Abstract
Background: Glioblastoma multiforme (GBM) is a form of highly malignant brain tumour. Temozolomide (TMZ) is the standard agent for GBM, but TMZ-resistance is common and accounts for many treatment failures. MicroRNA-21 (miR-21) is a non-coding RNA that plays critical roles in many biological processes in cancer, including chemoresistance. We investigated miR-21 expression and the effect of miR-21 inhibition in GBM with acquired TMZ resistance. Materials and Methods: Human GBM cell line D54MG was treated with TMZ chronically to develop a chemoresistant subclone. MiR-21 inhibition was achieved by transfection with anti-mir-21 oligonucleotide. Results: Chronic TMZ exposure resulted in acquired TMZ-resistance and elevated miR-21 expression. Concomitant treatment with miR-21 inhibitor and TMZ resulted in a significantly higher apoptotic rate than TMZ treatment alone. Conclusion: MiR-21 may have a potential for use as a biomarker of acquired TMZ resistance. MiR-21 inhibition can be further explored as a potential chemotherapy adjunct in the treatment of TMZ-resistant GBM.
Glioblastoma multiforme (GBM) is one of the most lethal forms of primary brain tumour. The alkylating agent temozolomide (TMZ) has been shown to provide significant survival benefits for patients with GBM (1). The standard treatment for GBM consists of maximal surgical resection, concomitant TMZ therapy and irradiation, and six-cycles of adjuvant TMZ (2). The overall prognosis, however, remains unsatisfactory due to the tumour's intrinsic or acquired resistance to TMZ (3). The molecular mechanisms underlying TMZ resistance are incompletely understood, and therapies aimed at overcoming it have attracted considerable research effort (4, 5). An area under investigation concerns the roles of microRNAs (miRNAs) (6). microRNAs belong to a class of small (~21 nucleotides) non-coding RNAs that regulate gene expression post-transcriptionally (7). miRNAs play many important roles in normal biological processes such as cell growth, proliferation, differentiation and apoptosis (8). Their roles in determining the biological behaviour of brain tumours have been described (9, 10). Amongst the oncogenic miRNAs, miRNA-21 (miR-21) was found to be consistently overexpressed in GBM cells (11). miR-21 is anti-apoptotic (12), and may promote glioma invasion (13) and proliferation (14). The role of miR-21 in modulating tumour response to TMZ has been described (15, 16). In this study, we further investigated the effects of specific miR-21 inhibition on a subclone of TMZ-resistant GBM cells. Our hypothesis was that miR-21 inhibition would re-sensitize chemoresistant GBM cells to TMZ.
Materials and Methods
TMZ-resistant GBM cells. We have previously described the development of isogenic subclones of TMZ-resistant GBM cells by means of chronic TMZ exposure (17). Briefly, human GBM cell line D54MG (Duke University Medical Center, Durham, NC, USA) was maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen, Carlsbad, CA, USA), supplemented with 10% foetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37°C in 5% carbon dioxide and 95% air atmosphere. The parental TMZ-sensitive cells (designated as P-D54MG) were exposed to 100 μM TMZ (TEMODAL®, Schering-Plough, NJ, USA) for two weeks, and then repeatedly to TMZ at the 50% inhibitory concentration (IC50) of 500 μM for 12 months. The TMZ-resistant subclone produced (designated as R-D54MG) was isolated and maintained in DMEM/F12 with low-dose TMZ (100 μM).
miRNA expression. Total RNA was obtained using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA concentrations were measured with a NanoDrop ND1000 Spectrophotometer (Thermo Scientific, MA, USA). Quantitative reverse transcription-PCR (qRT-PCR) of mature miRNAs was conducted. The quantities of mature miRNAs were evaluated using specific TaqMan real-time PCR primers and probes (Applied Biosystems, CA, USA). Real-time PCR was performed using GeneAmp Fast PCR Master Mix (Applied Biosystems) and an ABI 7900HT real-time PCR instrument. The expression levels of mature miRNAs were evaluated using the comparative CT method (2−ΔΔCT). All experiments were performed in triplicates.
Levels (mean±SD) of miR-21 expression in R-D54MG cells and their parental control cells P-D54MG were assessed by TaqMan-based quantitative reverse transcription-PCR (qRT-PCR). The former showed significantly up-regulated miR-21 expression (**p<0.01).
miR-21 inhibition. Transfection with anti-miR-21 inhibitor (AMI17000, ID No. AM10206) (Applied Biosystems, CA, USA) was performed using Lipofectamine™ RNAi Max Transfection Agent (Invitrogen, Carlsbad, CA, USA). Briefly, cells were seeded at 5000 cells/well in 96-well plates, and different amounts of the inhibitor (1 nM to 160 nM) were tested. The lowest possible concentration that achieved the most significant inhibition was chosen as the optimal dosage. Cells transfected with this optimal dosage (60 nM) were subjected to miRNA expression confirmation assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. To study the concomitant effects of miR-21 inhibition and TMZ, cells were seeded onto 8-well Chamber Slide System (Thermo Scientific, MA, USA) at 10,000 cells/well, and then transfected with anti-miR-21 at 60 nM, with or without TMZ treatment at IC30 for 72 hours.
