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. 2014 Jun 5;5(6):e1279.
doi: 10.1038/cddis.2014.245.

miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

S Haemmig  1 , U Baumgartner  1 , A Glück  1 , S Zbinden  1 , M P Tschan  1 , A Kappeler  1 , L Mariani  2 , I Vajtai  1 , E Vassella  1
Affiliations

miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

S Haemmig et al. Cell Death Dis. .

Abstract

Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor κB (NF-κB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-κB is in part owing to downregulation of negative regulators of NF-κB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-κB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-κB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFα- and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-κB confirming that miR-125b is implicated in NF-κB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy.

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Figures

Figure 1
Figure 1
miR-125a and miR-125b directly target TNFAIP3 and NKIRAS2 in GBM cells. Luciferase constructs containing the wild-type (TS) or mutated (mTS) miR-125a/b target site from TNFAIP3 or NKIRAS2 were co-transfected with pre-miR-125a/b or pre-control into GBM cells. Luciferase activity is presented for TNFAIP3 (a) and NKIRAS2 (b) relative to the activity obtained with the construct Luc containing no target site (n=3)
Figure 2
Figure 2
TNFAIP3 and NKIRAS2 are regulated by miR-125a/b. (a) TNFAIP3 or NKIRAS2 mRNA levels by real-time PCR of cells transiently transfected with pre-miR-125a/b relative to cells transfected with pre-control (n=3). (b) Western blot of GBM cells transiently transfected with pre-miR-125b relative to pre-control. (c) mRNA levels (n=3) and (d) protein levels of TNFAIP3 and NKIRAS2 in cells lentivirally transduced with anti-miR-125b relative to cells transduced with anti-control
Figure 3
Figure 3
miR-125b induces NF-κB activity. (a) NF-κB reporter activity of GBM cells overexpressing miR-125b or anti-miR-125b relative to control cells (n=6). (b) NF-κB reporter activity of LN-18 cells overexpressing anti-miR-125b. Cells were induced with 10 ng/ml TNFα for 4 h beginning 20 h post transfection (n=6). Expression of (c) nuclear p65 and (d) cytoplasmic p65, IκBα and phospho IκBα in LN-18 cells transiently transfected with pre-miR-125b or pre-control by Western blotting. Cells were induced with 10 ng/ml TNFα beginning at 48 h post transfection. The ratio of pIκBα to IκBα protein levels is presented below the corresponding Western blot
Figure 4
Figure 4
miR-125b induces the expression of NF-κB responsive genes. mRNA levels of Bcl-2 (a), c-IAP2 (b) and CCND1 (c) by real-time PCR in GBM cell lines overexpressing miR-125b relative to control transfected cells (n=3)
Figure 5
Figure 5
miR-125b induces proliferation and apoptosis resistance in GBM cells. (a) Population growth of U87 and LN-18 cells overexpressing miR-125b or anti-miR-125b relative to the control using the resazurin assay (n=3). (b) Cell cycle analysis of nocodazole-treated cells by flow cytometry (n=3). (c) Apoptosis. Cells were transfected with pre-miR-125b or pre-control and treated with 10 ng/ml TNFα or 250 ng/ml TRAIL for 48 h beginning 24 h post transfection. Apoptosis was assessed using the ApoTox-Glo Triplex assay (n=3) (d) LN-18 cells overexpressing anti-miR-125b or anti-control were induced with 10 ng/ml TNFα for 24 h and subjected to the ApoTox-Glo Triplex assay (n=3)
Figure 6
Figure 6
miR-125b induces NF-κB activity by targeting TNFAIP3 and NKIRAS2. (a) NF-κB activity of GBM cells retrovirally transduced with miR-125b-refractory TNFAIP3, NKIRAS2 or pBABE control relative to untransduced cells (n=6). (b) NF-κB activity of retrovirally transduced cells overexpressing miR-125b relative to pre-control transfected cells (n=6). (c) Western blot analysis of nuclear (upper panel) and cytoplasmic (lower panel) fractions of retrovirally transduced cells overexpressing miR-125b
Figure 7
Figure 7
miR-125b confers resistance to apoptosis and TMZ by targeting TNFAIP3 and NKIRAS2. (a) Retrovirally transduced LN-18 cells were transfected with pre-miR-125b or pre-control and induced with 10 ng/ml TNFα for 24 h (n=3). Apoptosis was assessed using the ApoTox-Glo Triplex assay. (b) Cell viability of GBM cells overexpressing miR-125b in the presence of 200 μM TMZ. Cell viability was assessed 3 days after treatment with TMZ using resazurin assay (n=3). (c) Cell viability of GBM cells overexpressing anti-miR-125b or anti-control in the presence of TMZ (n=6). (d) Cell viability of LN-18 cells retrovirally transduced with miR-125b-refractory TNFAIP3, NKIRAS2 or pBABE control following transfection with pre-miR-125b or pre-control in the presence of TMZ (n=6)
Figure 8
Figure 8
miR-125b expression is directly correlated with NF-κB activity in GBM tissues and with the patient's overall survival. (a) Comparison of the overall survival of patients expressing >120% or <120% miR-125a/b in the tumor tissue relative with the level of miR-125a/b in U87 cells using Kaplan–Meier survival plots. The threshold was defined using a density blot, which gave rise to a bimodal distribution with best separation at 120%. All patients were treated with TMZ. Significance of correlation was assessed using log-rank (Mantel–Cox) test. (b) Correlation of miR-125a/b expression and nuclear p65 in GBM tissues. A tumor with >25% nuclear p65 was considered as constitutive active for NF-κB
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