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Cited by 0MicroRNA in disease regulationCancer-related molecular mechanisms researchGlioma Diagnosis and Treatment

Functional interaction between miR-505-3p and miR-21 contributes to GBM suppression

Open access
Banadkooki, Mohammadreza Zare, Soltani, Bahram Mohammad, Mowla, Seyed Javad·2026·Scientific Reports
DOI 10.1038/s41598-026-59926-5
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Abstract

Recently, studies have shown that non-coding RNAs (ncRNAs) play an important role in cancer. An important group of these ncRNAs are micro RNAs (miRs), which are important regulators in the cell. miR-21 is one type of miR that plays an oncogenic role in various cancers, especially glioblastoma (GBM). On the other hand, according to the studies, miR-505-3p is known as a tumor suppressor (TSG). Other studies conducted in recent years indicate that miRs may target other ncRNAs, including other miRs. This mode is called miR-miR interaction. Therefore, this study aims to investigate the relationship of these two miRs in GBM. Bioinformatic analyses predicted a potential interaction between miR-505-3p and pri-miR-21. RNA hybridization modeling suggested thermodynamically feasible binding. Overexpression of miR-505-3p in GBM cell lines (U87-MG and A172) was associated with reduced levels of mature miR-21-5p. Concomitantly, increased mRNA and protein expression of known miR-21 target genes, including PDCD4, HNRNPK, and RECK, was observed. Functional assays demonstrated reduced cell proliferation and increased sub-G1 cell population following miR-505-3p overexpression. Dual luciferase reporter assays using a pri-miR-21 construct suggested that miR-505-3p may interfere with miR-21 processing. Our findings suggest that miR-505-3p may modulate miR-21 expression and is associated with restoration of tumor suppressor gene expression in GBM cells. While the data support a potential regulatory relationship, the precise molecular mechanism remains to be elucidated. Further studies are required to determine whether miR-505-3p directly interacts with pri-miR-21 and to clarify the biological relevance of this axis in GBM.

AI snapshot

Written from the paper’s full text.

TL;DR

Overexpression of miR-505-3p in U87-MG and A172 glioblastoma cells was associated with reduced mature miR-21-5p, increased expression of miR-21 target tumor suppressors PDCD4, HNRNPK, and RECK, and reduced proliferation. The authors propose a miR-505-3p/pri-miR-21 interaction that may interfere with miR-21 processing, but direct binding and the precise mechanism remain unproven.

Key findings

  • Bioinformatic prediction suggested a potential interaction between miR-505-3p and pri-miR-21, with RNAhybrid reporting a minimum free energy of −21.6 kcal/mol.
  • miR-505-3p overexpression in U87-MG and A172 cells significantly decreased miR-21-5p expression and significantly increased mRNA and protein levels of PDCD4, HNRNPK, and RECK compared with mock.
  • MTT assays showed significantly reduced cell proliferation at 24 h and 48 h after miR-505-3p overexpression; cell cycle assays showed a significant increase in sub-G1 population cells.
  • Dual luciferase assays using a pri-miR-21 construct suggested that miR-505-3p may prevent miR-21 maturation and restore Renilla expression compared with mock, but the authors state direct interaction cannot be conclusively established without binding-site mutation controls.

Methods

  • In silico analysis used ENCORI for Ago CLIP-seq-based miRNA–miRNA interaction prediction, RNAhybrid for pri-miR-21/miR-505-3p binding prediction, and MXfold2 for secondary structure prediction.
  • U87-MG, A172, and HEK293T cells were cultured and transfected with plasmid constructs: pPri miR-505 in pcDNA 3.1(+) and pPri miR-21 in psiCHECK-2; validation was by Sanger sequencing.
  • Molecular and functional assays included qRT-PCR for miR-21, miR-505, and target genes; western blot for PDCD4, HNRNPK, and RECK; MTT proliferation assays at 24, 48, and 72 h; propidium iodide cell cycle analysis; and dual luciferase reporter assays. Statistical analysis used GraphPad Prism with t-tests, significance at p < 0.05, and at least three independent biological replicates unless otherwise stated.

Limitations

  • The paper states that the precise molecular mechanism remains to be elucidated and that definitive evidence of direct interaction between miR-505-3p and pri-miR-21 is lacking; no binding-site mutation control was performed.
  • The authors note that the dual luciferase assay was performed in an unconventional manner and that many additional assays are needed to prove the proposed Drosha-blocking hypothesis.
  • The paper states that GBM is highly heterogeneous and that validation in additional models, including patient-derived glioma stem cells and in vivo systems, is required; suggested but not performed experiments include antagomir knockdown of miR-505-3p, pri-miR-21 pull-down, and nuclear/cytoplasmic fractionation, RNA-FISH, or AGO-associated nuclear localization studies.

