10 research outputs found

    Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells.

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    Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells

    RT-qPCR for Sonic Hedgehog (A) and TGF-β pathways analysis (B) in SIMA CSC-like cells.

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    <p>The graphs represent the 2<sup>−ΔΔCt</sup> values obtained by RT-qPCR for neurospheres. The dotted line indicates the 2<sup>−ΔΔCt</sup> control (cell line) value equal to 1. Significance against control: p<0.05 (*); p<0.01 (**); p<0.001 (***) and p<0.0001 (****). Selected genes cover the most relevant components of each pathway. Results confirmed aberrant activation of Hh (A) and TGF-β (B) signalling pathways in CSC-like cells.</p

    PANTHER classification by signalling pathway.

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    <p>The differentially expressed genes in both SK-N-DZ and SIMA CSC-like cells were classified by PANTHER and graphed. The percentage represents the number of genes altered against total number of genes involved in each pathway.</p

    Gene expression by RT-qPCR.

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    <p>The graphs represent the 2<sup>−ΔΔCt</sup> values obtained by RT-qPCR for neurospheres. The dotted line indicates the 2<sup>−ΔΔCt</sup> control (cell line) value equal to 1. Significance against control: p<0.05 (*); p<0.01 (**); p<0.001 (***) and p<0.0001 (****). <b>Corroboration of expression array (A).</b> Both SK-N-DZ (▪) and SIMA (▴) CSC-like cells showed a similar expression profile. <b>Stem Cell Markers expression (B).</b> Results confirmed a high overexpression of CSC markers in SIMA cell line after the neurosphere formation assay.</p

    Whole chromosome plots.

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    <p>Array-CGH from SK-N-DZ (A) and SIMA (B) cell lines. The X-axis represents the chromosomes while the Y-axis represent the normalize log2 ratio fluorescence intensity thresholds −1 (loss) and 1 (gain). The results show gains and losses of small chromosomal regions.</p

    Overview on gained (G) and lost (L) chromosomal regions detected by array-CGH.

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    <p>Chromosome:Chromosomal start position:Chromosomal end position (Cr:start:end). Number of polymorphisms or Copy Number Variants (CNV) in the region. Number of miRNA (miRNA) contained in the region. CpG islands (CpGIsl).</p><p>Overview on gained (G) and lost (L) chromosomal regions detected by array-CGH.</p

    DAVID generated categories.

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    <p>The differentially expressed genes in both cell lines were grouped in categories classified as significantly (*) and very significantly (**) enriched in neurosphere samples. Genes were categorized according to the biological processes they are involved in and to the molecular functions they code for.</p><p>DAVID generated categories.</p
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