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. 2015 Mar;17(3):419-29.
doi: 10.1093/neuonc/nou220. Epub 2014 Sep 12.

Suppression of miR-184 in malignant gliomas upregulates SND1 and promotes tumor aggressiveness

Luni Emdad  1 , Aleksandar Janjic  1 , Mohammad A Alzubi  1 , Bin Hu  1 , Prasanna K Santhekadur  1 , Mitchell E Menezes  1 , Xue-Ning Shen  1 , Swadesh K Das  1 , Devanand Sarkar  1 , Paul B Fisher  1
Affiliations

Suppression of miR-184 in malignant gliomas upregulates SND1 and promotes tumor aggressiveness

Luni Emdad et al. Neuro Oncol. 2015 Mar.

Abstract

Background: Malignant glioma is an aggressive cancer requiring new therapeutic targets. MicroRNAs (miRNAs) regulate gene expression post transcriptionally and are implicated in cancer development and progression. Deregulated expressions of several miRNAs, specifically hsa-miR-184, correlate with glioma development.

Methods: Bioinformatic approaches were used to identify potential miR-184-regulated target genes involved in malignant glioma progression. This strategy identified a multifunctional nuclease, SND1, known to be overexpressed in multiple cancers, including breast, colon, and hepatocellular carcinoma, as a putative direct miR-184 target gene. SND1 levels were evaluated in patient tumor samples and human-derived cell lines. We analyzed invasion and signaling in vitro through SND1 gain-of-function and loss-of-function. An orthotopic xenograft model with primary glioma cells demonstrated a role of miR-184/SND1 in glioma pathogenesis in vivo.

Results: SND1 is highly expressed in human glioma tissue and inversely correlated with miR-184 expression. Transfection of glioma cells with a miR-184 mimic inhibited invasion, suppressed colony formation, and reduced anchorage-independent growth in soft agar. Similar phenotypes were evident when SND1 was knocked down with siRNA. Additionally, knockdown (KD) of SND1 induced senescence and improved the chemoresistant properties of malignant glioma cells. In an orthotopic xenograft model, KD of SND1 or transfection with a miR-184 mimic induced a less invasive tumor phenotype and significantly improved survival of tumor bearing mice.

Conclusions: Our study is the first to show a novel regulatory role of SND1, a direct target of miR-184, in glioma progression, suggesting that the miR-184/SND1 axis may be a useful diagnostic and therapeutic tool for malignant glioma.

Keywords: SND1; intracranial injection; invasion; malignant glioma; miR-184.