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

Integrity signals

Registry

Crossref · 10.1038/s41598-026-59926-5

Cites

33 of 34 references verified0 citation errors · 1 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

11 of 15 cited sources checked · 2 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    The expression level of miR-505-3p is reduced in human GBM tissues and cell lines compared to control groups.

    “The expression level of miR-505-3p is reduced in human GBM tissues and cell lines compared to control groups (18).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Cheng, Z., Wang, B. & Zhang, C. MicroRNA–505–3p inhibits development of glioma by targeting HMGB1 and regulating AKT expression. Oncol. Lett. 20 (2), 1663–1670 (2020). source

      The Results (page unavailable) report reduced miR-505-3p in 44 glioma tissues versus normal tissues and in U251, A172, and LN229 cells versus normal human astrocytes. The Patients and methods section (page unavailable) shows that the tissue cohort included low- and high-grade gliomas, so the tissue finding is not established specifically for GBM. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
  • Premisecitation

    miR-505-3p is known to have a tumor suppressor role.

    “miR-505-3p is one of the miR types known to have a tumor suppressor role (14)(15)(16)(17).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [14] Chen, S., Sun, K. X., Liu, B. L., Zong, Z. H. & Zhao, Y. MicroRNA-505 functions as a tumor suppressor in endometrial cancer by targeting TGF-α. Mol. Cancer. 15, 11 (2016). source

      The Abstract Conclusions (page unavailable) state that miR-505 acts as a tumor suppressor in endometrial cancer, supported by the reported cell and mouse experiments. Our copy calls it miR-505, not miR-505-3p, so it does not establish the claim’s strand-specific designation. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [15] Tang, H., Lv, W., Sun, W., Bi, Q. & Hao, Y. miR–505 inhibits cell growth and EMT by targeting MAP3K3 through the AKT–NFκB pathway in NSCLC cells. Int. J. Mol. Med. 43 (3), 1203–1216 (2019). source

      The Abstract and Discussion state that miR-505 has a tumor-suppressor role in non-small cell lung cancer cells. The paper does not identify the tumor-suppressing form as miR-505-3p; the only exact mention of that form in the retrieved passages is a reference about macrophages. Thus, the tumor-suppressor role is supported, but the claim’s 3p specificity is not established by this copy. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [16] Ninomiya, M. et al. Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers. PloS one. 8 (6), e66086 (2013). source

      The Discussion identifies reduced serum miR-505-3p as a potential biomarker for primary biliary cirrhosis, but does not describe it as a tumor suppressor. Searches for “miR-505-3p tumor suppressor” and “tumor suppressor cancer miR-505” found no passage addressing that role. Page numbers were not available in our copy. (LLM, conf=0.73)

      Experimental — accuracy not yet measured
  • Findingown data

    Overexpression of miR-505-3p in U87-MG and A172 cells increased mRNA expression of PDCD4, HNRNPK, and RECK compared to mock.

    “qRT-PCR results indicated that expression of PDCD4, HNRNPK and RECK increases significantly in RNA level (Fig. 3A and Fig. 3B).”
    Quote in the paperSubstantial claim
  • Findingown data

    ENCORI analysis identified a binding site for miR-505-3p on Pri miR-21.

    “Also, AGO CLIP-seq data were extracted from the ENCORI platform, and the binding regions of miR-505-3p were explored, and it was found that there is a binding site on the Pri miR-21 (Table 1S).”
    Quote in the paperSubstantial claim
  • Findingown data

    ENCORI predicted a miRNA-miRNA interaction between miR-505-3p and Pri miR-21.

    “According to Ago CLIP-seq data, ENCORI predicted a miRNA-miRNA interaction between miR-505-3p and Pri miR-21.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 8
  • May combine two claims. 10

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

    Premisecitation

    HNRNPK inhibits GBM cells by inhibiting the proliferation of cancer cells.

    “HNRNPK plays an important role in inhibiting GBM cells by inhibiting the proliferation of cancer cells (31).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [31] Mucha, B. et al. Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation. Nat. Commun. 13 (1), 6614 (2022). source

      The Results section (“SCFFbxo4-hnRNPK-c-Myc regulates cell proliferation”; page not provided) reports that hnRNPK expression promoted proliferation in mouse embryonic fibroblasts, while hnRNPK knockdown prevented it—the opposite of the attributed inhibitory effect. The cited passages do not report an experiment in GBM cells. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    miRs can target other ncRNAs, including other miRs, a mode called miR-miR interaction.

