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. 2014 Apr 15;9(4):e95060.
doi: 10.1371/journal.pone.0095060. eCollection 2014.

MiR-330-mediated regulation of SH3GL2 expression enhances malignant behaviors of glioblastoma stem cells by activating ERK and PI3K/AKT signaling pathways

Yilong Yao  1 , Yixue Xue  2 , Jun Ma  2 , Chao Shang  2 , Ping Wang  2 , Libo Liu  2 , Wenjing Liu  3 , Zhen Li  1 , Shengtao Qu  1 , Zhiqing Li  4 , Yunhui Liu  1
Affiliations

MiR-330-mediated regulation of SH3GL2 expression enhances malignant behaviors of glioblastoma stem cells by activating ERK and PI3K/AKT signaling pathways

Yilong Yao et al. PLoS One. .

Abstract

MicroRNAs are currently considered as an active and rapidly evolving area for the treatment of tumors. In this study, we elucidated the biological significance of miR-330 in glioblastoma stem cells (GSCs) as well as the possible molecular mechanisms. SH3GL2 is mainly distributed in the central nervous system and considered to be a tumor suppressor in many tumors. In the present study, we identified miR-330 as a potential regulator of SH3GL2 and we found that it was to be inversely correlated with SH3GL2 expression in GSCs which were isolated from U87 cell lines. The expression of miR-330 enhanced cellular proliferation, promoted cell migration and invasion, and dampened cell apoptosis. When the GSCs were co-transfected with the plasmid containing short hairpin RNA directed against human SH3GL2 gene and miR-330 mimic, we found that miR-330 promoted the malignant behavior of GSCs by down-regulating the expression of SH3GL2. Meanwhile, the ERK and PI3K/AKT signaling pathways were significantly activated, leading to the decreased expression of apoptotic protein and increased expression of anti-apoptotic protein. Furthermore, in orthotopic mouse xenografts, the mice given stable over-expressed SH3GL2 cells co-transfected with miR-330 knockdown plasmid had the smallest tumor sizes and longest survival. In conclusion, these results suggested that miR-330 negatively regulated the expression of SH3GL2 in GSCs, which promoted the oncogenic progression of GSCs through activating ERK and PI3K/AKT signaling pathways. The elucidation of these mechanisms will provide potential therapeutic approaches for human glioblastoma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Isolation and identification of GSCs.
(A) a: U87 cell lines attached and grew as a monolayer in flasks. b: The spheres formed and reached 100–200 cells each in the serum-free medium. c: Subsphere-forming assay of single-cell suspensions from the cell spheres. (B) Immunofluorescence staining of Nestin (green) and CD133 (red) in cell spheres. (C) Glioblastoma sphere-differentiated progenies were stained with GFAP (green) and beta-tubulin III (red). Scale bars represent 20 µm.
Figure 2
Figure 2. Expression of miR-330 in GSCs and non-GSCs.
Relative expression levels of miR-330 in GSCs and non-GSCs. The expression of miR-330 in non-GSCs accounted for 45.26% of that in GSCs. Values represent the mean ± SD from five independent experiments. *P<0.05 vs. non-GSCs group.
Figure 3
Figure 3. Proliferation, apoptosis, migration and invasion of GSCs transfected with miR-330 mimics (or miR-330 inhibitors).
(A) CCK8 assay showed that the ability of GSCs proliferation with the expression of miR-330 changed. (B) Incidence of apoptotic cells was studied by flow ctometry. The cells were stained with annexin V-fluorescein isothiocyanate and counterstained with PI. (C) The ability of GSCs migration and invasion with the expression of miR-330 changed. The photographs about cells on the membrane and accompanying statistical plots were presented. Values represent the mean ± SD from five independent experiments. *P<0.05 vs. mock 1 group, #P<0.05 vs. mock 2 group.
Figure 4
Figure 4. Expression of SH3GL2 in GSCs and non-GSCs, and SH3GL2 expression with expression of miR-330 changed.
