The Expression Of Stathmin And Impact Of Stathmin On Chemosensitivity In Human Glioma

Gliomas are the most common primary tumor of the central nervous system and two-thirds of gliomas are greatly malignant neoplastic that result in a disproportionate share of morbidity and mortality in brain disease. optimal therapy including surgery,chemotherapy and radiation have been the main of therapy for most glioma patients, By optimal therapy including surgery,chemotherapy and radiation two-year survival for patients with glioblastoma is less than 30%. patients with low-grade gliomas have a relatively good prognosis, however,treatment is almost incurative. it is important to elucidate the mechanisms of glioma development and find key molecular targets for the development of effective therapies.Some studies had reported a over-expression of Stathmin has been represented in many malignant tumors. These results suggested that Stathmin plays a role in the oncogenesis of some malignancies. In our previous microarray study, we found expression of Stathmin at a higher level in gliomas more than normal compartment . However, whether Stathmin expression causes glioma development and progression is unknown.This study includes three parts: the first is to explore the expression level of Stathmin gene in gliomas of different grades and normal brain tissues, correlation of Stathmin expression and patient clinical characteristics was investigated;the second is to explore the expression pattern of Stathmin in glioma cell lines ;finally, the effects of inhibbited Stathmin expression on tumor proliferation ,apoptosis and its impact on chemosenstivity are evaluated in U251 glioma cell.PartⅠExpression of Stathmin in Gliomas and Survival rate of the patients analysis Objective: To explore the expression level of Stathmin gene in gliomas of different grades and normal brain tissues and Stathmin expression status of the tumors and the survival time of the patients with glioblastoma. evaluatedMethods: The expression levels of Stathmin mRNA were evaluated by real-time quantitative PCR, and expression levels of Stathmin protein were assessed in 63 gliomas of different grades and 5 normal brain by immunohistochemistry and western blot. Further, Stathmin immunoreactivity was evaluated by 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 showed high expression levels of Stathmin in high-grade gliomas compared to low-grade tumors (p<0.01) or normal counterparts (p<0.01). Further, Stathmin Statining was mainly observed in the cytoplasma of gliomas cells, however, no immunoreactivity for Stathmin was detected in normal brain. Statistical analysis showed increased Stathmin staining in high-grade glioams compared to low-grade (p<0.01) or normal counterparts(p<0.01). Kaplan-Meier survival curves domenstrated that higher expression of Stathmin was negative associated with mean survival time of GBM patients (P = 0.022).Conclusion: we demonstrate that Stathmin mRNA is present in either gliomas or normal compartments examined, and its expression positively correlates with the degree of malignancy at both RNA and protein levels. Overexpression of Stathmin was strongerly associated with clinical characteristics and GBM patients'survival.PartⅡExpression of Stathmin in U87 and U251 glioma cell linesObjective: To explore the expression of Stathmin in U87 and U251 glioma cell lines.Methods: The expression level of Stathmin mRNA and protein were evaluated by RT-PCR,Immunohistochemistry and Western blotting in U87 and U251 glioma cell lines. Results: RT-PCR analysis demonstrated showed higher expression levels of Stathmin /β-actin in U87 and U25 glioma cells compared to normal counterparts (p<0.01). Western blot analysis showed higher Stathmin expression levels in glioma cells compared to normal counterparts (P<0.05)Conclusion: Stathmin is present in glioma cell lines examined, and its expression levels significantly increase at both mRNA and protein levels compared to normal brain tissues..PartⅢRNA interference targeting Stathmin inhibits glioma cell proliferation, enhance cell apotosis and has its effect on chemosensitivity in vitroObjective: To investigate the effects of nhibited Stathmin expression on tumor proliferation apoptosis and its effect on chemosensitivity in U251 glioma cell.Methods: The Stathmin siRNA were transfected into U251 cell-line. After inhibiting Stathmin, the cell proliferative activity and its effect on chemosensitivity for CCNU/TMZ were assessed by MTT assay and cell apoptosis was analyzed by flow cytometry and Hoechst 33258 staining. The expression levels of TrailR1 , TrailR2 , Bax , Bcl-2,,P53,suriviin,MDR1,MRP Protein were measured by Western blotting.Results: The growth of inhibited-Stathmin expressed cells was significant lower in inhibited-Stathmin expressed cell than in control cell and Scrambled cell by MTT assay (P <0.05). The data of flow cytometric analysis shown the apoptosis percentage of inhibbtied-Stathmin expressed cell was increased relative to control cell. Similar results were obtained with Hoechst 33258 staining (P <0.05). Levels expression of p53,Bax andTrailR1/2 by Western blot were changed in cells campared to control or scrambled cells. In inhibited-Stathmin expressed cell, levels of p53,Bax and TrailR1/2 expression were significantly increased, However, no significant difference for the expression levels of Bcl-2,Survivin,MDR1,MRP was observedConclusion: Overall, our results indicate that inhibited-Stathmin expression by RNA interference targeting Stathmin can inhibit cell proliferation and induce apoptosis. Knock-down of Stathmin expression may effect on chemosensitivity for CCNUand TMZ.