    “It has also been shown that miRs can have other targets, including the miRs themselves.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [22] Hill, M. & Tran, N. miRNA interplay: Mechanisms and consequences in cancer. Dis. Models Mech. 14 (4), dmm047662 (2021). source

  • May combine two claims.

    Premisecitation

    Reduced expression of miR-505-3p enhances tumorigenic properties, including cell proliferation, colony formation, cell cycle progression, tumor cell migration, metastasis, and tumor growth in vivo.

    “Reduced expression of miR-505-3p enhances tumorigenic properties, including cell proliferation, colony formation, cell cycle progression, tumor cell migration, metastasis, and tumor growth in vivo (19)(20)(21).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [19] Guo, K., Fang, L., Li, M., Li, A. & Liu, N. Long Non-coding RNA KTN1-AS1 Targets miR-505 to Promote Glioblastoma Progression. Behav. Neurol. 2023, 4190849 (2023). source

      Results §3.4 (page not specified) reports that inhibiting miR-505 restored proliferation, migration, and invasion in GBM cells when KTN1-AS1 was silenced. The paper does not report that reduced miR-505-3p enhances colony formation, cell-cycle progression, metastasis, or tumor growth in vivo. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [20] Chung, E., Yang, H., Xing, H., Carroll, R. & Johnson, M. MiR-505 downregulates 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 4 to promote cell death in glioblastoma. Cancer Translational Med. ;1(5). (2015). source
    • cites [21] Zhang, C., Yang, X., Fu, C. & Liu, X. Combination with TMZ and miR-505 inhibits the development of glioblastoma by regulating the WNT7B/Wnt/β-catenin signaling pathway. Gene 672, 172–179 (2018). source

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

    Premisecitation

    PDCD4 inhibits cell proliferation by targeting AKT.

    “Previous studies show that PDCD4 inhibits cell proliferation by targeting AKT (30).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [30] Pin, G. et al. Down-regulation of PDCD4 promotes proliferation, angiogenesis and tumorigenesis in glioma cells. Front. Cell. Dev. Biology. 8, 593685 (2020). source

      The Introduction and the in-vivo Results describe PDCD4 as suppressing growth in glioma. But the Results section on AKT2 reports that AKT2 interacts with and suppresses PDCD4, not that PDCD4 inhibits proliferation by targeting AKT. The proposed AKT mechanism is therefore not supported as stated. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    PDCD4, HNRNPK, and RECK are tumor suppressor genes and direct targets of miR-21-5p.

    “Previous studies have proven that PDCD4, HNRNPK and RECK are TSGs among the direct targets of miR-21-5p (13,(27)(28)(29).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [13] Papagiannakopoulos, T., Shapiro, A. & Kosik, K. S. MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells. Cancer Res. 68 (19), 8164–8172 (2008). source

      The MiR-21 target validation section reports direct-target evidence for HNRPK (the paper’s abbreviation for HNRNPK) and identifies PDCD4 as a known miR-21 target. The Discussion links HNRPK to tumor-suppressive activity, and the References identify PDCD4 as a tumor suppressor. Our copy does not establish RECK as a target or specifically identify the targets as miR-21-5p targets; no page numbers are available. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [27] Chen, Y. et al. MicroRNA-21 down-regulates the expression of tumor suppressor PDCD4 in human glioblastoma cell T98G. Cancer Lett. 272 (2), 197–205 (2008). source
    • cites [28] Gabriely, G. et al. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol. Cell. Biol. 28 (17), 5369–5380 (2008). source

      The Results (page unavailable) identify PDCD4 as a tumor suppressor and a validated direct miR-21 target, and report experimental evidence that RECK is a direct miR-21 target. They describe RECK as an inhibitor of cancer invasion, but our copy does not call it a tumor suppressor gene. No retrieved passage identifies HNRNPK as a tumor suppressor or miR-21 target; the paper also uses “miR-21” rather than the specific designation “miR-21-5p.” (LLM, conf=0.87)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The precursor form of miR-21 is targeted by miR-122 in the nucleus in human hepatocyte cells.

    “A study in human hepatocyte cells found that the precursor form of miR-21 is targeted by miR-122 in the nucleus (23).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [23] Wang, D. et al. Nuclear miR-122 directly regulates the biogenesis of cell survival oncomiR miR-21 at the posttranscriptional level. Nucleic Acids Res. 46 (4), 2012–2029 (2018). source

  • May combine two claims.

    Interpretationown data

    miR-505-3p overexpression is associated with reduced miR-21 expression and increased levels of miR-21 target tumor suppressor genes in GBM cell lines.

