| Objective :to explore the pathogenesis of gliomas and the effective methods of treatment is one of the most important and the hot research topic of neurosurgery . In recent years,some reserachers have displayed that the control developmental signal pathway is closely related to the growth of brain tumors. In order to regulate the abnormal expression of the relevant signaling molecules of developmental signaling pathway in brain tumors , to inhibit tumor growth and to reduce the damage of the central nervous system with the most great degree, it is necessary to study on the developmental signaling pathway more comprehensive and in-depth. Wnt signaling pathway not only plays an important role in the development of different species, cell differentiation and proliferation, but also participates in the formation of certain tumors. Though Wnt /β-catenin signaling pathway is considered to be classical Wnt signaling pathway, the abnormal Wnt signaling pathway can result in cancer,which is the key procedure to accumulate free variation ofβ-catenin protein intracellular and then enter the nucleus.β-catenin protein accumulative expression is closely related to tumor of the invasion, metastasis and prognosis. Therefore, some scholars believe that the controling Wnt signaling pathway by targete therapy can inhibit tumor formation and growth. Our pre-research shows that the 94 cases of all levels glioma and the six cases of normal brain tissue by immunohistochemistry study, and found thatβ-catenin gene in glioma than normal brain tissue significantly increased expression, and with the higher and higher levels of glioma. Therefore, we consider thatβ-catenin is an ideal marker of glioma molecular biology and target of gene therapy. This study was to use RNA interference technology to constructe targetingβ-catenin gene RNA interference plasmid, further transfecte in U251 human glioma cells, to study of the impact the gene in glioma cell proliferation in the process,We expect for providing a basis and means of targeting therapy in the diagnosis and treatment of glioma.Methods :According to the principle of siRNA design, through software design and reference literature, the sequence of the coding region ofβ-catenin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 19-base oligonucleotides were designed and synthesized to form hairpin construct as the DNA templates for the target shRNA. The shRNA templates were cloned into shRNA expression vector pGPU6/GFP/Neo, obtain interference plasmid of targeting inhibited the expression ofβ-catenin.the sequence of the plasmid was identified by DNA sequencer and restriction endonuclease digestion. Thus, the resulted vector pGPU6/GFP/Neo-shRNA-β-catenin was transfected into U251 cells. RT-PCR and Western blotting were performed to evaluateβ-catenin gene silencing induced by siRNA transfection at the protein levels and the mRNArna levels, preliminary study of its growth effect on glioma cell U251 were measured by observe the changes in cell morphology,MTT and FCM.Results:It was verified that the specific DNA oligonucleotide was cloned into the vector successfully after digestion,sequencing The protein and mRNA expression ofβ-catenin gene in U251 cells was significantly reduced after transfecting the recombinant plasmid, compared to the controls.Through the cell morphology observed after 48 hours show the some vacuoles in nucleus, slow down cell proliferation, the death of cells gradually increased, the performance of the collapse, such as necrosis and broken, increase floating cells and cellular debris surrounding,showing a clear time dependence. MTT method illustrate pGPU6/GFP/Neo-shRNA-β-catenin plasmid transfected cells after 24 hours , 48 hours and 72 hours later , pGPU6/GFP/Neo-shRNA-β-catenin plasmid transfection group were inhibited ,compared to the blank control group and negative control group (P <0.01), U251 cell group of transfection to pGPU6/GFP/Neo-shRNA-β-catenin increased inhibition rate gradually with the time, and the impact of a higher inhibition rate in 72 hours, while the blank control group and negative plasmid transfection group is no statistical difference. Apply FCM to detect apoptosis showed that pGFP/Neo-shRNA-β-catenin transfection cell significantly increased apoptosis rate compared to the blank control group and negative plasmid control group after 72 hours, with statistical significance (P <0.01 ). Apply FCM to detect cell cycle showed that the cell cycle of pGPU6/GFP/Neo-shRNA-β-catenin transfected group changes were statistically significant differences after 72 hours, (P <0.05), cells are mostly gathered in the G0/G1 phase, while S phase, G2 / M phase cells significantly decreased , the activities in DNA replication decrease and a in the number of cells of activities and DNA replication decreased, Proliferation of U251 cells was inhibited.Conclusion:It indicates the shRNA expression vector has been successfully established which can inhibitβ-catenin gene expression specifically,and the study shows the initial evidence of interference plasmid of targetingβ-catenin promoted apoptosis of glioma cell, inhibited cell differentiation and proliferation. Malignant glioma growth is closely related to activation ofβ-catenin in Wnt signaling pathway,β-catenin is an ideal marker of glioma molecular biology and targets of gene therapy, and those provides the preconditionfor the further study ofβ-catenin by Wnt signaling pathway in glioma pathogenesis.
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