The Effect Of Ezrin On Invasive Of U251and U87Cell

Glioma is the tumor in the neural ectoderm, is the most common type of primaryintracranial tumors, it has high incidence and mortality, the rate of incidence is3~10per100thousand, accounted for46%of all intracranial tumors,1%~3%of systemicmalignant tumors,5-year survival rate is20%~30%[12]，and is a difficulty pointsfor the central nervous system diseases. Surgical resection and radiotherapy (RT),supplemented by chemotherapy is the current treatment standard, but gliomatreatment resistance, making very poor prognosis in glioma, average age12-15months[2-4]. Although in recent years, with the development and application of neuralimaging and intraoperative tumor tracer technology, it makes progress in the treatmentof glioma, but also difficult to ensure that predominantly surgical treatment cancompletely eradicate the tumor, the basic reason is the growth of gliomacharacteristics.Recent years base on the research of gliomas molecular targets, targeted therapydrugs the development, clinical trial has certain therapeutic effect, but far fromexpected results. Moreover, although workers have done many studies on themechanism of invasive tumor growth, but still can’t solve the mechanism of gliomainvasive growth, so further study of the mechanism and finding the effective way oftreatment, is the key to treatment of glioma and improve the quality of survival.Tumor metastasis is the biological characteristics of malignant tumor, cellmovement ability enhanced lead to the migration and invasion, cytoskeletonremodeling is a direct reason, and Ezrin is actin bridge connected to the cellmembrane and membrane surface receptor[18], involved in many kinds of actinfunctions such as cell adhesion[20], cell movement and morphogenesis[21].Up-regulated the expression of variety of tumor tissue[8-15], including glioma. But therole of Ezrin in infiltrating growth of gliomas is not clear.Combine with the current situation of biological characteristics, treatment ofglioma and the role of Ezrin genes in tumor migration and invasion, this paper studiesthe relationship of invasive growth between Ezrin and glioma cells U87、U251, and discuss the mechanism. The main research results:1.The shRNA’s expression vector of Ezrin gene was constructed successfully,and U87cells were transfected by liposomes Lipofectimine2000, RT-PCR andWestern blot results showed that shRNA-Ezrin-2can be used to be silent theexpression of Ezrin, and made the expression levels of mRNA and protein decreased,it was identified to be valid shRNA vector.2. The pEGFP-C1/Ezrin that was the expression vector of Ezrin gene wasconstructed successfully. Western blot detect the recombinant vector pEGFP-C1/Ezrinturn transfection group that Ezrin expression levels was higher than the empty vectorpEGFP-C1transfection group and blank control group.3. After U87cells were transfected by shRNA vectors and expression vectors ofEzrin gene respectively, scratch experiments show that Ezrin shRNA can block themigration of U251and U87cells, and its over-expression can promote the cellmigration. The result of Brain tumor model in nude mice show that，compared withthe control group, the number of transfection shRNA-Ezrin-2on cell migration isless than transfection pEGFP C1/Ezrin. It show that Ezrin gene silencing can blockthe invasive growth of U251and U87, over expression can promote migration,increase its infiltration growth, so Ezrin participation in the invasive growth of U251and U87.4. To study the effects of Ezrin on migration of U251and U87, invasive growthmechanism, rt-pcr and Western blot analysis of shRNA Ezrin-2and over expressionvector pEGFP C1/Ezrin transfection cells the expression of Rac1, Results show thatexpression level of pEGFP-C1/Ezrin transferred group Rac1is higher thanshRNA-Ezrin-2transferred group and the control group, and shRNA-Ezrin-2transfected group is lower than control group. The results indicate that Ezrin canactivate the Rac1, the effects of Ezrin on U251and U87cells are achieved throughRac1.5. Transwell found the ability of U87cells migration is stronger than the U251cells, to know the reason of this difference, the structure of Ezrin was analyzed, andresults show that the Ezrin protein of U87cells in the66th,258,265and577-bit isvariable, and the C-terminus amino acid of34is the binding site of Ezrin and F-actin, N terminus43amino acids is the functional region，it is connected to the Cterminus107amino acids[26], we do not know in this area amino acid variations, whether affect the Ezrin and F-actin or combination of membranes and influence itsfunction, and then affects the cell migration. This study analysed Ezrin protein andfunction on the different infiltration ability of glioma cells with U251and U87throughbioinformatics methods. the U251cells Ezrin protein "matched" has362，and the U87cell Ezrin protein has1503，Ezrin structure function showed that,the number of PDBin Ezrin of U87is more than U251.According to the analysis, while the SCOP andCATH have the same hierarchical structure of the domain structure and hierarchicalclassification; Domain organization in Ezrin of U87cells had45, while the U251hadonly5. The further analysis of SWISS-MODEL, Coloring by residue error, the Ezrinof U87and U251cells had8difference, QMEAN4rate that the Ezrin of U87cellswas less than U251cells; SWISS-PDB Viewer showed that the258th,265th aminoacids was relatively in the inside of spatial structure. We speculated the function、domain and spatial structure of U87cells’ Ezrin was different from them of U251cells, resulting in the differences of U251and U87cells migration and invasioncapacity.6. To test and verify the differences of the Ezrin protein’s structure which lead tothe differences of U251and U87cell migration and invasion ability, we used Ezrin ofU251cells to transfect U87cells, and then used Ezrin of U87cells to transfect U251cells, the results showed that U251cells of pEGFP-C1/Ezrin87migrate to the wound,the wound heal faster, while U87cells was transfected pEGFP-C1/Ezrin251whosenumber of migration was less than the pEGFP-C1/Ezrin87transfection group, and thewound healed slowly. The animal model of brain tumors in nude mice also confirmedthis result. The expression level of Rac1is also consistent with this result. It showedthat Ezrin can cause the differences of U251and U87cells migration and invasion,and achieve through activation of Rac1.