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PLoS One. 2012;7(6):e39818. doi: 10.1371/journal.pone.0039818. Epub 2012 Jun 22.
miRNA expression profiling in migrating glioblastoma cells: regulation of cell migration and invasion by miR-23b via targeting of Pyk2.
- 1
- Department of Biochemistry and Molecular Biology, Mayo Clinic Arizona, Scottsdale, Arizona, United States of America. loftus.joseph@mayo.edu
Abstract
BACKGROUND:
Glioblastoma (GB) is the most common and lethal type of primary brain tumor. Clinical outcome remains poor and is essentially palliative due to the highly invasive nature of the disease. A more thorough understanding of the molecular mechanisms that drive glioma invasion is required to limit dispersion of malignant glioma cells.
METHODOLOGY/PRINCIPAL FINDINGS:
We investigated the potential role of differential expression of microRNAs (miRNA) in glioma invasion by comparing the matched large-scale, genome-wide miRNA expression profiles of migrating and migration-restricted human glioma cells. Migratory and migration-restricted cell populations from seven glioma cell lines were isolated and profiled for miRNA expression. Statistical analyses revealed a set of miRNAs common to all seven glioma cell lines that were significantly down regulated in the migrating cell population relative to cells in the migration-restricted population. Among the down-regulated miRNAs, miR-23b has been reported to target potential drivers of cell migration and invasion in other cell types. Over-expression of miR-23b significantly inhibited glioma cell migration and invasion. A bioinformatics search revealed a conserved target site within the 3' untranslated region (UTR) of Pyk2, a non-receptor tyrosine kinase previously implicated in the regulation of glioma cell migration and invasion. Increased expression of miR-23b reduced the protein expression level of Pyk2 in glioma cells but did not significantly alter the protein expression level of the related focal adhesion kinase FAK. Expression of Pyk2 via a transcript variant missing the 3'UTR in miR-23b-expressing cells partially rescued cell migration, whereas expression of Pyk2 via a transcript containing an intact 3'UTR failed to rescue cell migration.
CONCLUSIONS/SIGNIFICANCE:
Reduced expression of miR-23b enhances glioma cell migration in vitro and invasion ex vivo via modulation of Pyk2 protein expression. The data suggest that specific miRNAs may regulate glioma migration and invasion to influence the progression of this disease.
Figure 1miRNA analysis of migration active and migration restricted glioma cells in vitro.
Glioma cell lines were seeded onto 10-well glass slides coated with 10 µg/ml laminin and allowed to migrate for 24 hours. Total RNA, including small RNA, was isolated from microdissected migrating (rim) and migration-restricted (core) cell populations. Total RNA (100 ng) was end-labeled with Cy3 and hybridized to Agilent human miRNA microarrays.
PLoS One. 2012;7(6):e39818.
Figure 2miRNA analysis of glioma cell migration.
miRNA expression in paired migration-restricted and migrating cell populations from seven different glioma cell lines was profiled using Agilent human miRNA microarrays. Hierarchical cluster dendrogram of significant differentially expressed miRNAs (p<0.01). Red and green color scale represents high and low expression respectively.
PLoS One. 2012;7(6):e39818.
Figure 3Constitutive miR-23b expression suppresses glioma cell migration and invasion.
A. Effect of constitutive miR-23b expression on glioma migration. Glioma cells stably transduced with empty vector (ctrl) or miR23b were seeded onto 10-well glass slides pre-coated with 10 µg/ml human laminin. Cell migration was assessed over 24 hours. Data represent the average of three independent experiments and is depicted as mean ± SD (*: p<0.01). B. Effect of constitutive miR-23b expression on glioma invasion in ex vivo murine brain slice assay. Control cells or cells constitutively expressing miR-23b were implanted onto the bilateral putamen of mouse brain slices and observed at 48 hr. Depth of invasion was calculated from Z-axis images collected by confocal laser scanning microscopy. The mean value of the depth of invasion was obtained from six independent experiments and is depicted as mean ± SD (*: p<0.01, **: p<0.05).
PLoS One. 2012;7(6):e39818.
Figure 4Suppression of miR-23b by anti-miR-23b oligonucleotides enhances glioma cell migration.
A. Effect of anti-miR-23b on miR-23b expression. T98G glioma cells were transfected with 100 nM anti-miR-23b oligonucleotide or a random sequence control oligonucleotide. 48 hours after transfection, expression of miR-23b was determined by quantitative PCR. B pre-coated with 10 µg/ml human laminin 16 hours after transfection. Cell migration was ass. Effect of anti-miR-23b expression on glioma migration. Cells were seeded onto 10-well glass slides essed over 24 hr. Bars represents the average of three independent experiments (*: p<0.05).
PLoS One. 2012;7(6):e39818.
Figure 5Differential expression of miR-23b in GB patient specimens.
Surgical glioblastoma biopsy samples were sectioned, stained, and the tumor core or invasive rim were delineated by histological examination. Cells from the invasive rim and tumor core populations were microdissected and total RNA, including small RNA, was isolated. The relative abundance of miR-23b in each cell population was determined by quantitative PCR. Results are depicted relative to control and normalized to 18S ribosomal RNA.
PLoS One. 2012;7(6):e39818.
Figure 6Pyk2 is a target of miR-23b in glioma cells.
A. Predicted pairing of Pyk2 3' UTR target region (top) and hsa miR-23b (bottom). Box highlights the exact match of positions 515-522 of Pyk2 3' UTR with positions 2-8 (the seed plus position 8) of the mature miRNA. B. Immunoblot analyses of Pyk2 and FAK in control transduced U87 and SF767 glioma cells (ctrl) or in U87 and SF767 cells constitutively expressing miR-23b. Cell lysates were immunoblotted with anti-actin antibody to ensure equivalent protein loading in each lane.
PLoS One. 2012;7(6):e39818.
Figure 7miR-23b targets the 3′ UTR of Pyk2.
Wild-type T98G and U87 glioma cells or T98G and U87 cells constitutively expressing miR-23b were infected with recombinant adenoviruses expressing FLAG epitope-tagged wild-type Pyk2 (Pyk WT) or Pyk2 lacking the 3' UTR (Pyk 3Kb). Cells were infected at the indicated multiplicity of infection (MOI). 24 hr after infection, cells were lysed, and the cell lysates were immunoblotted with an anti-FLAG monoclonal antibody. Lysates were immunoblotted with anti-actin antibody to ensure equivalent protein loading in each lane.
PLoS One. 2012;7(6):e39818.
Figure 8Re-expression of Pyk2 stimulates migration of miR-23b overexpressing cells.
A. T98G cells transduced with empty vector (ctrl) were left uninfected while T98G cells overexpressing miR-23b were infected with adenoviruses encoding ß-galactosidase, or FLAG epitope-tagged wild-type Pyk2 (Pyk WT) or Pyk2 lacking the 3' UTR (Pyk 3Kb) at MOI = 5. Sixteen hours after infection, cells were plated onto laminin coated slides and the migration rate determined after 24 hours. Values are mean ± SD of 10 replicate samples (*: p<0.05). B. Whole cell lysates were immunoblotted with the indicated antibodies.
PLoS One. 2012;7(6):e39818.
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