The Research On Effect Of Leucine-rich Repeats And Immunoglobulin-like Domains 2 (LRIG2) On The Biological Characterization Of Glioblastoma Cell Line GL15 And Its Molecular Biological Mechanism

PartⅠLRIG2—the New Transcriptional Regulatory Target ofEpidermal Growth Factor Receptor(EGFR) Signaling Network inHuman Glioma CellObjective To confirm that LRIG2 is the new regulatory target of epidermal growth factorreceptor(EGFR) signaling network in human glioma cell line GL15.Methods After the human glioma cell line GL15 was exposed to the concentrations of EGF100ng/ml,AG1478 10μM and Cycloheximide 10μg/ml in vitro,changes of mRNA andprotein levels of LRIG2 were measured by reverse transcriptional—polymerase chainreaction(RT-PCR) and Western Blotting.Results The levels of LRIG2 mRNA in GL 15 cell rised after 15 minutes of EGFstimulation,the protein levels of LRIG2 rised after 30 minutes of EGF sitmulation.Cycloheximide and AG1478 were added before the exposure of EGF at designed time on GL15,the protein levels of LRIG2 weren't change significantly.Conclusion The activation of EGFR could increase the expression of mRNA and protein ofLRIG2.LRIG2 is the new transcriptional regulatory target of EGFR signaling network inhuman glioma cell.PartⅡEstablishment of Stably Transfected Cell Clone ExpressingLRIG2 Specific Short-hairpin RNA and the Research on Effect ofDownregulation of LRIG2 on EGFR Signaling PathwayObjective To construct effective short-hairpin RNA(shRNA) expression vector encodingshRNA targeting LRIG2 gene and to establish the stably transfected cell clone.Toinvestigate the effect ofdownregulation of LRIG2 on EGFR signaling pathway.Methods Design and synthesize two shRNAs sequences based on the sequence of LRIG2mRNA in the GenBank and one scrambled shRNA sequence as negative control.Thesynthesized sequences were cloned into shRNA expression vector pGenesil2.Accordingthe fatal dose of G418 to GL15 cell,the selection concentration of G418 for GL15 cell wasdetermined.The three shRNA vectors were transfected into GL 15 by Metafectinerespectively.The stably transfected cell clones were obtained after the transfected cell werecultured in medium containing G418.Reverse transcriptase-polymerase chain reaction(RT-PCR) and Western Blotting were performed to examine the inhibitory effect on theRNA level and protein level of LRIG2.After cell stimulated by EGF,Western Blotting wasperformed to detect the levels of EGFR and pEGFR protein.Results The recombinant plasmids containing shRNA were analysized by doubleendonuclease digestion and DNA sequencing.The selective concentration of G418 forGL15 cell was 600μg/ml.LRIG2 expression was significantly down-regulated by siRNA as validated by RT-PCR and Western Bloting.After 30 min of EGF treatment,there was verylittle EGFR detected in the LRIG2-siRNA2 cells.The relative expression level of EGFRwas 0.16 in LRIG2-siRNA2 cell and 0.62 in control.After 5 min and 30 min of EGFstimulation,the relative expression level of pEGFR increased to 5.5 and 4.2 in LRIG2siRNA2 cells respectively,while relative expression level of pEGFR only increased to 3.8and 2.6 in control cells respectively.Conclusion RNA interfering(RNAi) mediated by the shRNA expression vector couldsignificantly down-regulate the expression of LRIG2 in glioma cell line GL15.The stabletransfected cell clone was obtained for further study.Know-down of LRIG2 could decreasethe level of EGFR and inhibit the phosphorylation of EGFR in GL15 cell.PartⅢThe Research on Effect of LRIG2 on the Proliferation,CellCycle,Apotosis,Adhesion and Invasion of Human Glioma Cells byRNA InterferenceObjective To determine the effects of LRIG2 on the proliferation,cell cycle,apotosis,adhesion and invasion of glioma cells by RNA interference.Methods The growth curves were determined by the methyl thiazolyl tetrazolium(MTT)assay.Cell cycles and apoptosis were analyzed by flow cytometry.The ability of adhesionand invasion were measured by Cell-matrix adhesion assay and Transwell chamber assay.The staining of PCNA and localization of LRIG2 were performed byimmunocytochemistry.Results The LRIG2-siRNA cell had less proliferation rate than control cell.The PCNAprotein was less stained cell for LRIG2-siRNA group than that in the control After silencingLRIG2 expression,most cells accumulated at G0/G1(p＜0.01),and the proportionof LRIG2-siRNA cells in S and G2/M phase decreased(p＜0.01).Down-regulation of LRIG2 increased the level of spontaneous apoptosis about three-fold compared to control(p＜0.01).The capabilities of adhesion and invasion were enhanced after knockdown ofLRIG2.LRIG2 was localized in the cytoplasmic area.Conclusions Down-regulation of LRIG2 decreased proliferation;G0/G1 arrest;increasedspontaneous apoptosis;enhanced cell adhesion and increased invasion capability of GL15cells in vitro.These findings validate the attractiveness of LRIG2 as a target in gliomatherapy.