MiR-196a exerts its oncogenic effect in glioblastoma multiforme by inhibition of IκBα both in vitro and in vivo

Abstract

Background: Recent studies have revealed that miR-196a is upregulated in glioblastoma multiforme (GBM) and that it correlates with the clinical outcome of patients with GBM. However, its potential regulatory mechanisms in GBM have never been reported.

Methods: We used quantitative real-time PCR to assess miR-196a expression levels in 132 GBM specimens in a single institution. Oncogenic capability of miR-196a was detected by apoptosis and proliferation assays in U87MG and T98G cells. Immunohistochemistry was used to determine the expression of IκBα in GBM tissues, and a luciferase reporter assay was carried out to confirm whether IκBα is a direct target of miR-196a. In vivo, xenograft tumors were examined for an antiglioma effect of miR-196a inhibitors.

Results: We present for the first time evidence that miR-196a could directly interact with IκBα 3'-UTR to suppress IκBα expression and subsequently promote activation of NF-κB, consequently promoting proliferation of and suppressing apoptosis in GBM cells both in vitro and in vivo. Our study confirmed that miR-196a was upregulated in GBM specimens and that high levels of miR-196a were significantly correlated with poor outcome in a large cohort of GBM patients. Our data from human tumor xenografts in nude mice treated with miR-196 inhibitors demonstrated that inhibition of miR-196a could ameliorate tumor growth in vivo.

Conclusions: MiR-196a exerts its oncogenic effect in GBM by inhibiting IκBα both in vitro and in vivo. Our findings provide new insights into the pathogenesis of GBM and indicate that miR-196a may predict clinical outcome of GBM patients and serve as a new therapeutic target for GBM.

Keywords: IκBα; apoptosis; glioblastoma; miR-196a; tumor growth.

Fig. 1.

Clinical significance of miR-196a in GBM patients. (A) miR-196a expression in 132 FFPE GBM specimens. NBTs refer to normal brain tissues. (B) Correlation of miR-196a expression with overall survival in GBM patients.

Fig. 2.

Effect of miR-196a on cell proliferation and apoptosis in vitro. (A) miR-196a expression was quantified by qRT–PCR in U87MG and T98G cells 48 h after transfection of miR-196a mimics, AMO-miR-196a, or negative control oligo. (B) Cell viability was determined by MTT assay in U87MG and T98G cells 48 hours after transfection. (C) Cell apoptosis was analyzed by flow cytometry in U87MG and T98G cells 48 hours after transfection. (D) Graphical representation of the apoptosis analysis in (C). (E) Hoechst 33258 staining of U87MG and T98G cells 48 hours after transfection of fluorescent dye labeled FAM-miRNA. Arrows indicate apoptotic cells. Original magnification is 400×. Data are presented as mean ± SEM for 3 separate experiments performed in duplicate. *P < .05; *P < .001.

Fig. 3.

IκBα is a direct target of miR-196a. (A) The potential interaction between miR-196a and putative binding sites in the IκBα 3′-UTR. The mutant sequences are equivalent to the wild-type ones, except mutations at the 3′ end of target site are underlined. (B) Relative IκBα mRNA expression was determined by qRT-PCR in U87MG and T98G cells 48 hours after transfection. (C) IκBα protein expression was determined by Western blot in U87MG and T98G cells 48 hours after transfection. β-actin was used as a loading control. (D) Photomicrographs showing representative results of hematoxylin and eosin staining and immunohistochemical analysis of IκBα protein expression in human GBM specimens and NBTs. Original magnification ×200. (E) A chi-square table showing a significant inverse correlation between miR-196a and IκBα expression (P < .001). (F) Luciferase activities were analyzed in 293 T cells 48 hours after cotransfection of miR-196a mimics and either wild-type or mutant IκBα 3′-UTR. Data are presented as mean ± SEM for 3 separate experiments performed in duplicate. *P < .05; **P < .001.

Fig. 4.

Inhibition of IκBα is essential for miR-196a–induced proliferation and inhibited apoptosis. (A) IκBα protein expression was determined by Western blot in U87MG and T98G cells 48 hours after transfection of specific IκBα siRNA. (B) Cell viability was determined by MTT assay in U87MG and T98G cells 48 hours after transfection of IκBα siRNA. (C) Cell apoptosis was analyzed by flow cytometry in U87MG and T98G cells 48 hours after transfection of IκBα siRNA. (D) Graphical representation of the apoptosis analysis in (C). (E) IκBα protein expression was determined by Western blot in U87MG and T98G cells 48 hours after transfection of empty vector + NC oligo, pc-IκBα + NC oligo, or pc-IκBα + miR-196a mimics. (F) Cell viability was determined by MTT assay in U87MG and T98G cells 48 hours after transfection. (G) Cell apoptosis was analyzed by flow cytometry in U87MG and T98G cells 48 hours after transfection. (H) Graphical representation of the apoptosis analysis in (G). Data are presented as mean ± SEM for 3 separate experiments performed in duplicate. * P < .05; **P < .001.

Fig. 5.

Inhibition of MiR-196a ameliorates tumorigenesis of GBM cells in vivo. (A) Expression of apoptosis-related proteins was determined by Western blot in U87MG and T98G cells 48 hours after transfection of miR-196a mimics, AMO-miR-196a, or negative control. (B) Photograph showing excised tumors in representative mice in each group implanted with U87MG or T98G cells (n = 8 mice per treatment group) on day 28 after treatment. (C) Graph showing growth inhibition of human tumor xenografts in nude mice treated with AMO-antagomiR-196a. (D) Graph illustrating the excised tumor weight on day 28 after tumor implantation. (E) Relative miR-196a expression was determined by qRT-PCR in xenograft tumor specimens.