Effects Of Protein PTEN-Long On Proliferation And Migration Of Glioma Cells

BACKGROUND pten,Phosphatase and TENsin homolog deleted on chromosome 10 gene,located at 10q23.3,it's product of transcription is a 515 kb m RNA which be Longs to the PTP(protein tyrosine phosphatases)gene family members.Since PTEN was discovered as a tumor suppressor in 1997,scientists have widely researched on it around the world.It is known well that PTEN is a dual specific phosphatase,containing both lipid and protein phosphatase activities specificity.The lipid phosphatase activity of PTEN can hydrolyze PIP3 into PIP2 in the PI3K/AKT signaling pathway,thereby inhibiting the signaling pathway and preventing the proliferation of cell by decreasing the phosphorylation level of AKT.In addition,PTEN also has protein phosphatase activity.It mainly dephosphorylates the serine,threonine and tyrosine of its substrate proteins.In most cancer cells,the phosphorylation levels of many key factors are not normal.PTEN may play an anti-cancer effect by dephosphorylating these proteins.At present,as a tumor suppressor,many biological functions of PTEN are not clearly understood,for example,whether PTEN has other activities besides the phosphatase activity,and it has been reported that PTEN in the nucleus can maintain the stability of chromosomal.In addition,PTEN-Long as a translation variant of PTEN,begins at CTG as a selective translation start site prior to the normal initial translation site 519 bp to form a protein longer than classic PTEN with additional 173 amino acids.This protein can be secreted out of cells,enters into and acts on the adjacent cells.This secretory characteristic makes it different from the classic PTEN and attracts the interest of many clinical researchers.PTEN-Long has the same phosphatase activities as the classical PTEN,which can inhibit the PI3 K signal pathway.After secretion,PTEN-Long can mediate the paracrine regulation of cell growth and apoptosis.In previous studies,PTEN deletions and abnormalities are often associated with the development of tumors,and many literature and reports indicated that mutation of PTEN occurs in a variety of sporadic and two autosomal dominant hamartomas.The traditional treatment is committed to restoring the expression of the PTEN gene,but this is usually difficult to achieve.Now we can take advantage of the secretory function of PTEN-Long to achive treatment.PTEN-Long can accurately targeted to and affect cancer cells,which will guide future clinical treatment.CAPITAL In this study,we will construc PTEN-Long expression vector and express PTEN-Long in cultured cells,exploring effect of PTEN-Long on the glioma cell line U87 cells.we will analyze the inhibitory effect of PTEN-Long on cancer cells and explore the potential therapeutic effects of PTEN-Long in the clinical patients with PTEN default.METHOD Construction of PTEN overexpression and control lentiviral vector.U87 cells were infected with PTEN overexpression and control lentiviral vector,and then selected by puromycin to form stable infected U87 cell lines.Western blot was used to verify PTEN overexpression in U87 cell lines.At the same time,the vector of PTEN-Long was constructed and expressed in cultured cells.The purified PTEN-Long proteins were added to the wild-type U87 cells which are PTEN-null,and its cell biological function was measured by growth curve,wound healing and transwell invasion assay with PTEN-overexpressed cells as positive control.RESULT1.PTEN-Long prokaryotic expression vector was constructed successfully,the conditions of protein-induced expression is currently optimizing.2.The PTEN-Long eukaryotic expression vector was successfully constructed,successfully expressed and purified.3.In PTEN-null U87 cells,the function of PTEN-Long was similar to classical PTEN,It was observed to inhibit proliferation and migration of U87 cell.CONCLUSION1.The PTEN-Long and 6×His fusion proteins constructed in eukaryotic expression vector were successfully expressed and purified using the tag affinity purification.2.PTEN-Long has the same effect as PTEN on tumor cellproliferation,migration and invasion.