Análisis de la implicación de los micrornas en la diferenciación de las células madre tumorales de glioblastoma multiforme

Résumé

Background: Brain tumor stem cells (BTSC) are cancer cells with neural stem cell (NSC)-like properties found in the fatal glioblastoma multiforme (GBM). These BTSC account for less than 1% of total tumor cells, are poorly differentiated and are believed to be involved in tumor development, progression, therapeutic resistance and relapse. BTSC are considered key targets of effective treatment. Therapeutic, drug-induced differentiation of BTSC is viewed as a promising approach to defeat GBM. MicroRNAs (miRNAs) are essential regulators of gene expression and development. Specific miRNAs play important roles in modulating the proliferation and differentiation of stem cells. In this study we searched for miRNAs controlling differentiation in GBM-BTSCs through high throughput screening miRNA expression profiling. Methods: BTSC derived from five GBM obtained after surgery were cultured in vitro as neurospheres (in presence of EGF, b-FGF and B27 supplement) and subsequently differentiated (removing B27 complement and adding FBS to the medium) for 4 and 14 days. RNA was extracted using mirVana miRNA Isolation Kit (Ambion, USA). We performed miRNA miRCURY¿ LNA Array version 5th Generation (Exiqon, Denmark) that contains capture probes targeting 2090 miRNAs (all miRNAs for human, mouse or rat registered in the miRBASE version 15.0 at the Sanger Institute). Lowess normalization was applied to the array data and 3 comparisons were made (stem vs 4 days, stem vs 14 days and stem vs 4 and 14 days) using the LIMMA software. MicroRNAs showing values of B>0 in any of the 3 comparisons performed were selected. Cy3 raw data values were scrutinized and miRNAs with values greater than 200 were selected. Visual images were generated through hierarchical clustering. Validation of candidate miRNAs was carried out with the use of TaqMan MicroRNA assays (Applied Biosystems, USA) by quantitative retrotranscriptase PCR (q-RT-PCR). Results and conclusions: The LIMMA analysis resulted in 36 differentially expressed miRNAs. Further selection by Cy3 raw data value >200 yielded 21 candidate miRNAs. Ten of them were downregulated in the differentiated status: hsa-miR-103, hsa-miR-106a, hsa-miR-106b, hsa-miR-15b, hsa-miR-17, hsa-miR-19a, hsa-miR-20a, hsa-miR-25, hsa-miR-301a and hsa-miR-93. The remaining eleven miRNAs were found to be upregulated in the differentiated status: hsa-miR-100, hsa-miR-1259, hsa-miR-21, hsa-miR-22, hsa-miR-221, hsa-miR-222, hsa-miR-23b, hsa-miR-27a, hsa-miR-27b, hsa-miR-29a and hsa-miR-29b. Most of the miRNAs obtained belong to clusters and various paralogous clusters. Expression of 11 of the 21 miRNAs obtained was examined by qPCR and 7 of them were validated. Validation of candidate miRNAs rendered 5 upregulated miRNAs (hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222) and 2 downregulated miRNAs (hsa-miR-93 and hsa-miR-106a) in the differentiated status (4 and 14 days) as compared to the stem-like (neurosphere) status. Among these miRNAs, miR-29a/29b and miR-15/16-2 clusters are pro-apoptotic. Others, such as miR-21, miR-221/222, mir-93/106b and miR17/92 clusters are involved in cell cycle progression and proliferation. Functional studies are being carried out at the moment to further examine the specific roles of the validated miRNAs in BTSC differentiation.