    “Our results suggest that miR-505-3p overexpression is associated with reduced miR-21 expression and increased levels of miR-21 target tumor suppressor genes in GBM cell lines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    PDCD4, HNRNPK, and RECK are not directly related to miR-505-3p; their increased expression following miR-505-3p overexpression is due to reduced miR-21-5p.

    “However, according to the investigations carried out, these three genes are not directly related to miR-505-3p, and only by reducing the expression of miR-21-5p, which targets these genes, can their expression increase significantly.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In dual luciferase assays, miR-505-3p prevents maturation of miR-21 from its precursor, restoring Renilla expression compared to mock.

    “However, in the presence of miR-505-3p, the maturation of miR-21 from its precursor form cloned at the end of Renilla is prevented, and Renilla expression is largely restored compared to the Mock state (Fig. 6).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: In the dual luciferase construct, pri-miR-21 cloned at the end of Renilla is processed to mature miR-21, causing loss of the Renilla 3'UTR and impaired Renilla expression.

  • May combine two claims.

    Interpretationown data

    Overexpression of miR-505-3p followed by PDCD4 and HNRNPK leads to cell cycle inhibition, as examined and proved by MTT and cell cycle assay.

    “Overexpression of miR-505-3p followed by PDCD4 and HNRNPK leads to cell cycle inhibition, which is examined and proved by MTT and cell cycle assay.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    miR-505-3p may inhibit miR-21, and there is a negative co-expression between these two miRs.

    “This shows that miR-505-3p may inhibit miR-21, and there is a negative co-expression between these two miRs.”
    Quote in the paperSubstantial claim

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

    Study design and conductreasoning

    The study's findings are limited to two established GBM cell lines and may not generalize to the heterogeneity of GBM.

    “Although two established GBM cell lines were used, GBM is highly heterogeneous.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    By increasing miR-505-3p expression, miR-21 may be targeted in some way, and as a result, expression of TSGs previously suppressed by miR-21 can be restored.

    “So, by increasing miR-505-3p expression, miR-21 may be targeted in some way, and as a result, the expression of TSGs previously suppressed by miR-21 can be restored.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    All vectors were validated by Sanger sequencing.

    “All these vectors are validated by performing Sanger sequencing using the Sanger Sequencing 3500 Dx Genetic Analyzer (Applied Biosystems; Cat.#A27772).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The dual luciferase assay was performed in an unconventional manner.

    “The dual luciferase assay was performed in an unconventional manner, different from its usual form.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All experiments were performed with at least three independent biological replicates unless otherwise stated.

    “All experiments were performed with at least three independent biological replicates unless otherwise stated.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    No binding-site mutation control was performed, so direct interaction cannot be conclusively established.

    “However, because no binding-site mutation control was performed, direct interaction cannot be conclusively established.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The dual luciferase assay result showed a significant relationship between Pri miR-21 and miR-505-3p, and miR-505-3p may play an inhibitory role in miR-21.

    “The result of the dual luciferase assay showed a significant relationship between Pri miR-21 and miR-505-3p, and it shows that miR-505-3p may play an inhibitory role in miR-21.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: In the dual luciferase construct, pri-miR-21 cloned at the end of Renilla is processed to mature miR-21, causing loss of the Renilla 3'UTR and impaired Renilla expression.
18 of 18

  • The paper relies on this without support.

    The relationship between miR-505-3p and miR-21 in GBM cell lines had not been investigated before this study.

    “To date, the harnessing of miR-21 by miR-505-3p has not been investigated, and the study conducted is the first to investigate this relationship in GBM cell lines.”

  • The paper relies on this without support.

    In the dual luciferase construct, pri-miR-21 cloned at the end of Renilla is processed to mature miR-21, causing loss of the Renilla 3'UTR and impaired Renilla expression.

    “Because Pri miR-21 is converted to the mature form of miR, the 3'UTR area of the Renilla gene is lost, and Renilla protein expression is impaired.”
2 of 2

Cites

Flagged first, then by citations

1 of the 33 references couldn’t be matched to a known paper.

Authors
Banadkooki, M. Z., Soltani, B. M., & Mowla, S. J.
Affiliations
Tarbiat Modares University (IR)
Corresponding author
Mowla, Seyed Javad
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2026-06-25
Volume
16
Issue
1
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-026-59926-5
OpenAlex
W7165888909
Semantic Scholar
589f9ae4efe4aa08e32afb336f34d084e3192e91
Cited by
0
References
33
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
  • Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
Keywords
miR-21, glioblastoma, microRNA processing, tumor suppressor microRNA, PDCD4, hnRNP K, RECK, cell proliferation, cell cycle arrest, RNA hybridization

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