Inhibition Of SH-SY5Y Cell Growth By TMEFF2 Gene

Objective(1)To clone the full length TMEFF2 from human brain cDNAs,then to construct eukaryotic gene expression vector;(2)To construct TMEFF2 gene specific RNAi vector;(3)To determine the subcellular localization of TMEFF2 protein;(4)To isolate SH-SY5Y cell strains with downregulated and upregulated TMEFF2 genes with G418,then to investigate the influence of TMEFF2 gene expression on cell growth and conclude the function of TMEFF2 protein.Methods The experiment was consisted of two parts:(1)Plasmid pEGFP-TMEFF2 is constructed and transfected into SH-SY5Y cells using Lipofectamine to express TMEFF2-EGFP fusion protein in cells.Cells stably expressing TMEFF2 were selected with G418.Cells stably transfected with pEGFP-N2 were selected with the same method.Proliferation of pEGFP-TMEFF2 transfected cells,pEGFP-N2 transfected cells and SH-SY5Y cells were evaluated with MTT.Subcellular localization of TMEFF2 protein was determined with laser confocus microscopy.(2)Two RNAi vectors targeting to TMEFF2 gene were constructed and transfected into SH-SY5Y cells.Stable SH-SY5Y cell strains with downregulated TMEFF2 genes were isolated with G418.The efficiency of two vectors were estimated with real-time PCR.Proliferation of cell strain with downregulated TMEFF2 gene,pSuper-EGFP transfected cells and SH-SY5Y cells were evaluated with MTT.Results(1)The pEGFP-TMEFF2,pEGFP-RNAil,pEGFP-RNAi2 eukaryotic expression vector was constructed successfully.Plus,pEGFP-RNAil was selected through Real-Time PCR as a RNAi vector with higher interfering efficiency;(2)The TMEFF2 fusion protein was located in cell membrane;(3)SH-SY5Y cell strains with downregulated and upregulated expressed TMEFF2 genes were isolated;(4) When inoculated with high density,the proliferation of pEGFP-TMEFF2 transfected cells were decreased than pEGFP-N2 transfected cells and SH-SY5Y cells.While the proliferation activity of pEGFP-RNAil transfected cells were increased than pSuper-EGFP transfected cells and SH-SY5Y cells. Conclusions(1)TMEFF2 protein was located in membrane;(2)TMEFF2 significantly inhibit the proliferation of SH-SY5Y cells,and the inhibition was density dependent.