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. 2010 May;20(3):539-50.
doi: 10.1111/j.1750-3639.2009.00328.x. Epub 2009 Sep 19.

Identification and functional characterization of microRNAs involved in the malignant progression of gliomas

Bastian Malzkorn  1 , Marietta Wolter, Franziska Liesenberg, Michael Grzendowski, Kai Stühler, Helmut E Meyer, Guido Reifenberger
Affiliations

Identification and functional characterization of microRNAs involved in the malignant progression of gliomas

Bastian Malzkorn et al. Brain Pathol. 2010 May.

Abstract

Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma WHO grade III or secondary glioblastoma WHO grade IV. We explored the role of microRNAs (miRNAs) in glioma progression by investigating the expression profiles of 157 miRNAs in four patients with primary WHO grade II gliomas that spontaneously progressed to WHO grade IV secondary glioblastomas. Thereby, we identified 12 miRNAs (miR-9, miR-15a, miR-16, miR-17, miR-19a, miR-20a, miR-21, miR-25, miR-28, miR-130b, miR-140 and miR-210) showing increased expression, and two miRNAs (miR-184 and miR-328) showing reduced expression upon progression. Validation experiments on independent series of primary low-grade and secondary high-grade astrocytomas confirmed miR-17 and miR-184 as promising candidates, which were selected for functional analyses. These studies revealed miRNA-specific influences on the viability, proliferation, apoptosis and invasive growth properties of A172 and T98G glioma cells in vitro. Using mRNA and protein expression profiling, we identified distinct sets of transcripts and proteins that were differentially expressed after inhibition of miR-17 or overexpression of miR-184 in glioma cells. Taken together, our results support an important role of altered miRNA expression in gliomas, and suggest miR-17 and miR-184 as interesting candidates contributing to glioma progression.

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Figures

Figure 1
Figure 1
Results of validation experiments performed for the six selected microRNA (miRNA) candidates in an independent series of 14 diffuse astrocytomas (AII), 13 secondary glioblastomas (sGBIV) and nine non‐neoplastic brain tissue samples (NB). Expression levels of the individual miRNAs were determined by using TaqMan‐based polymerase chain reaction assays. Box plots are depicted indicating median, lower and upper quartile, as well as sample maximum and sample minimum of normalized expression values. Asterisks indicate significant expression differences (P < 0.05).
Figure 2
Figure 2
In vitro effects of miR‐184 overexpression (A–D) and miR‐17 inhibition (E,F) in glioma cells. (A,E) Cell viability was determined using an MTT assay. (B) Cell invasion was quantified using a transwell assay with Matrigel‐coated membranes. (C) Proliferation was measured using a BrdU assay. (D,F) For the comparison of apoptotic activity, a fluorometric caspase‐3/‐7 assay was used. The results of the in vitro assays were converted into arbitrary units (a.u.), setting the average result in the corresponding control cells as 100 a.u. Error bars show standard deviations; asterisks indicate significant differences (P < 0.05). Pre‐NC and Anti‐NC, cells transfected with negative controls; Pre‐184, cells transfected with miR‐184 precursors; Anti‐17, cells transfected with miR‐17 inhibitors.
Figure 3
Figure 3
Proteome changes in T98G glioma cells following miR‐184 overexpression (A) or miR‐17 inhibition (B). (A) Expression of nucleophosmin 1 (Npm1) was found to be significantly (P < 0.05) down‐regulated in T98G cells transfected with miR‐184 precursors (Pre‐184) as compared to control‐transfected cells (Pre‐NC). (B) Protein expression of Pold2 was significantly (P < 0.05) up‐regulated in T98G cells transfected with miR‐17 inhibitors (Anti‐17) as compared to control‐transfected cells (Anti‐NC). Npm1 is a predicted _target of miR‐184, while Pold2 is a predicted _target of miR‐17 according to the Sanger Institute mirBase _target database (http://microrna.sanger.ac.uk/index.shtml).
Figure 4
Figure 4
Down‐regulation of Akt2 in glioma cells overexpressing miR‐184. A172 and T98G cells were transfected with miR‐184 precursors (+) or corresponding controls (−). Western blots were probed with antibodies against Akt2 and α‐tubulin as a loading control. As a further control, glioma cells transfected with inhibitors of miR‐17 (−) or respective controls (+) were probed. Note markedly reduced Akt2 protein levels in A172 and T98G cells transfected with miR‐184 precursors, but not with miR‐17 inhibitors or controls.
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