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

Ordóñez R, Gallo-Oller G, Martínez-Soto S, Legarra S, Pata-Merci N, Guegan J, Danglot G, Bernheim A, Meléndez B, Rey JA, Castresana JS - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona, Spain.

ABSTRACT
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.

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Related in: MedlinePlus

Gene expression by RT-qPCR.The graphs represent the 2−ΔΔCt values obtained by RT-qPCR for neurospheres. The dotted line indicates the 2−ΔΔCt control (cell line) value equal to 1. Significance against control: p<0.05 (*); p<0.01 (**); p<0.001 (***) and p<0.0001 (****). Corroboration of expression array (A). Both SK-N-DZ (▪) and SIMA (▴) CSC-like cells showed a similar expression profile. Stem Cell Markers expression (B). Results confirmed a high overexpression of CSC markers in SIMA cell line after the neurosphere formation assay.
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pone-0113105-g005: Gene expression by RT-qPCR.The graphs represent the 2−ΔΔCt values obtained by RT-qPCR for neurospheres. The dotted line indicates the 2−ΔΔCt control (cell line) value equal to 1. Significance against control: p<0.05 (*); p<0.01 (**); p<0.001 (***) and p<0.0001 (****). Corroboration of expression array (A). Both SK-N-DZ (▪) and SIMA (▴) CSC-like cells showed a similar expression profile. Stem Cell Markers expression (B). Results confirmed a high overexpression of CSC markers in SIMA cell line after the neurosphere formation assay.

Mentions: RT-qPCR was performed in order to corroborate the expression microarray results. In both cell lines, we confirmed the decrease in the expression of SMAD6, SMAD7 and BAMBI, as the increase observed in JAG1. The results suggested that the CSC-like subpopulation of both cell lines could have a similar expression profile after enrichment with the neurosphere assay (Figure 5A).


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

Ordóñez R, Gallo-Oller G, Martínez-Soto S, Legarra S, Pata-Merci N, Guegan J, Danglot G, Bernheim A, Meléndez B, Rey JA, Castresana JS - PLoS ONE (2014)

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

License
Show All Figures
getmorefigures.php?uid=PMC4231109&req=5

pone-0113105-g005: Gene expression by RT-qPCR.The graphs represent the 2−ΔΔCt values obtained by RT-qPCR for neurospheres. The dotted line indicates the 2−ΔΔCt control (cell line) value equal to 1. Significance against control: p<0.05 (*); p<0.01 (**); p<0.001 (***) and p<0.0001 (****). Corroboration of expression array (A). Both SK-N-DZ (▪) and SIMA (▴) CSC-like cells showed a similar expression profile. Stem Cell Markers expression (B). Results confirmed a high overexpression of CSC markers in SIMA cell line after the neurosphere formation assay.
Mentions: RT-qPCR was performed in order to corroborate the expression microarray results. In both cell lines, we confirmed the decrease in the expression of SMAD6, SMAD7 and BAMBI, as the increase observed in JAG1. The results suggested that the CSC-like subpopulation of both cell lines could have a similar expression profile after enrichment with the neurosphere assay (Figure 5A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona, Spain.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus