Downregulation Of Diacylglycerol Kinase ? Inhibites The Proliferation And Migration Of Rat Neural Stem Cells

Objective:1.To construct lentiviral expression vector of rat DGK?-shRNA.2.To investigate the effect of DGK? on the proliferation and migration of neural stem cells(NSCs).Methods:1.Homologous recombination was used to construct lentiviral vector of DGK?-shRNA.The primary NSCs of rat were infected with lentivirus,which was packaged with DGK?-shRNA vector and Scramble vector.RT-qPCR and Western blotting were used to analyse DGK? mRNA and proteinexpression.2.CCK-8 assay and neurosphere counting were carried out to detect NCSs proliferation after treatment with DGK?-shRNA,DGK inhibitor R59949 and PKC agonist PMA.And the migration distance of NSCs in each group was measured under microscope.Results :1.Construction and identification of the DGK?-shRNA lentiviral vector: After co culture with lentivirus for 48 h,NSCs were infected effectively and GFP-positive in both DGK?-shRNA group and Scramble group.RT-qPCR and western blotting showed that the DGK? mRNA and protein expression of the DGK?-shRNA group was remarkably lower than that of the Scramble group(all P < 0.001).2.The effect of DGK?-shRNA on NSCs proliferatio: The CCK-8 assay showed expression downregulation of DGK? in NSCs caused by shRNA interference obviously inhibited the cell proliferation(P < 0.05).DGK inhibitor R59949 also obviously inhibited the cell proliferation(P < 0.05).But PKC agonist PMA significantly promoted the cellproliferation(24h,P < 0.05;48h and 72 h,P < 0.001).The neurosphere counting showed that both expression downregulation of DGK? and R59949 remarkably confered supression in forming of neurospheres with diameter of 50~100?m(P < 0.001),while PMA markedly promoted the forming(P < 0.05).3.The microscopic measurement showed that lower expression of DGK?,R59949 and PMA treatment decreased the migration distance of NSCs(all P < 0.001).The migration distance of PMA group was shortest and lower than that of R59949 group(P <0.05).Conclusion:The rat DGK?-shRNA lentiviral vector has been successfully constructed,then interfere the gene expression of DGK? in NSCs.The downregulation of DGK? expression inhibits the proliferation and migration of NSCs.In addition,the pathway of DGK?accelerate the proliferation of NSCs is not associated with PKC,while DGK? may be able to enhance the migration of NSCs by PKC pathway.