Effects Of RNA Interference-mediated Of LRIG1 Silencing On Glioma Cells (GL15 Cell Lines)

PartⅠConstruction of LRIG1 Specific RNAi Expressing Vector andScreening of Stably Transfected Cell ClonObjective To construct eukaryotic expression vector encoding RNA interferencesequences specific for LRIG1 gene, to screen the stably transfected cell clone, and toobserve its effect on the expression of LRIG1.Methods Designed two shRNAs sequences based on the sequence of LRIG1 mRNA in theGenBank and one scrambled shRNA sequence as negative control. The synthesizedsequences were inserted into shRNA expression vector pGenesil2 and sequenced. TheshRNA vectors were transfected into GL15 by Metafectine. The stably transfected cellclones were obtained after being screened with G418.Western Blotting was performed toexamine the inhibitory effect at the protein level.Results The recombinant plasmids containing shRNA were analysized by restrictionenzyme digest analysis and DNA sequencing. The screening concentration of G418 toGL15 cell was 600μg/ml. LRIG1 expression was significantly down-regulated by siRNA as validated by Western Bloting.Conclusion RNA interfering (RNAi) mediated by the shRNA expression vector couldsignificantly down-regulate the expression of LRIG1 in glioma cell line GL 15. The stabletransfected cell clone was obtained for further study.PartⅡThe effect of Down-regulation of LRIG1 Gene Expression onCell Cycle,Apoptosis and Proliferation inGlioblastoma Cell Line GL15Objective To investigate the effect of down-regulation of LRIG1 gene expression on cellcycle, apoptosis and proliferation in glioblastoma cell line GL 15.Methods The cell cycle in control and experimental group was detected by flowcytometry. Annexin V-FITC/PI double-labeled flow cytometry analyzed apoptosis. Methylthiazolyl tetrazolium (MTT)detected cell proliferation.Results Flow cytometry analysis showed that the G2 / M phase cell percentage inexperimental group was higher than control group significantly. Cell proliferation indexincreased significantly (p＜0.01); In shRNAi group, GL15 cells in early apoptotic decreasedsignificantly after down regulation of LRIG1 (p＜0.01) The role of LRIG1 onanti-apoptotic is remarkably (p＜0.01); results of MTT showed that cell proliferation ratein interfered group was higher than control (p＜0.01 ) .Conclusion The expression of LRIG1 is down regulated significantly in GL15 cell afterRNA interference, which enhances cell growth. PartⅢThe Effect of RNA interference of Gene LRIG1 on EGFRExpression in Glioblastoma Cell Line(GL15) andthe Molecular MechanismsObjective To explore the interaction between the leucine-rich repeat Ig-like protein 1(LRIG1) and epidermal growth factor receptor (EGFR) in human glioma cells and theeffect of LRIG1 on the tumor cell growth.Method The interaction of LRIG1 and EGFR in human glioma cell line GL15 wasconfirmed by co-immunoprecipitation. Western-blot was used to analysis the level ofEGFR,pAkt,pMAPK protein after silencing LRIG1.Results Immune co-precipitation method showed that the GL15 glioma cell lines inexistence LRIG1 interaction with EGFR. The LRIG1 protein level inpGenesil2-LRIG1-shRNA (siRNA) transfected cells was significantly silenced by47.9%and the EGFR,Akt,MAPK protein level in it rised by 57.1%,43.6 %,52.3 %.Conclusion Knowdown of LRIG1 could enhance the functions of EGFR signalingpathway and promote tumor proliferation. LRIG1 protein level could be one index to adjustmalignant degree and prognose of glioma, and provides a new treatment for glioma...