The Significance Of Ezrin Expression In Astrocytic Glioma And The Effect Of Its Expression Inhibited By Baicalein On SHG-44 Cell Proliferation And Invasiveness

Part One.The expression and significance of ezrin in human brain astrocytic gliomaObjective:To investigate the expression and significance of cytoskeletal organizer ezrin in human brain astrocytic glioma.Methods:Expression of ezrin in normal human brain tissues from 10 autopsies and tissues from 107 cases of human astrocytic glioma was examined by immunohistocheinical staining.The analysis of clinicopathologic data revealed that the associativity between ezrin and prognosis.Results:The expression of ezrin was significantly higher in human astrocytic glioma tissues of the brain than that in normal tissues(P＜0. 05),In the 107 cases human astrocytic glioma,ezrin expression of 96 cases were positive,and the positive rate was 89.7%,The increase of ezrin correlated positively with tumor grade(r=0.551,P＜0.01).The analysis of clinicopathologic data revealed that The disease-free survival times after operation were significantly different between the group with Strong positive ezrin expression and another with weak negative ezrin expression(x²=29.85,P=0.000).Conclusion:Statistical analysis revealed a stronger association with increasing malignancy for ezrin immunoreactivity.Enhanced ezrin expression maybe played important role in the development of astrocytic tumors.The results indicate that ezrin may provide a useful tool for the grading of astrocytic tumors,and a valuable molecular marker of the prognosis. Part Two.The influence on cell proliferation and invasiveness of human astrocytic glioma cell line SHG-44 by baicaleinObjective:To investigate the influence on cell cycle,tumor growth, and cell invasiveness after hum astrocytic glioma cell line SHG44 were treated by baicalein with the valious inhibitory concentrations.Methods:Hum astrocytic glioma cell line SHG44 were cultured in vitro and treated in various concentrations of baicalein,then MTT method, flow cytometer(FCM)analysis,Transwell assay and Western Blot were applied to measure cell growth,cell cycle,cell invasiveness,expression of MMP₂,MMP₉,survivin,and cyclin D.Results:1.Inhibitory effects of baicalein on proliferation of SHG-44 cells observed by MTT colorimetric survival assay,was that treatment with Bacalein inhibited growth of SHG-44 cells in a time and dose-dependent manner.2.Baicalein-induced apoptosis in a dose-dependent manner was determined by flow cytometry.Compared with the control,Baicalein caused a significant G₀/G₁ phase arrest (P＜0.05)and a decrease in the percentage of cells in S-phase by approximately18%(P＜0.05)in a dose-dependent manner.No significant change for G₂/M phase cells was observed.3.The result of Transwell assay exhibited that cell number through the artificial basal membrane became less compared with the control after 48 hours(P＜0.05).4. Baicalein inhibited the expression of MMP₂,MMP₉,survivin,and cyclin D in a dose-dependent manner.Conclusion:1.Statistical analysis revealed treatment with bacalein inhibited growth of SHG44 cells in a time and dose-dependent manner, and IC₅₀was 13.77μM for 48 hours.2.Baicalein can inhibit cell invasion and migration of human astrocytic glioma cell line SHG-44.3.Baicalein can inhibit expression of MMP₂,MMP₉,survivin,and cyclin D.4. Bacalein may be a useful therapeutic strategy for hum astrocytic glioma Part Three.Baicalein induces influence of cell movement on SHG-44 cell via inhibiting the expression and activation of ezrinObjective:To compare the change of cell motor ability between SHG-44 cell being downregulated the expressing of ezrin by siRNA and the cell treated by baicalein with the eligible inhibitory concentration,to investigate the mechanism on antineoplastic effectiveness whether baicalein inhibiting expression and activation of ezrin..Methods:Human astrocytic glioma cell line SHG44 were cultured in vitro and treated in various concentrations of baicalein,then western blot were applied to observe the expression of ezrin and phosphorylation ezrin, and RT—PCR to measure the level mRNA of ezrin.Further,the expression of ezrin in SHG-44 cell were downregulated by siRNA, Wound healing assay and scanning electron microscope were used to observe the change of cell movement and pseudopods..Results:Baicalein inhibited the expression and activation of ezrin, and degraded the level of ezrin-mRNA in a time and dose-dependent manner.After SiRNA downregulated the expression of ezrin in SHG-44 cell,cell movement and pseudopods were distinctly decreased,the change is at equal pace with inhibition of baicalein.Conclusion:Baicalein maybe induces partly antineoplastic effectiveness via inhibiting expression and phosphorylation of ezrin.