Impact On Chemosensitivity In Glioma By ShRNA Mediated Down-regulation Of AKT2

Gliomas are the most common primary tumor of the central nervous system in adults, Currently, the treatment of glioma include surgery, radiation therapy, chemotherapy, and comprehensive treatment, although in recent years the treatment has made great progress, but because of the invasive growth of glioma was, effects are not good enough. New treatments such as gene therapy of glioma, although expected to become one of the ways to cure, but there is a lack of reliable ideal therapeutic target. So look for regulating the malignant behavior of glioma cells molecular target, a clear process in glioma pathogenesis pathological mechanism of the field of neurosurgery has become one of the focus.In this study, we will construct AKT2 shRNA lentivirus vector and infect glioma cell lines to study the process of AKT2 in the development of glioma in and explore the possible role AKT2 and Temozolomide chemotherapy sensitivity. Experiments were divided into three parts:Part I: Construction and identification of lentiviral vector of RNA Interference of AKT2Objective: To construct lentiviral vector plasmid containing AKT2 and provide the basis for further experiments.Methods: The vector was obtained from pMAGic 7.0 plasmid digested with EcoR I and Age I restriction enzymes. Then AKT2 gene was inserted into the vector to construct pLV-AKT2, which was subsequently confirmed by PCR and DNA sequencing analysis.After these identification, the resultant pLV-AKT2 plasmid, pCD/NL-BH*DDD and pLTR-G were transfected into 293T cell to produce a replication incompetent lentivirus. Finally the titer of lentivirus was determined.Results: PCR analysis and DNA sequencing confirmed that the AKT2 sequences were successfully inserted into the lentiviral vectors. The titer of concentrated virus was 2.97×10⁸TU/ml.Conclusion: The lentiviral vector plasmid containing AKT2 has been successfully constructed, which will provide a foundation for the further study on function of AKT2 gene and its influence on chemosensitivity in glioma. mediated down-regulation of AKT2Objective: To study the effect on proliferation and apoptosis of U87 cell line by targeted inhibition of AKT2 gene expression on glioma cell.Methods: Infect U87 cell line by shRNA mediated down-regulation of AKT2. Determination of cell proliferation MTT, flow cytometry cell cycle change, AV / PI stain cells, RT-PCR and Western blot measured expression of AKT2 in U87-AKT2 shRNA. And determinate the expression of Bcl-2,Caspase-3 on mRNA and protein levels.Results: Transfection of AKT2 shRNA can effectively reduce the U87 cell lines of AKT2 expression. With low expression of AKT2 , the proliferation of U87 cells was significantly lower compared with the control group (P <0.05). By flow cytometry after transfection AKT2shRNA U87 in the apoptosis rate was significantly increased compared with the control group (p <0.01), cell cycle arrest in G0/G1. U87 cells after inhibition of AKT2 expression, Bcl-2 expression was significantly reduced compared with control, but significantly increased the expression of Caspase-3 in mRNA level and protein level.Conclusions: It can inhibit proliferation of glioma cell and lead to tumor cell apoptosis by targeted inhibition of AKT2 gene expression on U87 cell line. In mRNA and protein levels, AKT2 interference can lead to Bcl-2 expression decreased, Caspase-3 increased.Partâ…¢: Impact on Chemosensitivity In Glioma by shRNA mediated down-regulation of AKT2Objective: To study the interference of AKT2 on glioblastoma cell lines U87MG and U251MG chemotherapy sensitivity.Methods: Infection U87, U251 cell lines, the establishment of different expression levels of AKT2 U87, U251 glioma cell lines. The glioma cell lines were cultured with different concentrations of VM-26 three days, MTT inhibition rate determined on the third day, calculated IC₅₀;Results: U87, U87-GFP (interference negative control), U87-shRNA (interference) 3 groups of IC₅₀ : 6.42±0.38μg/ml,6.46±0.42μg/ml,3.95±0.22μg/ml; U251, U251-GFP negative control, U251-shRNA (interference) IC₅₀: 7.27±0.53μg/ml,7.30±0.78μg/ml,4.22±0.51μg/ml. shRNA interference joint VM-26 more than any single treatment significantly enhance the chemosensitivity.Conclusions: AKT2 expression in glioma cells is correlated with VM-26 sensitivity. Partâ…¡: Effects on proliferation and apoptosis of U87 glioma cell line by shRNA VM-26 combined with AKT2 shRNA may provide an effective treatment method for gliomas.