TUNEL analysis. Fluorescent in situ hybridization was performed using the In situ Cell Death Detection Kit, Fluorescein, (Roche, Schweiz, Germany) according to the manufacturer's instructions. Omission of the terminal deoxynucleotidyl transferase enzyme during processing was used as a negative control. The chamber slides were observed with fluorescent microscope (Nikon Eclipse 80i, Nikon Intenselight c-HGFI, Kanagawa, Japan). Three high-power fields (×200) were randomly selected to count both the apoptotic cells [Fluorescein Isothiocyanate (FITC) stained] and the total cell population [counterstained by 4’,6-diamidino-2-phenylindole (DAPI)]. At least 100 cells in total were counted in each field. The apoptotic rate was calculated by dividing the total cell number by the number of apoptotic cells.
Transfection with miR-21 inhibitor reduced miR-21 expression in R-D54MG cells in a dose-dependent manner. In comparative CT fold-change analysis, blank transfection control (BC) was used as the control. MC, Mock transfection control (RNAiMax).
Annexin V assay. Cells were seeded at a density of 1×105 cells/ml in 6-well plates, and harvested after treatments, as described above. Annexin V-FITIC/PI kit (BD Biosciences, San Jose, USA) was used to label apoptotic cells. In analyzing the results of flow cytometery (BD FACSCalibur, CA, USA), annexin V-positive cells were considered as apoptotic regardless of propidine iodide (PI) staining. Cells stained positively for PI were considered as dead cells regardless of annexin V staining.
Statistical analysis. Data were expressed as mean±standard error. Student's t-test was used to examine statistical correlation. A p-value <0.05 was considered to be statistically significant. Statistical analysis was performed with SPSS 16.0 software (SPSS Inc., IL, USA).
Results
miR-21 in TMZ-resistant cells. We measured the expression of miR-21 using TaqMan-based qRT-PCR (Figure 1). GBM cells with acquired TMZ-resistance (R-D54MG) exhibited significantly higher levels of miR-21 expression than their parental control cells (mean fold change=3.2, p<0.01). We then proceeded to knock-down endogenous miR-21 expression. The inhibition effect was assessed by qRT-PCR at 72 hours after transfection. To achieve the optimal effect of silencing with minimal off-target effects, different inhibitor concentrations (from 1 nM to 160 nM) were tested. We found that miR-21 inhibition suppressed miR-21 expression in R-D54MG cells in a dose-dependent manner. The effect was maximal at the optimal concentration of 60 nM, which resulted in around an 80% decrease in miR-21 expression (Figure 2). Mock control transfection did not have the same effect. Moreover, miR-21 inhibition alone led to increased apoptosis on TUNEL analysis (Figure 3).
Concomitant use of TMZ and miR-21 inhibition. We then investigated whether miR-21 inhibition would increase the response of TMZ-resistant cells to TMZ re-challenge. R-D54MG cells were transfected with miR-21 inhibitor for 24 hours and then treated with TMZ. TUNEL analysis was performed after 72 hours of TMZ treatment. For cells pretreated with miR-21 inhibitor, TMZ treatment at IC30 resulted in a 52.9% apoptosis rate; cells treated with TMZ only without prior miR-21 inhibition exhibited only a 10.8% rate. Few apoptotic cells were observed in the corresponding mock controls (Figure 4 A to C). We also used annexin V-PI staining to quantify apoptosis. Treatment with miR-21 inhibitor (96 hours) plus TMZ (72 hours) resulted in a significant increase in the apoptosis rate when compared with TMZ treatment alone. The corresponding mock control cells again exhibited low levels of apoptosis (Figure 4E).
Discussion
GBM is characterized by its resistance towards a broad spectrum of structurally unrelated cytotoxic drugs with different modes of action (18). Despite the positive impact of TMZ on survival, the majority of patients with GBM continue to suffer from progressive or recurrent disease (1, 3). The mechanisms underlying TMZ resistance may include contributions from cancer stem cells and genetic mutations, as well as epigenetic phenomena such as the silencing of O-6-methylguanine-DNA-methyltransferase (MGMT) (4, 19, 20).
