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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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Cell differentiation and apoptosis. The levels of the neural marker βIII Tubulin and of the antiapoptotic protein Bcl2 were analyzed by western blot. The experiment was performed twice using two different cell extracts. a) The higher level of βIII Tubulin detected in knockdown (KD) vs control (Ctrl) indicates that Msi1 may play a role in inhibiting cell differentiation. In addition, the reduced levels of Bcl2 detected in the knockdown (b) suggested a potential role for Msi1 in preventing apoptosis. c) β-Actin was used as loading control.
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Figure 6: Cell differentiation and apoptosis. The levels of the neural marker βIII Tubulin and of the antiapoptotic protein Bcl2 were analyzed by western blot. The experiment was performed twice using two different cell extracts. a) The higher level of βIII Tubulin detected in knockdown (KD) vs control (Ctrl) indicates that Msi1 may play a role in inhibiting cell differentiation. In addition, the reduced levels of Bcl2 detected in the knockdown (b) suggested a potential role for Msi1 in preventing apoptosis. c) β-Actin was used as loading control.

Mentions: In order to determine Msi1's possible involvement in cell differentiation and apoptosis we analyzed the levels of the neural marker βIII Tubulin and the antiapoptotic protein Bcl2 by western blot in the cell line Daoy (Figure 6). On average, a 5-fold increase (p < 0.05) in βIII Tubulin and a 8-fold decrease (p < 0.05) in Bcl-2 were detected when Msi1 was knocked down. The higher levels of βIII Tubulin detected in the knockdown indicated that Msi1 arrests cell differentiation. Decline in both Bcl2 protein (Figure 6) and BCL2 transcript (Figure 4) levels were also detected in the knockdown suggesting that Msi1 may block apoptosis in Daoy cells. As a more important reduction was detected at protein level than at mRNA level (~30% down-regulation; Figure 4), additional post-trancriptional mechanisms might be mediating the decrease of Bcl2 protein in our model system. Overall, the results obtained in our model system are consistent with a recent report by Dobson et al. [25] which demonstrates that Msi1 has an inhibitory effect upon oligodendrocyte precursor differentiation and apoptosis.


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)

Cell differentiation and apoptosis. The levels of the neural marker βIII Tubulin and of the antiapoptotic protein Bcl2 were analyzed by western blot. The experiment was performed twice using two different cell extracts. a) The higher level of βIII Tubulin detected in knockdown (KD) vs control (Ctrl) indicates that Msi1 may play a role in inhibiting cell differentiation. In addition, the reduced levels of Bcl2 detected in the knockdown (b) suggested a potential role for Msi1 in preventing apoptosis. c) β-Actin was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Cell differentiation and apoptosis. The levels of the neural marker βIII Tubulin and of the antiapoptotic protein Bcl2 were analyzed by western blot. The experiment was performed twice using two different cell extracts. a) The higher level of βIII Tubulin detected in knockdown (KD) vs control (Ctrl) indicates that Msi1 may play a role in inhibiting cell differentiation. In addition, the reduced levels of Bcl2 detected in the knockdown (b) suggested a potential role for Msi1 in preventing apoptosis. c) β-Actin was used as loading control.
Mentions: In order to determine Msi1's possible involvement in cell differentiation and apoptosis we analyzed the levels of the neural marker βIII Tubulin and the antiapoptotic protein Bcl2 by western blot in the cell line Daoy (Figure 6). On average, a 5-fold increase (p < 0.05) in βIII Tubulin and a 8-fold decrease (p < 0.05) in Bcl-2 were detected when Msi1 was knocked down. The higher levels of βIII Tubulin detected in the knockdown indicated that Msi1 arrests cell differentiation. Decline in both Bcl2 protein (Figure 6) and BCL2 transcript (Figure 4) levels were also detected in the knockdown suggesting that Msi1 may block apoptosis in Daoy cells. As a more important reduction was detected at protein level than at mRNA level (~30% down-regulation; Figure 4), additional post-trancriptional mechanisms might be mediating the decrease of Bcl2 protein in our model system. Overall, the results obtained in our model system are consistent with a recent report by Dobson et al. [25] which demonstrates that Msi1 has an inhibitory effect upon oligodendrocyte precursor differentiation and apoptosis.

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