous leukemia exosomes, Cancer Res. source

  • May combine two claims.

    Interpretationown data

    Engineered exosomes have the potential to suppress miR-21 and consequently upregulate miR-21 target genes PDCD4 and RECK.

    “Our data revealed that the engineered exosomes have the potential to suppress miR-21 and consequently to upregulate miR-21 target genes, PDCD4 and RECK.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Tumor size was measured by FSL with threshold-determining and manually voxel counting.

    “The size of the tumors were then measured by FSL (FMRIB Software Library), with a threshold-determining and manually voxel counting.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    5S rRNA was used as an internal control for miR-21 data analysis and GAPDH for PDCD4 and RECK expression analysis.

    “5S rRNA (HGNC: 1380) was used as an internal control for miR-21 data analysis and GAPDH (HGNC: 4141) was also employed as the internal control for PDCD4 and RECK expression analysis (Supplementary Table 1).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The accuracy of cloning procedures was confirmed by DNA sequencing.

    “The accuracy of cloning procedures was confirmed by DNA sequencing (Macrogene, South Korea).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Two independent replicates were used for each flow cytometry experiment.

    “Two independent replicates were used for each experiment.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Engineered exosomes containing miR-21-sponge have therapeutic potential to combat glioblastoma, suggesting a new therapeutic strategy.

    “Our data revealed that the exosomes containing miR-21-sponge have a therapeutic potential to combat glioblastoma.”
    • Also stated in: § Abstract · hedged
      “In brief, our findings suggest a new therapeutic strategy to use engineered exosomes to deliver a miR-21-sponge construct to GBM cells, in order to block its malignant behavior.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The C6 rat glioma xenograft model is a valid model of human glioblastoma for testing therapeutic effects.
    • Rests on an assumption: The U87-MG and C6 cell lines are valid models of human glioblastoma.
    • Rests on an assumption: Effects observed in cell lines and the rat model predict therapeutic effects in human glioblastoma.

  • May combine two claims.

    Interpretationown data

    In cells treated with miR-21-sponge exosomes, there is a decline in proliferation and an elevation in apoptotic rates.

    “Interestingly, in cells treated with miR-21-sponge exosomes we observed a decline in proliferation and also an elevation in apoptotic rates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    The engineered exosomes have the potential to successfully cross the BBB and transfer their contents within the brain.

    “It is safe to suggest that the engineered exosomes have the potential to successfully cross the BBB and transfer their contents within the brain.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    Whether yield of exosomes was actually increased by use of the bioreactor is not clear, since the obtained sample contained larger vesicles (200-800 nm in diameter).

    “However, whether yield of exosomes was actually increased by use of the bioreactor is not clear, since the obtained sample contained larger vesicles (200–800 nm in diameter).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The latter results approved the suitability of the miR-21-sponge packaged exosomes to combat GBM, and probably other types of cancers.

    “The latter results approved the suitability of the miR-21-sponge packaged exosomes to combat GBM, and probably other types of cancers.”
    Quote in the paperSubstantial claim
45 of 45

  • The paper relies on this without support.

    The C6 rat glioma xenograft model is a valid model of human glioblastoma for testing therapeutic effects.

    “we generated a glioblastoma xenograft rat model by stereotaxically injecting 1 million C6 cells in 10 μl of PBS at Caudate Putamen striatum (CPu, 2 mm up and right from bregma, in 4 mm depth) of Wistar male rats (250–300 g).”

  • The paper relies on this without support.

    The reduction in tumor volume in the rat model is mediated by miR-21-sponge inhibition of miR-21.

    “Finally, in a rat model of glioblastoma, administrating exosomes loaded with a miR-21-sponge construct leads to a significant reduction in the volume of the tumors.”

  • The paper relies on this without support.

    The U87-MG and C6 cell lines are valid models of human glioblastoma.

    “The glioblastoma cell line, U87-MG, is used to examine a potential effect of secreted miR-21 and miR-21-sponge in a co-culture system with the HEK-293T stably transfected cells.”

  • The paper relies on this without support.

    Effects observed in cell lines and the rat model predict therapeutic effects in human glioblastoma.

    “In brief, our findings suggest a new therapeutic strategy to use engineered exosomes to deliver a miR-21-sponge construct to GBM cells, in order to block its malignant behavior.”
4 of 4

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.

  • Viable and early-apoptotic quadrants are reversed

    Minor

    The results identify Q4 as viable cells and Q3 as early-apoptotic cells, but the Figure 6 caption assigns viability to Q3 and early apoptosis to Q4. This reverses the interpretation of the reported cell-death results.

  • Replication count conflicts with flow-cytometry methods

    Minor

    The statistical-analysis section says all data were repeated at least three times, whereas the flow-cytometry method specifies two independent replicates per experiment.

2 of 2

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

  • Cites a retracted work: 10.1111/cns.12785

    Info

    The cited work was retracted on 2021-01-25 (notice 10.1111/cns.13614), not before this paper was published (2019-08-20).

    Cited work 10.1111/cns.12785 · retracted 2021-01-25 · notice 10.1111/cns.13614 · this paper published 2019-08-20

1 of 1

Cites

1 retracted

Flagged first, then by citations

Cited by

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Related

Authors
Monfared, H., Jahangard, Y., Nikkhah, M., Mirnajafi-Zadeh, J., & Mowla, S. J.
Affiliations
Tarbiat Modares University (IR)
Corresponding author
Mowla, Seyed Javad
ORCID
Venue
Frontiers in Oncology
Publisher
Frontiers Media SA
Published
2019-08-20
Volume
9
Pages
782-782
ISSN
2234-943X (ISSN-L)
DOI
10.3389/fonc.2019.00782
PMC
PMC6710330
OpenAlex
W2964477143
Semantic Scholar
3305717fd9383d24f9e7a85bc1cf9517c61dcdc7
Cited by
123
References
70
Citations by year
2026: 9 · 2025: 18 · 2024: 27 · 2023: 17 · 2022: 15 · 2021: 18 · 2020: 18 · 2019: 1
FWCI
3.72
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Oncology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
glioblastoma, exosomes, engineered exosomes, microRNA delivery, extracellular vesicles, PDCD4, RECK, tumor cell proliferation, apoptosis

About these checks

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 2026-10-21.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-21.

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Statistics and arithmetic are recomputed, not judged.

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