| Part I The expression of zinc finger protein X-linked in gliomas of humanObjective Zinc finger protein X-linked (Zfx) is a zinc finger protein of the Zfy family. As a shared transcriptional regulator of embryonic and adult stem cells, Zfx may be a common molecular basis of stem cells self-renewal. In this study, we investigated the expressions of zinc finger protein X-linked (Zfx) in gliomas of human and its relationship with malignant degree.Methods 52 cases of gliomas and 8 cases of normal brain tissues were collected and were divided into several groups by malignant degree. The expressions of Zfx were measured by real-time quantitative PCR in mRNA level and by western blot and immunohistochemisty in protein level. Zfx levels between normal brain tissues and glioma tissues were analyzed by statistics.Result The positive particles of Zfx were distributed in the neuronal nucleus. In both mRNA and protein level, the expression of Zfx was much higher in glioma tissues than that in normal brain tissues. The more malignant the glioma was, the higher the Zfx level was. Conclusions Zinc finger protein X-linked (Zfx) was overexpression in glioma of human and was direct correlation with malignant degree. Zfx may control the ability to self-renew in brain tumor stem cell of glioma. Understanding Zfx-induced self-renewal mechanism is essential to encompass the full complexity of the highly dynamic and complex nature of glioma biology as we seek to better treat and potentially cure the human disease.PartⅡThe expression of zinc finger protein X-linked in brain tumor stem cells of human gliomaObjective Zinc finger protein X-linked (Zfx) is a zinc finger protein of the Zfy family. As a shared transcriptional regulator of embryonic and adult stem cells, Zfx may be a common molecular basis of stem cells self-renewal. In this study, we investigated the expressions of zinc finger protein X-linked (Zfx) in brain tumor stem cells (BTSCs) of human glioma.Methods We isolated and identified brain tumor stem cells from two glioma cell lines U251 and U87. The expressions of Zfx were measured by western blot in protein level and analyzed by statistics.Result The ratio of CD133-positive cells of U251 cells and U87 cells was 1.97%±0.53%; 1.32%±0.34%. The isolated CD133-positive cells formed tumor spheres in serum-free medium. Undifferentiated spheres from both U251 and U87 were immunostained for characteristic neural stem cell marker nestin. Under differentiation conditions, the tumor spheres were differentiated in complete medium and expressed neuronal markerβ-tubulin and astrocytic marker GFAP, similar to those in the original glioma cell lines. The expression of Zfx in undifferentiated spheres was significantly increased approximately when compared to that in differentiated spheres and original glioma cell lines.Conclusions Zinc finger protein X-linked (Zfx) was overexpression in brain tumor stem cells of human glioma. Zfx may control the ability to self-renew and anti-apoptosis in brain tumor stem cells of human glioma.PartⅢConstruction and identification of lentiviral shRNA vectors targeting human Zfx geneObjective To construct and identify lentiviral shRNA vectors targeting human Zfx gene.Methods The target shRNA against human Zfx gene (GenBank accession NM003410) for RNAi was designed, and the pFU-GW-iRNA lentiviral vectors were constructed. Then human glioma cell line U251 was transfected with specific or negative control lentiviral vectors. Cells were observed under fluorescence microscope. The silencing of Zfx was analyzed by real-time quantitative PCR and western blot analysis.Result Assessment of infection rate was accomplished with the determination of the rate of positive expression of GFP by fluorescent microscopy. Infection efficiency of Zfx-RNAi and mock groups were more than 90% in U251 cells 7days after transfection. The silencing efficiency of lentiviral vector was determined by real-time quantitative PCR and western blot analysis. We found that the expression of Zfx was predominantly knocked down in R2 group compared to control, mock and other Zfx-RNAi groups.Conclusions The lentiviral shRNA vectors targeting human Zfx gene has been successfully constructed, which provides a basis for further study of mechanisms that Zfx may be an important transcriptional regulator of glioma for the proliferation behavior.Part IV The function of Zinc finger protein X-linked in brain tumor stem cells of human glioma Objective Zinc finger protein X-linked (Zfx) is a zinc finger protein of the Zfy family. As a shared transcriptional regulator of embryonic and adult stem cells, Zfx may be a common molecular basis of stem cells self-renewal. In this study, we investigated the function of Zinc finger protein X-linked (Zfx) in brain tumor stem cells of human glioma.Methods We knocked down the expression of Zfx gene in glioma cells lines U251 and U87 by lentiviral vector-mediated RNA interference and isolated brain tumor stem cells from cell lines, then investigated the function of Zfx in brain tumor stem cells of human glioma by colony-forming assay, apoptosis and western blot analysis.Result We established stable Zfx silencing glioma cells with lentiviral vector-mediated RNA interference, and found that the deficient of Zfx suppressed proliferation and induced apoptosis of glioma. Downregulation of Zfx significantly suppressed self-renewal and induced apoptosis of brain tumor stem cells (BTSCs). The deficient of Zfx also induced change in activating transcription factor 4 (ATF4) pathways.Conclusions Our results supposed that transcription factor Zfx silencing suppressed self-renewal and induced apoptosis of brain tumor stem cells in human gloma, Zfx might be an important transcriptional regulator of glioma for the proliferation behavior. |
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