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Figures

Fig. 1.
Fig. 1.
miR-184 significantly downregulates SND1 by potentially targeting its 3′-UTR. (A) Real-time PCR analysis of SND1 in multiple glioma cell lines including U87, T98G, H4, and GBM6 following transfection of either Con mimic or miR-184 mimic. RNA was isolated at 48 hours after transfection. The average SND1 expression was normalized to GAPDH and presented as relative fold-changes. *P < .05 compared with Con mimic. (B) Upper panel, Western blotting analysis of SND1 in U87 and GBM6 cells following transient transfection of either Con mimic or miR-184 mimic. Protein was isolated 48 hours after transfection. Lower panel, SND1 level analyzed by Western blotting in GBM6 cells stably transfected with miR-184. Actin was used as loading control. Right panel, immunofluorescence staining of SND1 (red) in GBM6 cells stably overexpressing GFP-miR-184 (green). Note that the green cells express low levels of SND1 as compared with the non-GFP-expressing cells. Correlation score of red and green is 0.157. Scale bar, 50 μM. (C) Luciferase assay of GBM6 cells transfected with the SND1-3′UTR reporter (WT) or the SND1-3′UTR mutant (Mut) reporter with Con mimic or miR-184 mimic or mock control. *P <.05 compared with mock control (basal). Bar represents the mean ± SD of 3 independent experiments. (D) miR-184 significantly inhibits the invasive ability of glioma cells, which can be rescued by overexpression of SND1. Matrigel invasion assay of H4, U87, and GBM6 cells after transfecting with Con mimic, miR-184 mimic, or combination of miR-184 mimic with SND1. Upper panel, representative images of invasion assay. Lower panel, quantification of invaded cells. *P < .05 versus Con mimic; #P < .05 versus 184 mimic. (E) Western blotting analysis of SND1 in Con mimic, miR-184 mimic, or combination of miR-184 mimic with SND1-transfected cells. Cell lysates were collected at 48 hours after transfection. Actin was used as loading control.
Fig. 2.
Fig. 2.
Analysis of SND1 expression in glioma tissues and cell lines. (A) SND1 detected by immunohistochemistry in a human glioma tissue microarray; scale bar, 50 μM. Lower panel, bar graph shows staining intensity quantified by 3 independent investigators. (B) The expression of SND1 was examined in primary glioma tissues including astrocytoma and GBM (Tumor) and normal brain (Normal) from 5 individual patients in each group. EF-1α was used as loading control. Lower panel, densitometric values of SND1 normalized by EF-1α. *P < .05 compared with normal brain. (C) SND1 expression at RNA level (upper panel) and protein level (lower panel) was analyzed in IM-PHFA and glioma cell lines including T98G, GBM6, H4, and U87. The average SND1 mRNA was normalized to GAPDH and expressed as relative fold-changes. *P < .05 compared with IM-PHFA. (D) Improved survival of patients in intermediate SND1 group (light gray line, n = 80) as compared with SND1 amplified group (dark gray line, n = 99). Data source: https://caintegrator.nci.nih.gov/rembrandt/kmGraph.do?method=redrawKMPlot.
Fig. 3.
Fig. 3.
Stable overexpression of SND1 in IM-PHFA cells significantly enhances invasion (B), colony formation in monolayer culture (C), and in soft agar (D). *P < .05.
Fig. 4.
Fig. 4.
Stable KD of SND1 recapitulates the biological phenotype observed following overexpression of miR-184. (A) Real time PCR (upper) and Western blotting analysis of SND1 in SND1-knocked down GBM6 cells. Actin was used as a loading control in Western blotting assays. (B and C) KD of SND1 significantly inhibits invasion (B) and colony formation (C) in GBM6 cells. *P < .05 compared with control. (D and E) KD of SND1 improves the chemoresistance in primary glioma cells. Control or SND1-SH GBM6 cells were treated with 200 μM of TMZ. Apoptosis (D) was quantified by Annexin V-APC at 72 hours after treatment. Colonies (E) were quantified at 2 weeks after treatment, as described in Materials and Methods section.
Fig. 5.
Fig. 5.
SND1 facilitates glioma progression by decreasing senescence and increasing invasion. (A) KD of SND1 dramatically inhibits STAT3 phosphorylation in T98G and U87 cells. (B) Knockdown of SND1 promotes senescence-induced cell death. Reactivation of STAT-3 by IL-6 partly abrogates the SND1-mediated senescence phenotype. Upper panel, beta- galactosidase assay; lower panel, γ-H2AX staining. (C) KD of SND1 significantly decreases invasion by STAT-3-dependent pathway that can be partially rescued by IL-6-mediated STAT-3 reactivation in glioma cells.
Fig. 6.
Fig. 6.
Stable expression of miR-184 or KD of SND1 decreases in vivo glioma invasion and improves survival in nude mice. (A) Hematoxylin and eosin staining of mice brains from GBM6-control, GBM6 SND1-SH, and GBM6 miR-184 group. Note the highly invasive tumor margin in the control group indicated by arrows. (B) Survival time for mice injected with GBM6 control, GBM6 SND1-SH, and GBM6 miR-184 cells. In both SND1-SH and miR-184 group, the survival is significantly improved versus control mice. P = .0216. (C) SND1, Ki-67, cleaved caspase 3, CD31, P-STAT3 and cyclinD1 expression in brain sections from GBM6 control, GBM6 SND1-SH, and GBM6 miR-184 mice. Scale bar, 50 μM.
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References

    1. Ostrom QT, Gittleman H, Farah P, et al. CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2006–2010. Neuro Oncol. 2013;15(Suppl 2):ii1–ii56. - PMC - PubMed
    1. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med. 2008;359(5):492–507. - PubMed
    1. Lefranc F, Brotchi J, Kiss R. Possible future issues in the treatment of glioblastomas: special emphasis on cell migration and the resistance of migrating glioblastoma cells to apoptosis. J Clin Oncol. 2005;23(10):2411–2422. - PubMed
    1. Schickel R, Boyerinas B, Park SM, et al. MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. Oncogene. 2008;27(45):5959–5974. - PubMed
    1. Chen CZ. MicroRNAs as oncogenes and tumor suppressors. N Engl J Med. 2005;353(17):1768–1771. - PubMed

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