The Effect Of PTEN-Long On The Biological Activity Of Glioma Cells

Objective:PTEN-Long is a translational variant of phosohatase and tensin homolgue deleted on chromosome 10(PTEN).This study explored the effects of PTEN-Long on proliferation,migration and invasion of glioma cells U251 and related molecular mechanisms,and examined the effect of PTEN-Long on glioma in vivo.Methods:1.The recombinant lentiviral vector carrying the PTEN-Long was transfected into U251 cell line.U251 which was not infected with lentivirus was named as control group(U251);cell line infected with LV-EGFP was named as empty group(U251-EGFP);cell line infected with LV-PTEN-Long-EGFP was named as overexpressed group(U251-PTEN-Long-EGFP).The expression of green fluorescent protein was observed by a fluorescence microscope,and the transfection efficiency was detected by RT-PCR and Western blot.2.The effect of PTEN-Long on the capacity of proliferative of each group was detected by CCK8 method,and the difference of cell cycle of each group was detected by flow cytometry.3.The effect of PTEN-Long on the ability of migration of each group was detected by scratch test,and the ability of invasion of each group was detected by transwell chamber method.4.The effect of PTEN-Long on the expression level of related molecules in PI3K-AKT-NF-κB signaling pathway was detected by RT-PCR and Western blot.5.Allogeneic tumors were implanted subcutaneously in nude mice by injecting cells of each group.The general condition of nude mice and the growth trend of tumor were observed.The volume and weight of subcutaneous tumors in nude mice were recorded and analyzed.The level of protein about PTEN-Long 、 p-Akt and NF-κB p65 in tumors was detected by immunohistochemical staining.Results:1.After the transfection of LV-PTEN-Long-EGFP into glioma cells,transfection was confirmed by fluorescence microscopy 、 RT-PCR(F=65.42,P ﹤ 0.01)and Western blot(F=103.23,P﹤0.01).And PTEN-Long was stably expressed in cells.2.The ability of proliferation(F=18.42,P﹤0.01;F=79.92,P﹤0.01;F=306.47,P﹤0.01)、migration(F=128.42,P﹤0.01;F=90.41,P﹤0.01)and invasion(F=632.01,P﹤0.01)of the overexpression group(U251-PTEN-Long-EGFP)compared with the control group(U251)and the empty group(U251-EGFP)was inhibited.And the cell cycle is blocked in the G0/G1 phase of the overexpression group(F=26.32,P﹤0.01).3.PTEN-Long may inhibit tumors by down-regulating the signaling pathway of PI3K-AKT-NF-κB.RT-PCR showed that the expression level of NF-κB p65(F=53.32,P<0.01)、bcl-xl(F=933.88,P<0.01)was decreased,and the IκBα(F=26.61,P<0.01)was increased in the overexpressed group compared with the control group and the empty group.Western blot showed that the expression levels of p-Akt(F=56.19,P <0.01)、NF-κB p65(F=43.65,P<0.01)、 p-NF-κB p65(F=114.68,P<0.01)、 bcl-xl(F=19.26,P<0.01)was decreased,and the IκBα(F=45.23,P<0.01)was increased in the overexpression group.4.Overexpression of PTEN-Long inhibited the proliferation of subcutaneous U251 cells in nude mice.This may be related to down-regulation of the signaling pathway of PI3K-AKT-NF-κB.Compared with the control group and the empty vector group,the overexpressed group was generally in slightly better condition,and the tumor volume(F=369.1,P < 0.01)and weight(F=27.71,P < 0.01)were smaller.The result of immunohistochemical staining showed that the expression of PTEN-Long(F=25.33,P<0.01)was increased,and the p-Akt(F=21.50,P<0.01)and NF-κB p65(F=10.96,P<0.01)were decreased in the overexpression group.Conclusions:1.PTEN-Long can inhibit the ability of proliferation、migration and invasion of glioma cells U251.The cell cycle is arrested in the G0/G1 phase and apoptosis can be induced.2.PTEN-Long can inhibit the growth of subcutaneous xenografts of U251 cells in nude mice.3.PTEN-Long inhibits tumors,and its mechanism may be related to down-regulation the signaling pathway of PI3K-AKT-NF-κB.PTEN-Long may become a new target for glioma about gene therapy.Provide a new direction for gene therapy.