Expression Of TPX2 In Gliomas And Effect Of Altered TPX2 On Progression Of Glioma

Astrocytoma remains the most common primary neoplasm of the central nervous system and accounts for approximately 45-55% of all brain tumors. It represents a heterogenous group of diseases with different degree of malignancy from relatively indolent pilocytic astrocytomas to highly aggressive glioblastomas. Unfortunately, current therapeutic modalities including surgical resections, chemotherapy, radiotherapy or combinations can not ensure a cure and, the molecular mechanisms underlying the initiation, maintenance and progression of astrocytomas still remain largely unclarified. Hence, identification and characterization of the regulatory molecules that involved in the astrocytoma tumorigenesis may offer important targets for treatment strategies.Targeting protein for Xklp2 (TPX2) is a cell cycle-associated human protein encoded by a gene located on human chromosome band 20q11.2. Its expression is tightly cell cycle regulated. Aberrant expression of TPX2 has been found in various malignant tumors. These results suggested that TPX2 plays a role in the oncogenesis of some malignancies. In our previous microarray study, we found expression of TPX2 at a higher level in human astrocytomas more than normal control. However, whether TPX2 expression contributes to glioma development and progression is unknown. In the present study, we sought to determine whether and, if so, how TPX2 regulates the growth of astrocytomas. we investigated expression of TPX2 in different grade human astrocytomas and the related cell lines, and the correlations between TPX2 protein levels and grade of differentiation. Hence, further investigation of the functional role of TPX2 in the carcinogenesis of glioma cells may offer a better understanding of malignant behavior of glioma cells.The aim of this study is to clarify the exact role for TPX2 in the oncogenic process of glioma cells by examining its expression pattern in glioma tissues of different grades and glioma cell lines and knocking down its expression level in glioma cell lines. This study is consisted of three main parts:the first part is to investigate the expression level of TPX2 gene in astrocytomas of different grades and normal brain tissues, correlation of TPX2 expression and patient clinical characteristics was investigated by statistic analysis.the second is to investigate the expression pattern of TPX2 in glioma cell lines and finally, the effects of inhibbited TPX2 expression on tumor proliferation and apoptosis are evaluated in U87 glioma cell. Objective:To investigate the expression level of TPX2 gene in astrocytomas of different grades and normal brain tissues and TPX2 expression status of the tumors and the survival time of the patients.Methods:The expression levels of TPX2 mRNA were evaluated by real-time quantitative PCR, and expression levels of TPX2 protein were assessed in 52 astrocytic tumors of different pathological grades and 5 normal brain controls by using immunohistochemistry and western blot. Further, TPX2 protein level was evaluated by the product-limit estimate of the survival function (Kaplan-Meier method), and differences between the survival functions were assessed with log-rank test and confirmed by the generalized Wilcoxon test..Results:Quantitative real time PCR analysis demonstrated elevated expression levels of TPX2/β-actin in high-grade astrocytomas versus low-grade (p<0.01) or normal brain tissues (p<0.01). Further, TPX2 immunoreactivity was predominantly detected in the nucleus of tumor cells, whereas no positive staining for TPX2 was observed in normal brain tissues. Statistical analysis showed increased TPX2 labelling index in high-grade astrocytomas versus low-grade tumors (p<0.01) or normal controls (p<0.01). Kaplan-Meier survival curves indicated that increased expression of TPX2 was significantly associated with poor overall survival of astrocytoma patients (P=0.000). Conclusion:In summary, we demonstrate thatTPX2 is present in either astrocytomas or normal brain tissues examined, and its expression positively correlates with the degree of malignancy at both RNA and protein levels. TPX2 overexpression was significantly associated with clinical stage and patient survival. Methods:The expression level of TPX2 mRNA and protein were evaluated by real-time quantitative PCR and Western blotting respectively in U87 and U251 glioma cell lines. Subcellular localization of TPX2 in U87 cells identified by immunofluorescent staining.Results:Quantitative real time PCR analysis demonstrated elevated expression levels of TPX2/β-actin in U87 and U25 glioma cells versus normal brain tissues (p<0.01). Western blot analysis showed increased TPX2 expression levels in glioma cells versus normal brain tissues (P<0.05). When the U87 cells were immunofluorescently stained, it was observed that TPX2 protein localized in nucleus and it associates with the mitotic spindle.Conclusion:TPX2 is present in glioma cell lines examined, and its expression levels significantly increase at both mRNA and protein levels versus normal brain tissues.Objective:To investigate the effects of inhibbited TPX2 expression on tumor proliferation and apoptosis of U87glioma cell.Methods:The TPX2 siRNA were transfected into U87 glioma eel by RNA interference. After the inhibition of the endogenous TPX2, the cell proliferative activity was assessed by 3H-TdR incorporation test and cell apoptosis was analyzed by flow cytometry and Hoechst 33258 stainning. The expression levels of Aurora A, Ran, c-Myc, p53, cyclin D1, cyclin B1 Protein were measured by RT-PCR and Western blotting respectively.Results:The growth of inhibbited-TPX2 expressed cells was significantly decreased Cmp was significant lower in inhibbited-TPX2 expressed cell than in control cell by 3H-TdR incorporation test (P=0.025). The data of flow cytometric analysis shown the apoptosis percentage of inhibbtied-TPX2 expressed cell was increased relative to control cell. Similar results were obtained with Hoechst 33258 staining (P<0.05). Levels expression of Aurora A, Ran, c-Myc, p53 and cyclin B1 by Western blot were changed in cells campared with control cells. In inhibbited-TPX2 expressed cell, levels of Aurora A, Ran and cyclin B1 expression were significantly decreased, respectively. Levels of c-Myc and p53 mRNA expression were increased in glioma cell, respectively. However, no significant difference for the expression levels of Cyclin D1 was noted. Conclusion:Overall, our results indicate that inhibbited-TPX2 expression can inhibit cell proliferation and induce apoptosis. Knock-down of TPX2 expression may effect cycle associated-protein.