Construction Of Lentiviral Vector With Over-expressing CASK And Exploration Of Its Effect On Cell Proliferation And Migration In Human Nsclc H1299 Cells

Objective Lung cancer is one of the most common maligancies in our country,in which non-small cell lung cancer(NSCLC)accounts for 80-85 percent and is the most commonly seen type in clinical practice.Because of its lack of obvious specific symptoms in early stage and easy transfer into hematogenous metastasis,most newly diagnosed patients have entered the advanced stages,which lead to a poor treatment effect and prognosis.By the means of bioinformatic screening,our previous study found 11 significant genes,in which CASK was invloved in the pathogenesis of lung cancer.In this study,we constructed the lentiviral vector with over-expressing CASK,and examine its ability to express the CASK gene in human non-small cell lung cancer(NSCLC)H1299 cells after transfection.After transfection,we explored its effect on proliferation and migration in H1299 cells.Methods1.Construction of the lentiviral vector with over-expressing CASK The CASK gene fragments were amplified by PCR and the in vitro cycling of the CASK gene products and the linearized GV358 vector was accomplished by recombinant reaction.After the transformation of the recombinant products,the monoclones were picked and identified by PCR,and the positive clones were sequenced.Finally,the correct clones were expanded and extracted to obtain high purified plasmids.The recombinant plasmids and packaging plasmids were co-transfected into 293 T cells.Western Blot was used to detect the protein expression level of 293 T cells after transfection.The virus titer was determined by ELISA.2.Efficiency detection on 293 T cells after transfection The recombinant lentivirus with over-expressing CASK(LV-CASK)and negetive control virus CON238 were transfected into human NSCLC H1299 cells.Real-time PCR and Western Blot were used to detect the expression level of CASK.3.Effects of over-expressing CASK on biological functions of H1299 cells The recombinant lentivirus with over-expressing CASK(LV-CASK)and negative control virus CON238 were transfected into human NSCLC H1299 cells.MTT and wound healing assay were used to evaluate the effect of over-expressing CASK on cell proliferation and migration of H1299 cells.Results1.Construction of the lentiviral vector with over-expressing CASK The CASK gene was successfully amplified by PCR and bound to GV358 lentivirus vector.The sequences of the recombinant plasmid were confirmed correct by PCR and DNA sequencing.The expression of GFP could be observed after recombinant lentiviruses were transfected into 293 T cells.The lentiviruses with over-expressing CASK were obtained and the titer of concentrated viruses was 2×108 TU/m L.2.Efficiency detection on 293 T cells after transfection According to the results of PCR and Western Blot,the m RNA and protein expression level of CASK significantly increased in H1299 cell of LV-CASK group,comparing to the CON238 group.3.Effects of over-expressing CASK on biological funtions of H1299 cells Compared to the CON238 group,the proliferation ability of the infected H1299 cells decreased while the over-expressing CASK had no significant effect on its migration ability.Conclusions The lentiviral vector with over-expressing CASK gene was constructed successfully,and the lentivirus could transfect the NSCLC H1299 cells and exogenous gene was over-expressed stably.Over-expressing CASK could suppress proliferation ability in H1299 cell.It constructs the foundation of CASK for regulation in the further research on lung cancer metastasis.