miRNA Expression Profiling in Migrating Glioblastoma Cells: Regulation of Cell Migration and Invasion by miR-23b via Targeting of Pyk2

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.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.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

Financial Impact of Incentive Spirometry

Despite largely unproven clinical effectiveness, incentive spirometry (IS) is widely used in an effort to reduce postoperative pulmonary complications. The objective of the study is to evaluate the financial impact of implementing IS. The amount of time nurses and RTs spend each day doing IS-related activities was assessed utilizing an online survey distributed to the relevant national nursing and respiratory therapists (RT) societies along with questionnaire that was prospectively collected every day for 4 weeks at a single 10-bed cardiothoracic surgery step-down unit. Cost of RT time to teach IS use to patients and cost of nurse time spent reeducating and reminding patients to use IS were used to calculate IS implementation cost estimates per patient. Per-patient cost of IS implementation ranged from $65.30 to$240.96 for a mean 9-day step-down stay. For the 566 patients who stayed in the 10-bed step-down in 2016, the total estimated cost of implementing IS ranged from $36 959.80 to$136 383.36. Using national survey workload data, per-patient cost of IS implementation costed $107.36 (95$97.88-$116.98) for a hospital stay of 4.5 days. For the 9.7 million inpatient surgeries performed annually in the United States, the total annual cost of implementing postoperative IS is estimated to be$1.04 billion (95% CI, $949.4 million-$1.13 billion). The cost of implementing IS is substantial. Further efficacy studies are necessary to determine whether the cost is justifiable

Down-regulated miRNAs in migrating glioma cells.

<p>Down-regulated miRNAs in migrating glioma cells.</p

miRNA analysis of glioma cell migration.

<p>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 (<i>p</i><0.01). Red and green color scale represents high and low expression respectively.</p

Differential expression of miR-23b in GB patient specimens.

<p>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.</p

Constitutive miR-23b expression suppresses glioma cell migration and invasion.

<p><b>A</b>. 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 (*: <i>p</i><0.01). <b>B</b>. Effect of constitutive miR-23b expression on glioma invasion in <i>ex vivo</i> 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 (*: <i>p</i><0.01, **: <i>p</i><0.05).</p

miR-23b targets the 3′ UTR of Pyk2.

<p>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.</p

miRNA analysis of migration active and migration restricted glioma cells <i>in vitro</i>.

<p>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.</p

qPCR analysis of differentially expressed miRNAs in migrating glioma cells.

<p>qPCR analysis of differentially expressed miRNAs in migrating glioma cells.</p