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Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy.

Sanchez-Diaz PC, Burton TL, Burns SC, Hung JY, Penalva LO - BMC Cancer (2008)

Bottom Line: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation.We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer.Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, TX, USA. sanchezdiaz@uthscsa.edu

ABSTRACT

Background: Musashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer.

Methods: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells.

Results: We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

Conclusion: Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

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Msi1 downstream effects. The RNA levels of a subset of Notch, Hedgehog and Wnt pathway components were quantified by quantitative RT-PCR in control and knockdown Daoy cells. Gene expression changes are represented (mean ± se) using semi-logarithmic scale. The plot in the upper part (a) shows the fold change in mRNA levels for the Msi1 knockdown normalized to the housekeeping gene 15S. The table (b) summarizes the ratio (in percentage) of mRNA levels detected in Msi1 knockdown vs control. * represents p < 0.05 and ** p < 0.01.
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Figure 4: Msi1 downstream effects. The RNA levels of a subset of Notch, Hedgehog and Wnt pathway components were quantified by quantitative RT-PCR in control and knockdown Daoy cells. Gene expression changes are represented (mean ± se) using semi-logarithmic scale. The plot in the upper part (a) shows the fold change in mRNA levels for the Msi1 knockdown normalized to the housekeeping gene 15S. The table (b) summarizes the ratio (in percentage) of mRNA levels detected in Msi1 knockdown vs control. * represents p < 0.05 and ** p < 0.01.

Mentions: Msi1 represses translation of NUMB [12], which antagonizes the activity of Notch and Hedgehog [18,19]. Recently, it has been shown that Msi1 also activates Notch and Wnt in mammary progenitors through an autocrine mechanism involving up-regulation of Proliferin-1 and down-regulation of Dickkopf-3 [23]. Notch, Hedgehog and Wnt pathways are central regulators of cell proliferation and their deregulation is frequently associated with medulloblastoma (reviewed in [22]). With the high levels of Msi1 reported for medulloblastoma, a connection among Msi1, Notch, Hedgehog and Wnt activities and tumor growth is likely. To investigate if Msi1 was modulating the activity of these pathways in Daoy, the expression of some Notch, Hedgehog and Wnt pathway components and downstream targets were evaluated by quantitative RT-PCR. The primers used for this analysis are listed in Additional file 1. In the knockdown, up-regulation of the Wnt inhibitor Dickkopf-1 (DKK1; >70% increase) was observed along with a down-regulation in the Wnt downstream target FOS (60% reduction) as shown in Figure 4. Down-regulation of a subset of Notch and Hedgehog related genes was also found. In this regard, lower levels of the Hedgehog component SMO (50% decrease) and of the downstream targets MYCN (90% decrease), CCND2, PPAP2B and CDKN1A (50% decrease) were detected in the Msi1 knockdown cells together with lower levels of the relevant Notch component NOTCH2 (40% decrease) and of the downstream target HEY2 (60% decrease). Intriguingly, up-regulation of the Wnt and Hedgehog common downstream target CCND1 (>60% increase) was observed. Overall it seemed that Msi1 modulates expression of a number of genes involved in cell proliferation, differentiation and survival processes. Therefore, our observation was in agreement with Wang et al recent findings using mammary progenitors [23].


Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy.

Sanchez-Diaz PC, Burton TL, Burns SC, Hung JY, Penalva LO - BMC Cancer (2008)

Msi1 downstream effects. The RNA levels of a subset of Notch, Hedgehog and Wnt pathway components were quantified by quantitative RT-PCR in control and knockdown Daoy cells. Gene expression changes are represented (mean ± se) using semi-logarithmic scale. The plot in the upper part (a) shows the fold change in mRNA levels for the Msi1 knockdown normalized to the housekeeping gene 15S. The table (b) summarizes the ratio (in percentage) of mRNA levels detected in Msi1 knockdown vs control. * represents p < 0.05 and ** p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572071&req=5

Figure 4: Msi1 downstream effects. The RNA levels of a subset of Notch, Hedgehog and Wnt pathway components were quantified by quantitative RT-PCR in control and knockdown Daoy cells. Gene expression changes are represented (mean ± se) using semi-logarithmic scale. The plot in the upper part (a) shows the fold change in mRNA levels for the Msi1 knockdown normalized to the housekeeping gene 15S. The table (b) summarizes the ratio (in percentage) of mRNA levels detected in Msi1 knockdown vs control. * represents p < 0.05 and ** p < 0.01.
Mentions: Msi1 represses translation of NUMB [12], which antagonizes the activity of Notch and Hedgehog [18,19]. Recently, it has been shown that Msi1 also activates Notch and Wnt in mammary progenitors through an autocrine mechanism involving up-regulation of Proliferin-1 and down-regulation of Dickkopf-3 [23]. Notch, Hedgehog and Wnt pathways are central regulators of cell proliferation and their deregulation is frequently associated with medulloblastoma (reviewed in [22]). With the high levels of Msi1 reported for medulloblastoma, a connection among Msi1, Notch, Hedgehog and Wnt activities and tumor growth is likely. To investigate if Msi1 was modulating the activity of these pathways in Daoy, the expression of some Notch, Hedgehog and Wnt pathway components and downstream targets were evaluated by quantitative RT-PCR. The primers used for this analysis are listed in Additional file 1. In the knockdown, up-regulation of the Wnt inhibitor Dickkopf-1 (DKK1; >70% increase) was observed along with a down-regulation in the Wnt downstream target FOS (60% reduction) as shown in Figure 4. Down-regulation of a subset of Notch and Hedgehog related genes was also found. In this regard, lower levels of the Hedgehog component SMO (50% decrease) and of the downstream targets MYCN (90% decrease), CCND2, PPAP2B and CDKN1A (50% decrease) were detected in the Msi1 knockdown cells together with lower levels of the relevant Notch component NOTCH2 (40% decrease) and of the downstream target HEY2 (60% decrease). Intriguingly, up-regulation of the Wnt and Hedgehog common downstream target CCND1 (>60% increase) was observed. Overall it seemed that Msi1 modulates expression of a number of genes involved in cell proliferation, differentiation and survival processes. Therefore, our observation was in agreement with Wang et al recent findings using mammary progenitors [23].

Bottom Line: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation.We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer.Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, TX, USA. sanchezdiaz@uthscsa.edu

ABSTRACT

Background: Musashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer.

Methods: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells.

Results: We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

Conclusion: Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

Show MeSH
Related in: MedlinePlus