Study On Diversity Regulation Mechanisms Of LRIG3 Gene For The Biological Behavior Of Glioma Cells

PartⅠConstruction of LRIG3 specific short-hairpin RNA expressingvector and screening of stably transfected cell clonesObjective To construct eukaryotic expression vectors of RNA interference specific forLRIG3 gene,and to screen the stably transfected cell clones.Methods Genomic sequences of LRIG3 gene was retrieved from Genbank and cDNAwas designed encoding shRNA (small hairpin RNAs) for LRIG3.The cDNA wassynthesized and inserted into plasmid pGenesil2.Recombinant vectors were thentransformed into competent E.coli.The positive clones were selected and recombinantplasmids were extracted.The plasmids were digested with SalⅠand loaded in agarose gelelectrophoresis.The three shRNA vectors were transfected into GL15 by Metafectene.Thestably transfected cell clones were obtained after being screened with G418.Reversetranscriptase-polymerase chain reaction (RT-PCR) and Western blotting were performed toexamine the inhibitory effect at the RNA level and protein level.Results The stably transfected pGenesil2-LRIG3-shRNA cell clones was significantlydown-regulated by siRNA as validated by RT-PCR and Western blotting.Conclusions RNA interfering (RNAi) mediated by the shRNA expression vector couldsignificantly down-regulate the expression of LRIG3 in glioma cell lines GL15.The stabletransfected cell clones was obtained for further study. PartⅡEffects of RNAi-mediated gene silencing of LRIG3 expression onproliferation,cell cycle and cell apoptosis of GL15 cell lines and itsmechanismsObjective To study the expression of proliferating cell nuclear antigen (PCNA),Ki-67 nuclear antigen,cell cycle and cell apoptosis in RNA interference (RNAi) - mediatedLRIG3 gene silencing Glioma cell and to investigate the correlation with tumor cellproliferation and its possible mechanisms.Methods The plasmids pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2were transfected into GL15 glioma cells respectively by Metafectine,and thetransfected-cells that stably suppressed LRIG3 expression were selected by G418.Thecontrol cells were transfected with negative shRNA (neg).The changes in LRIG3 mRNAand protein levels were measured by RT-PCR and Western blot.The expressions of PCNAand Ki-67 in GL 15 glioma cells were examined by SABC immunohistochemistry method.The apoptosis rate and cell cycle were analyzed by flow cytometry.Results As compared with the negative shRNA-transfected GL 15 cells,LRIG3 mRNAexpression in GL15 cells transfected with pGenesil2-LRIG3-shRNA1 andpGenesil2-LRIG3-shRNA2 was silenced by 52.4%,63.8%,and LRIG3 protein expressionwas reduced by 50.9% and 67.4% respectively.The average PCNA positive staining cellwas (72.13±5.64) % in LRIG31-siRNA targeted cell and ( 81.93±5.23) % inLRIG32-siRNA targeted cell compared to negative control LRIG3 expressing cell (35.40±5.69) %(P＜0.01).Ki-67 labeling index (Ki-67 L I) from (82.27±5.50) % inLRIG31 -siRNA targeted cell to( 88.67±3.52) % in LRIG32-siRNA targeted cell and (49.73±5.73 ) % in negative controls ( P＜0.01).There was a good correlation between thePCNA L I and Ki-67 L I.Cell cycle analysis showed that silencing LRIG3 increased thepercentage of G2/M phase cells and the proliferation index significantly (P＜0.01).Silencing LRIG3 could inhibit the apoptosis of GL15 cells (P＜0.05). Conclusions These findings suggest that the siRNA targeting LRIG3 gene shows adramatic inhibitory effect on RNA transcription and protein expression,then promoting theproliferation of GL15 cells,arresting GL15 cells in G2/M phase,and suppressing apoptosisof GL 15 cells. PartⅢEffects of Down-regulating LRIG3 gene expression on theadhesion,invasion,and EGFR signaling pathways of GL15 cell lines andits mechanismsObjective To explore the effects of RNA interference-mediated LRIG3 genesilencing on the adhesion and invasion of glioblastoma cell lines GL15 and to investigatethe correlation with EGFR signaling pathways.Methods The plasmid pGenesil2-LRIG3-shRNA (siRNA) was transfected intoGL15 glioma cells by Metafectine,and the cells (siRNA) that stably suppress LRIG3expression were selected by G418.The control cells were transfected with negative shRNA(neg).The changes in LRIG3,EGFR and P-EGFR expression levels were analyzed byRT-PCR and Western blot.The changes of the GL15 cells adhesive and invasive abilitywere measured by cell adhesion assay and Transwell chamber.Cell proliferation wasdetected by MTT assay.Results Compared with LRIG3 mRNA expression in the negative shRNA-treatedGL15 cells,the transcription after treatment with LRIG3-specific shRNA was silenced by52.4%,63.8%,and the expression of LRIG3 protein was reduced to 49.1%,32.6%.TheEGFR and P-EGFR protein level in pGenesil2-LRIG3-shRNA (siRNA) transfected cellswere significantly higher than that in negative shRNA (neg) transfected cells.Treatment ofGL15 cells with pGenesil2-LRIG3-shRNA can enhance adhesive and invasive ability.MTT assay showed that Cell proliferation was enhanced by LRIG3 shRNA transfection.Conclusions Silencing LRIG3 expression could result in up-regulating of EGFR,then affect the downstream pathways of EGFR signaling,accordingly enhance the adhesiveand the invasive ability of GL15 cell,and can improve the proliferation of glioma GL15cells.LRIG3 could be a targeted protein for gene therapy of glioma.