Treatment with miR-21 inhibitor alone increased the apoptosis rate in R-D54MG cells. Apoptotic nuclei are shown in green on the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Magnification: ×200. A: Transfection with miR-21 inhibitor; B: transfection with RNAiMax (mock control) alone. C: Bar chart showing the percentage (mean±SD) of apoptotic cells counted from at least three randomly selected fields of view. (p<0.01).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showing increased apoptosis after concomitant treatment with miR-21 inhibitor and temozolomide (TMZ) at 30% inhibitory concentration (IC30). Apoptotic nuclei are shown in green on TUNEL assay. Magnification: ×200. A: miR-21 inhibitor plus TMZ; B: TMZ alone; C: RNAiMax plus DMSO (mock control). D: Bar chart showing the percentage (mean±SD) of apoptotic cells counted from at least three randomly selected fields of view (p<0.01). E: Apoptotic quantification using annexin V-propidine iodide (PI) flow cytometric analysis, showing increased apoptosis after concomitant treatment with miR-21 inhibitor and TMZ (right), when compared with control (left) and TMZ alone (centre).
Recently, miRNAs have been shown to play critical roles in determining the biological behavior of GBM. One of the candidates, miR-21, has been identified as a potential target for anticancer therapies (14). Overexpression of miR-21 is implicated in many malignant conditions, including cancer of the breast (21), lung (22), and colorectum (23), as well as in hepatocellular carcinoma (24). In malignant glioma, in vitro studies demonstrated that miR-21 may down-regulate the expression of important tumour suppressor genes such as programmed cell death protein 4 (PDCD4) (23), tropomyosin alpha-1 (TPM1) (25), and phosphatase and tensin homolog (PTEN) (24). Clinically, miR-21 expression has been shown to correlate with tumour grading (13), as well as patient survival (26). Shi et al. reported that overexpression of miR-21 inhibited TMZ-induced apoptosis by reducing the BCL-2–associated X protein (BAX)/B-cell lymphoma 2 (BCL-2) ratio and caspase-3 activity in treatment-naive GBM cells (15). Our findings provide additional information on GBM cells that have previously been subjected to chronic TMZ treatment. Firstly, chronic TMZ exposure and the acquisition of TMZ resistance were associated with up-regulation of miR-21 expression. Secondly, miR-21 knock-down increased the cytotoxic effect of TMZ in resistant cells. To our knowledge, this is the first report that demonstrates the effect of miR-21 inhibition in TMZ-resistant GBM. Our findings suggest two potential clinical uses of miR-21 in the treatment of GBM. miR-21 is detectable in serum and has been shown to be a potential biomarker for oesophageal cancer (27). The elevated expression of miR-21 in our chemoresistant subclone may be exploited similarly as a biomarker of TMZ resistance during treatment or upon disease recurrence. miR-21 inhibition has been shown to enhance the chemosensitivity of human GBM to taxol (28), to activate caspase-3 and -9 (29), and to suppress growth in glioma cells independently of PTEN regulation (30). Our findings therefore indicate the potential use of miR-21 inhibition as a chemotherapy adjunct, for example, for patients who have elevated miR-21 levels serologically or within their tumours. Other miRNA candidates that are reportedly associated with TMZ resistance in GBM include miR-195, miR-455-3p, miR-10a, miR-181b and miR-181c (31, 32). These may be further explored for similar clinical applications alone or in combination with miR-21. A major obstacle to miRNA-based therapy is finding an effective mode of delivery for miRNAs and their inhibitors. Systemic administration is unlikely to be successful for glioma due to the blood-brain barrier. Although this barrier is disrupted in GBM, it is intact in the adjacent brain into which GBM cells may have infiltrated. Interestingly, miRNAs may be transferred between glioma cells, which can potentially reduce the threshold efficiency required (33). Other approaches such as convection-enhanced delivery with nanoparticles (34), and the use of viral vectors (35) can be explored. miRNA inhibition may carry the risk of undesirable side-effects in normal cells. But since the endogenous expression of targeted miRNAs may be normal within normal cells, the impact of off-target effects is likely to be small (36).
Conclusion
Using an in vitro model of acquired TMZ resistance, we have demonstrated that chronic TMZ exposure up-regulates the expression of miR-21 in GBM, and that inhibition of miR-21 can resensitize chemoresistant GBM cells to TMZ re-challenge. Further in vivo studies are required to demonstrate the roles of miR-21 as a biomarker and as a novel therapeutic target for the treatment of TMZ-resistant GBM.
Acknowledgements
We thank Dr. Stella Sun for her technical support and advice.
Footnotes
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Declaration of Interest
The Authors have no potential conflict of interest to report.
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Funding Support
The study was supported by the Seed Funding for Basic Research, The University of Hong Kong, Hong Kong.
- Received March 28, 2012.
- Revision received April 24, 2012.
- Accepted April 25, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
