miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells

Abstract

Background: Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells.

Methods: We used quantitative RT-PCR to assess microRNA expression in high-grade astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, -mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining, and cellular proliferation was determined using fluorescence-activated cell sorting.

Results: Our studies revealed that expression levels of microRNA-124 and microRNA-137 were significantly decreased in anaplastic astrocytomas (World Health Organization grade III) and glioblastoma multiforme (World Health Organization grade IV) relative to non-neoplastic brain tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neural stem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to 12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced morphological changes and marker expressions consistent with neuronal differentiation in mouse neural stem cells, mouse oligodendroglioma-derived stem cells derived from S100 beta-v-erbB tumors and cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969). Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 and SF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins.

Conclusion: microRNA-124 and microRNA-137 induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme-derived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest that targeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells may be therapeutically efficacious for the treatment of this disease.

Figure 1

miR-124 and miR-137 are down-regulated in anaplastic astrocytomas and glioblastoma multiformes and are up-regulated in glioblastoma multiforme cell lines following treatment with DNA demethylating agents. (A) Expression of high-grade astrocytomas-microRNAs in individual tumor samples measured relative to let-7a (black dots) and miR-16 (white dots). Sample classes are glioses, anaplastic astrocytomas and glioblastoma multiformes. (B) Glioblastoma multiforme cell lines (U87 and U251) were treated with 5-aza-dC at 1 μM (Aza.1) or 5 μM (Aza.5) alone, trichostatin A (100 ng/ml) alone, or combinations of both agents. MicroRNA expression was measured relative to let-7a and normalized to vehicle control (dimethyl sulfoxide). Error bars represent standard deviation of triplicate polymerase chain reactions from a single experimental set. Similar results were obtained in independent experiments (see Additional file 8).

Figure 2

MiRNA expression during differentiation of subventricular zone-neural stem cells. (A) Marker expression during adult neural stem cell neurogenesis. Photomicrographs are shown of proliferating subventricular zone-neural stem cells cultures in proliferation conditions (A)-(D), and following 1 day (E)-(H), 2 days (I)-(L), 3 days (M)-(P) and 4 days (Q)-(T) of mitogen deprivation. Phase images (A), (E), (I), (M), (Q) are shown with corresponding epifluorescent images showing 4'-6-diamidino-2-phenylindole-stained nuclei (B), (F), (J), (N), (R) and Tuj1 expression (C), (G), (K), (O), (S). Glial fibrillary acidic protein expression (D), (H), (L), (P), (T) is shown in parallel cultures. (B) Expression analysis of high-grade astrocytoma-microRNAs during a 5-day differentiation time-course of subventricular zone-neural stem cells.

Figure 3

miR-124 and miR-137 promote neuronal differentiation of subventricular zone-neural stem cells. (A) Epifluorescent images of subventricular zone-neural stem cells 72 hours after transfection with miR-124, miR-137 and control oligonucleotide. Cells were immunostained with Tuj1 and glial fibrillary acidic protein antibodies, nuclei were counterstained with 4'-6-diamidino-2-phenylindole- and images are merged. Scale bar is 10 μm. (B) Phase contrast images of subventricular zone-neural stem cells 48 hours post-transfection by miR124 and miR137 and Tuj1 immunostaining of the same cultures 72 hours post-transfection. (C) Quantification of percentage of Tuj1+ cells, Tuj1+ cells with neuronal morphology and glial fibrillary acidic protein+ cells 72 hours after transfection with miR-124, miR-137, both miR-124 and miR-137, control oligonucleotide or transfection reagent.

Figure 4

Induction of neuronal differentiation of tumor-derived neural stem cells by miR-124 and miR-137. (A) Epifluorescent images of tumor-derived neural stem cells 72 hours after transfection with miR-124, miR-137, control oligonucleotides and lipofectamine reagent alone. Cells were immunostained with Tuj1 and glial fibrillary acidic protein antibodies and DNA was stained with Hoechst 33258 reagent. The percentage of Tuj1- and glial fibrillary acidic protein-positive cells was quantified in each sample after transfection and staining and plotted against the total number of counted cells (n = 450). (C) Quantification of Tuj1+ and glial fibrillary acidic protein+ cells in primary glioblastoma multiforme cultures 10 days after transfection of miR-124, miR-137 or control oligonucleotides. The inset shows a Tuj1+ cell with neuronal morphology from a miR-124 and/or miR-137 cotransfection. (D) Immunostaining with neuronal markers Tuj1 and microtubule-associated protein 2 10 days after miR-137 or negative control miR-transfections in glioblastoma multiforme lines maintained as neurospheres.

Figure 5

miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme stem cells and induce cell G0/G1 cycle arrest. Cell cycle analysis was conducted by fluorescence-activated cell sorter at 48 hours after transfection of 100 nM (final total microRNA concentration) miR-124, miR-137, miR-124 and miR-137 together or negative control oligonucleotides (neg#1, neg#2) to U251 (A) and SF6969 (B) glioblastoma multiforme cells. Cells were treated with bromodeoxyuridine for 30 minutes, fixed, treated with fluorescein isothiocyanate-labeled antibromodeoxyuridine antibody and the DNA stain 7-amino-actinomycin D and subject to flow cytometry. Values represent mean ± standard deviation of replicate experiments; *P < 0.05.

Figure 6

CDK6 expression is inhibited by miR-124 and miR-137 in glioblastoma multiforme cells. (A) Transfection of 100 nM miR-124 or miR-137 reduces cyclin-dependent kinase 6 mRNA transcript levels by 50% in U251 cells at 48 hours relative to cells transfected with 100 nM control oligonucleotide. Cyclin-dependent kinase 6 expression was determined by TaqMan relative to control genes Gus (black bars), GAPDH (gray bars) and 18S (white bars). Values represent average +/- standard deviation of independent experiments. (B) Cyclin-dependent kinase 6 protein expression is dramatically reduced as determined by western blotting following transfection of miR-124 or miR-137. Levels of phosphorylated RB (pSer 807/811) are also markedly reduced in response to miR-124 or miR-137 transfection. (C) miR-137 sequence in relation to the pMIR-REPORT vector containing the predicted cyclin-dependent kinase 6 miR-137 binding site (wild type). Mutated bases (underlined) were also introduced into the miR-137 seed region (boxed) of the cyclin-dependent kinase 6-3'UTR (mutated). Vertical lines denote Watson-Crick base pairing. (D) Relative luminescence of U251 cells following transfection miR-137 or negative control microRNA in conjunction with wild type or mutated cyclin-dependent kinase 6 reporter constructs. *P < 0.0001.