(A) The expression levels of SH3GL2 in GSCs and non-GSCs, using GAPDH as an endogenous control. The IDVs of SH3GL2 protein levels are shown. Data represent means ± SD (n = 5, each). *P<0.05 vs. non-GSCs group. (B) Western blot analysis for miR-330 regulated the expression of SH3GL2 in GSCs, using GAPDH as an endogenous control. The IDVs of SH3GL2 protein levels are shown. Values represent the mean ± SD from five independent experiments. *P<0.05 vs. mock 1 group, #P<0.05 vs. mock 2 group.
Figure 5
Figure 5. Proliferation, apoptosis, migration and invasion of GSCs with the expression of miR-330 and SH3GL2 changed.
(A) The ability proliferation in GSCs with the expression of miR-330 and SH3GL2 changed. (B) Incidence of apoptotic cells was studied by flow ctometry. The cells were stained with annexin V-fluorescein isothiocyanate and counterstained with PI. (C) The ability of GSCs migration and invasion with the expression of miR-330 and SH3GL2 changed. Values represent the mean ± SD from five independent experiments. *P<0.05 vs. control group, #P<0.01 vs. SH3GL2 (−) & pre-miR-330 group. Scale bars represent 20 µm.
Figure 6
Figure 6. Expression of p-ERK/ERK and PI3K/AKT in GSCs with the expression of miR-330 and SH3GL2 changed.
(A) The expression levels of p-ERK/ERK with the expression of miR-330 and SH3GL2 changed. (B, C) The expression levels of PI3K/AKT pathway within the expression of miR-330 and SH3GL2 changed. GAPDH was used as an internal loading control. Accompanying graphs show densitometry analysis of protein expression. Values represent the mean ± SD from five independent experiments. *P<0.05 vs. control group, #P<0.01 vs. SH3GL2 (−) & pre-miR-330 group.
Figure 7
Figure 7. Expression levels of apoptotic proteins in GSCs with the expression of miR-330 and SH3GL2 changed.
(A) The expression levels of caspase-3 and TRAIL in GSCs with the expression of miR-330 and SH3GL2 changed. (B) The expression levels of XIAP and Bcl-2 in GSCs with the expression of miR-330 and SH3GL2 changed. Accompanying graphs show densitometry analysis of protein expression. Values represent the mean ± SD from five independent experiments. *P<0.05 vs. control group, #P<0.01 vs. SH3GL2 (−) & pre-miR-330 group.
Figure 8
Figure 8. Tumorigenicity assay in nude mice.
(A) Nude mice were subcutaneously injected in the right flank with control cells, SH3GL2 (+) stable transfected cells, miR-330 (−) stable transfected cells and SH3GL2 (+) & anti-miR-330 stable transfected cells. (B) A sample tumor from representative group at 42 days post-injection was shown. (C) Tumor growth curve in nude mice. After subcutaneous implantation, tumor volume was measured every 5 days in mm3 (n = 5). *P<0.05 vs. control group, #P<0.05 vs. SH3GL2 (+) group, ▴P<0.05 vs. miR-330 (−) group. (D) Numbers of survived mice for 60 days (n = 15).
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References

    1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, et al. (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352: 987–996. - PubMed
    1. Ji J, Black KL, Yu JS (2010) Glioma stem cell research for the development of immunotherapy. Neurosurg Clin N Am 21: 159–166. - PMC - PubMed
    1. Fukaya R, Ohta S, Yamaguchi M, Fujii H, Kawakami Y, et al. (2010) Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1. Cancer Lett 291: 150–157. - PubMed
    1. Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, et al. (2009) Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4: 568–580. - PubMed
    1. Garzon R, Calin GA, Croce CM (2009) MicroRNAs in Cancer. Annu Rev Med 60: 167–179. - PubMed

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Grants and funding

This work is supported by grants from the Natural Science Foundation of China (81172197, 81072056, 81171131, 81272564, 81272795 and 81372484), the special fund for Scientific Research of Doctor-degree Subjects in Colleges and Universities, (20102104110009), the Natural Science Foundation of Liaoning Province in China (No. 201102300), Liaoning Science and Technology Plan Projects (No. 2011225020), Shenyang Science and Technology Plan Projects (nos. F11-264-1-15, F12-277-1-05, F13-318-1-16 and F13-220-9-15), and Outstanding Scientific Fund of Shengjing Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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