Abstract
RNA interference (RNAi) technology is one of the main means in the study of stem cell differentiation. This study describes Rho-GDIγ function during the differentiation of neural stem cells by using RNAi. Rho-GDIγ belongs to the Rho-GDI protein family, which is expressed at high level throughout the brain. Although it exists in neuronal population, its physiological function is poorly understood. By using RNAi technology to downregulate expression of Rho-GDIγ, we found distinct morphological changes in neural stem cell line C17.2. More important, RT-PCR confirmed that RNAi-mediated downregulation of Rho-GDIγ decreased expression of Rho-GDIγ-regulated genes RhoA and slightly increased expression of Rac1. Further, immunochemical staining indicated that downregulation of Rho-GDIγ increased the tendency of C17.2 cells to differentiate. These data strongly suggest that Rho-GDIγ plays a key role in the differentiation of neural stem cells.
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References
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.
Gan, L., Anton, K. E., Masterson, B. A., Vincent, V. A., Ye, S., and Gonzalez-Zulueta, M. (2002) Specific interference with gene expression and gene function mediated by long dsRNA in neural cells. J Neurosci Methods 121, 151–157.
Wood, M. J., Trulzsch, B., Abdelgany, A., and Beeson, D. (2003) Therapeutic gene silencing in the nervous system. Hum Mol Genet 12(2), R279–284.
Krichevsky, A. M., and Kosik, K. S. (2002) RNAi functions in cultured mammalian neurons. Proc Natl Acad Sci USA 99, 11926–11929.
Nicchia, G. P., Frigeri, A., Liuzzi, G. M., and Svelto, M. (2003) Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes. Faseb J 17, 1508–1510.
Higuchi, H., Yamashita, T., Yoshikawa, H., and Tohyama, M. (2003) Functional inhibition of the p75 receptor using a small interfering RNA. Biochem Biophys Res Commun 301, 804–809.
Merkle, F. T., Mirzadeh, Z., and Alvarez-Buylla, A. (2007) Mosaic organization of neural stem cells in the adult brain, Science (New York, NY) 317, 381–384.
Lu, W., Wang, J., and Wen, T. (2008) Downregulation of Rho-GDI gamma promotes differentiation of neural stem cells. Mol Cell Biochem 311, 233–240.
Newman, M. B., and Bakay, R. A. (2008) Therapeutic potentials of human embryonic stem cells in Parkinson’s disease. Neurotherapeutics 5, 237–251.
Bar-Sagi, D., and Hall, A. (2000) Ras and Rho GTPases: A family reunion. Cell 103, 227–238.
Adra, C. N., Iyengar, A. R., Syed, F. A., Kanaan, I. N., Rilo, H. L., Yu, W., Kheraj, R., Lin, S. R., Horiuchi, T., Khan, S., Weremowicz, S., Lim, B., Morton, C. C., and Higgs, D. R. (1998) Human ARHGDIG, a GDP-dissociation inhibitor for Rho proteins: Genomic structure, sequence, expression analysis, and mapping to chromosome 16p13.3. Genomics 53, 104–109.
Ferland, R. J., Li, X., Buhlmann, J. E., Bu, X., Walsh, C. A., and Lim, B. (2005) Characterization of Rho-GDIgamma and Rho-GDIalpha mRNA in the developing and mature brain with an analysis of mice with targeted deletions of Rho-GDIgamma, Brain Res 1054, 9–21.
Wen, T., Gu, P., Minning, T. A., Wu, Q., Liu, M., Chen, F., Liu, H., and Huang, H. (2002) Microarray analysis of neural stem cell differentiation in the striatum of the fetal rat, Cell Mol Neurobiol 22, 407–416.
Chalisova, N. I., Pennijajnen, V. P., Baskova, I. P., Zavalova, L. L., and Bazanova, A. V. (2003) The neurite-stimulating activity of components of the salivary gland secretion of the medicinal leech in cultures of sensory neurons, Neurosci Behav Physiol 33, 411–414.
Acknowledgments
We thank Prof. Snyder, E.Y for kindly donating C17.2 for this research. This work was supported by grants from the Major State Basic Research and Development Program of China (973 program, Grant No. 2006CB500702), the National Natural Science Foundation of China (Grant No. 30570590, 30770695), the Shanghai Commission of Science and Technology Basic Research Fund (Grant No. 03JC14030), the Shanghai Commission of Education Science and Technology Innovation Fund (Grant No. 08 ZZ41).
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Wang, J., Lu, W., Wen, T. (2010). Silencing of Rho-GDIγ by RNAi Promotes the Differentiation of Neural Stem Cells. In: Zhang, B., Stellwag, E. (eds) RNAi and microRNA-Mediated Gene Regulation in Stem Cells. Methods in Molecular Biology, vol 650. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-769-3_9
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DOI: https://doi.org/10.1007/978-1-60761-769-3_9
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