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A role for the transcription factor HEY1 in glioblastoma.

Hulleman E, Quarto M, Vernell R, Masserdotti G, Colli E, Kros JM, Levi D, Gaetani P, Tunici P, Finocchiaro G, Baena RR, Capra M, Helin K - J. Cell. Mol. Med. (2008)

Bottom Line: In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways.Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture.Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

View Article: PubMed Central - PubMed

Affiliation: European Institute of Oncology, Via Ripamonti, Milan, Italy.

ABSTRACT
Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

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Specific inhibition of HEY1 expression by siRNA in glioblastoma cell lines results in slower growth. (A) mRNA levels of HEY1 in human glioblastoma cell lines as measured by Q-PCR. Expression of HEY1 in normal brain was taken to be 1 and relative expression levels were calculated. (B) Growth curves of glioblastoma cell lines after RNA interference. U87MG, T98G and U373MG cells were transfected with luciferase siRNA oligos (-♦-) or oligos specific for HEY1 mRNA (–▪–). HEY1 mRNA levels were detected by Q-PCR at the indicated times after transfection (left panels) and concomitant growth curves are presented (right panels).
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fig04: Specific inhibition of HEY1 expression by siRNA in glioblastoma cell lines results in slower growth. (A) mRNA levels of HEY1 in human glioblastoma cell lines as measured by Q-PCR. Expression of HEY1 in normal brain was taken to be 1 and relative expression levels were calculated. (B) Growth curves of glioblastoma cell lines after RNA interference. U87MG, T98G and U373MG cells were transfected with luciferase siRNA oligos (-♦-) or oligos specific for HEY1 mRNA (–▪–). HEY1 mRNA levels were detected by Q-PCR at the indicated times after transfection (left panels) and concomitant growth curves are presented (right panels).

Mentions: To test if HEY1 expression is required for the maintenance of glioblastoma cell proliferation and as such may represent a candidate drug target, we transfected various glioblastoma cell lines, expressing different levels of HEY1 (Fig. 4A), with a siRNA specific for HEY1. Although the endogenous levels of HEY1 mRNA were relatively high in some of the cell lines used, HEY1 expression was not detectable by Western blotting using the currently available antibodies. Thus, in order to check the efficiency of the RNA interference, HEY1 expression was examined by real time Q-PCR. As shown in Fig. 4B, HEY1 expression in the glioblastoma cell line U87MG was drastically decreased after 24 hrs of treatment with siRNA oligos, but levels increased rapidly at later time-points. Inhibition of HEY1 expression in the cell lines T98G and U373, which have relatively high levels of HEY1, was more effective and persisted for at least 72 hrs. The decrease in HEY1 expression correlated with a reduction in cell proliferation, demonstrating that HEY1 is required for the proliferation of glioblastoma cells with high expression of HEY1.


A role for the transcription factor HEY1 in glioblastoma.

Hulleman E, Quarto M, Vernell R, Masserdotti G, Colli E, Kros JM, Levi D, Gaetani P, Tunici P, Finocchiaro G, Baena RR, Capra M, Helin K - J. Cell. Mol. Med. (2008)

Specific inhibition of HEY1 expression by siRNA in glioblastoma cell lines results in slower growth. (A) mRNA levels of HEY1 in human glioblastoma cell lines as measured by Q-PCR. Expression of HEY1 in normal brain was taken to be 1 and relative expression levels were calculated. (B) Growth curves of glioblastoma cell lines after RNA interference. U87MG, T98G and U373MG cells were transfected with luciferase siRNA oligos (-♦-) or oligos specific for HEY1 mRNA (–▪–). HEY1 mRNA levels were detected by Q-PCR at the indicated times after transfection (left panels) and concomitant growth curves are presented (right panels).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823042&req=5

fig04: Specific inhibition of HEY1 expression by siRNA in glioblastoma cell lines results in slower growth. (A) mRNA levels of HEY1 in human glioblastoma cell lines as measured by Q-PCR. Expression of HEY1 in normal brain was taken to be 1 and relative expression levels were calculated. (B) Growth curves of glioblastoma cell lines after RNA interference. U87MG, T98G and U373MG cells were transfected with luciferase siRNA oligos (-♦-) or oligos specific for HEY1 mRNA (–▪–). HEY1 mRNA levels were detected by Q-PCR at the indicated times after transfection (left panels) and concomitant growth curves are presented (right panels).
Mentions: To test if HEY1 expression is required for the maintenance of glioblastoma cell proliferation and as such may represent a candidate drug target, we transfected various glioblastoma cell lines, expressing different levels of HEY1 (Fig. 4A), with a siRNA specific for HEY1. Although the endogenous levels of HEY1 mRNA were relatively high in some of the cell lines used, HEY1 expression was not detectable by Western blotting using the currently available antibodies. Thus, in order to check the efficiency of the RNA interference, HEY1 expression was examined by real time Q-PCR. As shown in Fig. 4B, HEY1 expression in the glioblastoma cell line U87MG was drastically decreased after 24 hrs of treatment with siRNA oligos, but levels increased rapidly at later time-points. Inhibition of HEY1 expression in the cell lines T98G and U373, which have relatively high levels of HEY1, was more effective and persisted for at least 72 hrs. The decrease in HEY1 expression correlated with a reduction in cell proliferation, demonstrating that HEY1 is required for the proliferation of glioblastoma cells with high expression of HEY1.

Bottom Line: In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways.Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture.Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

View Article: PubMed Central - PubMed

Affiliation: European Institute of Oncology, Via Ripamonti, Milan, Italy.

ABSTRACT
Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

Show MeSH
Related in: MedlinePlus