MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators - PubMed (original) (raw)
MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators
Galina Gabriely et al. Mol Cell Biol. 2008 Sep.
Abstract
Substantial data indicate that microRNA 21 (miR-21) is significantly elevated in glioblastoma (GBM) and in many other tumors of various origins. This microRNA has been implicated in various aspects of carcinogenesis, including cellular proliferation, apoptosis, and migration. We demonstrate that miR-21 regulates multiple genes associated with glioma cell apoptosis, migration, and invasiveness, including the RECK and TIMP3 genes, which are suppressors of malignancy and inhibitors of matrix metalloproteinases (MMPs). Specific inhibition of miR-21 with antisense oligonucleotides leads to elevated levels of RECK and TIMP3 and therefore reduces MMP activities in vitro and in a human model of gliomas in nude mice. Moreover, downregulation of miR-21 in glioma cells leads to decreases of their migratory and invasion abilities. Our data suggest that miR-21 contributes to glioma malignancy by downregulation of MMP inhibitors, which leads to activation of MMPs, thus promoting invasiveness of cancer cells. Our results also indicate that inhibition of a single oncomir, like miR-21, with specific antisense molecules can provide a novel therapeutic approach for "physiological" modulation of multiple proteins whose expression is deregulated in cancer.
Figures
FIG. 1.
miR-21 expression in glioma progression. qRT-PCRs for miR-21 and miR-10b have been performed with primers specific for mature miRNAs and normalized to levels for miR-19b uniformly expressed among control brain tissues and tumors. Normal brain tissues (Normal) and gliomas (grade II, grade III [AA, anaplastic astrocytoma; AOD, anaplastic oligodendroglioma; AOA, anaplastic oligoastrocytoma], grade IV [GBM]) were examined. The reactions were performed in duplicates, and the data are represented as means ± SEM.
FIG. 2.
Analysis of genes regulated by anti-miR-21. (A) Validation of miR-21 inhibitor. Glioma A172 cells were cotransfected with 50 nM miR-21 synthetic antisense inhibitors (CTRL, control oligonucleotide; 2′-_O_-Me, 2′-_O_-methyl) and the luciferase sensor of miR-21 activity containing a perfect miR-21 binding site. Anti-miR-21 activity was then measured using the luciferase reporter assay and normalized to Renilla levels. 2′-MOE ASO inhibits miR-21 ∼20-fold. (B) Microarray expression profiling confirms upregulation of endogenous targets. The hexamer composition of the 3′ UTRs of transcripts up- or downregulated (P < 0.05) by anti-miR-21 at 24 h in two replicate experiments was compared to the hexamer composition of the 3′ UTRs of all transcripts on the microarray. The levels of enrichment of miR-21 seed hexamers in the regulated transcripts are shown. (C) Biofunctions of genes regulated by anti-miR-21 identified by microarray profiling. Transcripts up- or downregulated (P < 0.05) by anti-miR-21 in two replicate experiments were mapped to Entrez genes annotated with Gene Ontology (GO) database biological process terms. GO terms were ranked by the hypergeometric P values for their enrichment in the regulated genes compared to the levels for all transcripts in the microarray. The levels of enrichment of the most significantly enriched distinct terms from either up- or downregulated signatures are shown.
FIG. 3.
Microarray expression profiling identifies genes regulated by both miR-21 and anti-miR-21. Expression profiles of cells transfected with the control mismatched anti-miR-21 oligonucleotide, the anti-miR-21 oligonucleotide, or the miR-21 mimetic duplex were compared. The top panel is a heat map representation of genes regulated in any one experiment and containing motifs matching the octamer (ATAAGCTA) and heptamer(s) (ATAAGCT and/or TAAGCTA) of miR-21 nucleotides 1 to 8. The bottom panel is a detail of the heat map, showing the cluster of seed-matched genes both upregulated by anti-miR-21 and downregulated by the miR-21 duplex.
FIG. 4.
miR-21 targets RECK and TIMP3. (A) Predicted miR-21 binding sites within RECK and TIMP3 3′ UTRs. The asterisks depict nucleotides mutated for the luciferase reporter assays. (B) RECK and TIMP3 mRNA expression in normal brain tissue and gliomas of different grades was analyzed by qRT-PCR and normalized to GAPDH mRNA levels. Per group, four control brains and grade II gliomas and eight grade III and grade IV gliomas were analyzed. The gray bars represent the data ranges. Mean expression levels are depicted by black dots and corresponding numbers. Since a few samples were analyzed in “normal” and “grade II” sets, the analysis reaches significance (P < 0.05) for the transition from grade III to IV only. (C) RECK and TIMP3 mRNAs are upregulated upon treatment of glioma cells with anti-miR-21. A172 cells were transfected with either the anti-miR-21 or the control oligonucleotide (control oligo) and grown for 48 h, mRNA was isolated, and qRT-PCR for RECK and TIMP3 was performed. The data were normalized to GAPDH mRNA levels. Data are represented as means ± SEM. (D) Western blot validation of RECK and TIMP3 protein upregulation by anti-miR-21 in glioma (A172 and LN229) and other (MCF7, breast carcinoma; U2OS, osteosarcoma) cell lines. The analysis was performed at 72 h posttransfection. (E) pMir-Report vectors containing the wild-type RECK 3′ UTR (RECK wt), RECK 3′ UTR with a mutated miR-21 binding site (RECK mut), the wild-type TIMP3 3′ UTR (TIMP3 wt), or the TIMP3 3′ UTR with both predicted miR-21 binding sites mutated (TIMP3 mut) were cotransfected with either miR-21 inhibitor (Anti-miR-21) or the mismatched control oligonucleotide into A172 and HeLa cells. The inhibition of miR-21 by the antisense inhibitors resulted in a significant increase in luciferase signals of RECK wt- but not RECK mut-transfected cells. No consistent effect of miR-21 inhibition on the TIMP3 3′ UTR was observed. Depicted are the averages of results from representative experiments performed in triplicate. Data are represented as means ± SEM.
FIG. 5.
miR-21 inhibition affects MMP activity in vitro and in vivo. (A) miR-21 inhibition reduces the activity of gelatinolytic enzymes in glioma A172 and U87 cells. Fresh media of cell cultures transfected with either anti-miR-21 or the corresponding control oligonucleotide were analyzed by gelatin zymography. (B) In vivo imaging of MMP activity. Nude mice were injected subcutaneously in the flank with U87 cells transfected with 50 nM anti-miR-21 (left side) or control oligonucleotides (right side). At 3 days postinjection, the MMP activity was measured using MMPsense (Visen) and fluorescence imaging. Shown are two representative mice of a group of eight mice. (C) Quantitation of the relative fluorescence intensities shown in panel B by use of ImageJ software (NIH). Quantitation is demonstrated for a representative experiment (n = 4). The experiment was repeated three times. Data are represented as means ± SEM.
FIG. 6.
Inhibition of miR-21 affects glioma cell motility. (A) Validation of RECK downregulation by siRNA. A172 cells were transfected with RECK siRNA or Lipofectamine only (mock). At 72 h following transfection, cells were harvested and subjected to Western blot analysis using anti-RECK antibodies. (B) Confluent A172 cells were transfected with either miR-21 inhibitor or the control oligonucleotide in combination with RECK siRNA or control siRNA. At 5 h posttransfection, the cell monolayer was scratched with a scratch spatula (time zero) and migration of the cells toward the “wound” was visualized. Images were taken at 0 and 72 h after the monolayer was scratched. The software program MetaVue was used to determine the migration distance. Depicted are representative images and indications of the migration distances (lines at monolayer fronts). (C) Confluent A172 cells were transfected in triplicate with the miR-21 inhibitor, the mismatched control oligonucleotide, or no oligonucleotide (mock). The cell monolayers were scratched, and migration of the cells toward the “wound” was visualized as described for panel B. Images were taken at various time points after the monolayer was scratched, and the software program MetaVue was used to determine the migration distance. The experiment was repeated three times. Depicted are the average values of results from a representative experiment. Data are represented as means ± SEM. (D) Quantification of the experiment whose results are shown in panel B. Images were taken at various time points after the monolayer was scratched, and the software program MetaVue was used to determine the migration distance. Inhibition of miR-21 resulted in a significant reduction in the migration distance of the monolayer front (P < 0.01), which was rescued by cotransfection with RECK siRNA. Depicted are the averages of results from representative experiments performed in triplicate. Data are represented as means ± SEM.
FIG. 7.
Inhibition of miR-21 reduces glioma cell invasiveness. The Matrigel invasion assay was performed on glioma cell lines (A172 and U87) transfected with either the inhibitor of miR-21 (anti-miR-21) or the control oligonucleotide (control oligo). Invaded cells were fixed, stained, and observed by optical microscopy. The experiment was performed in triplicate and repeated three times. (A) Pictures of representative cell fields for each treatment were taken by a camera connected to the microscope with ×10 magnification. (B) Numerical representation of the data obtained by counting average numbers of cells from three different fields for each treatment. Data are represented as means ± SEM. (C) A172 cells were cotransfected with the indicated oligonucleotides (anti-miR-21, RECK siRNA, or the corresponding control 2′-_O_-MOE and siRNA oligonucleotides). RECK siRNA increased the invasiveness and rescued the effect of anti-miR-21. Data are represented as means ± SEM.
References
- Asangani, I. A., S. A. Rasheed, D. A. Nikolova, J. H. Leupold, N. H. Colburn, S. Post, and H. Allgayer. 2008. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 272128-2136. - PubMed
- Bremer, C., C. H. Tung, and R. Weissleder. 2001. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 7743-748. - PubMed
- Chan, J. A., A. M. Krichevsky, and K. S. Kosik. 2005. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 656029-6033. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases