The Expression Of Livin In Brain Glioma And Its Effect In Cell Apoptosis Of Brain Glioma

Objects:To approach the expression of livin in brain glioma tissue and to research the mechanism of VM-26 in suppression of U-251 by studying the effect of livin in cell invasion metastasis and apoptosis.Materials and methods:30 cases of glioma specimen from patients accepted operation in Xiangya Hopital on the period from May 2004 to April 2005 have been collected.RT-PCR and Western blot were used to detect the mRNA and protein expression levels of livin in glioma tissues. With the combination of histopathological results,the relationship between the expression levels of livin and the clinicopathological characteristics have also been studied.60 cases of paraffin imbedding specimen have also been chosen and immunohistochemistry staining were applied to detect the expression of livin protein in brain glioma and to study the correlation between the livin protein expression and the prognosis of glioma patients.Glioma cell line U-251 were cultured in vitro and treated by VM-26.Then MTT was used to detect the suppression rate of glioma cells to estimate the sensitivity of U-251 cells to chemotherapy.Flow cytometry test was further used to detect the apoptosis rate of U-251 before and after the treat of VM-26.Wound healing assay and Transwell assay were also applied to study the change of cell migration of U-251 cells after VM-26 interfere. Results:The expression of livin mRNA can be detected in all 30 cases of glioma tissue specimens and the livin mRNA expression are significantly higher in glioma tissue than the normal brain tissue (2.13±0.41 vs.0.67±0.13,P=0.008).To concern the expression pattern of livin mRNA,we also used Western blot to detect the same specimens and the results showed that the protein expression of livin are also significantly higher in glioma tissue than the normal brain tissue (2.45±0.610 vs 0.991±0.22,P=0.001).In addition,the expression level of livin mRNA are closely correlated to the expression level of livin protein(R=8.321,P=0.015).The expression level of livin mRNA and protein have close correlations to the differentiation degree,PTBE,and tumor diameterof glioma,but have no correlation to age,gender,tumor location and pathological type.The positive staining of livin protein is the huffy or dark brown particle in cytoplasm.44 cases of 60 cases glioma have positive livin protein expression and the positive rate is 73.3%.With the combination of histopathological datum,we have found that the expression level of livin mRNA and protein have close correlations to the differentiation degree,PTBE,and tumor diameter of glioma,but have no correlation to age,gender,tumor location and pathological type(P＜0.05).The livin positive group had a high risk of recurrence/metastasis(P =0.011)and a shorter survival than weak positive group(590 days vs 389 days,P=0.024).Cox proportional hazards regression model revealed that the Mus81 expression was an independent significant variable(RR=5.74,P=0.0015)as well as tumor diameter and differentiation degree.After been treated by 3ug/ml,10ug/ml and 30ug/ml VM-26 for 12h,24h,48h and 72h,U-251 cells all have been suppressed compared to the controls.MTT shows that the suppressed cell number is increased with the treating time and the concentration of VM-26,which shows that the dose dependence and time dependence.The IC50 of VM-26 to U-251 cell is 12.23 ug/ml and the effect of VM-26 to U-251 is most obvious after been treated for 72 hours.Flow cytometry test shows that U-251 cells are blocked in G2/M phase after been treated by VM-26 and the blockage rate is 53.6%in 24 hours and 61.59%in 72 hours.The U-251 cells which have invased through the artificial basal membrane are integrated and have deep red cell nucleus and shallow red cytoplasm under HE staining.The number of U-251 cells which invased through the artificial basal membrane in a high power field are 115.2±11.3 for the treated group and 343.5±23.8 for the control group(P＜0.001).After been treated by VM-26,the expression level of livin mRNA and protein are increased.Wound healing assay shows that the U-251 cells without the VM-26 treating have crawled through the wound zone in the 24 hours but few of the cells treated by VM-26 can crawled through the wound zone and the wound still exist in 24 hours.Conclusion:The expression level of livin are closely correlated to recurrence,metastasis and prognosis of glioma,which indicates that livin may be a valuable molecular marker of glioma.VM-26 may suppress U-251 cell line by inducing the cell apoptosis,in which livin may play a key role.