Survivin Gene RNA Interference And Bax Overexpression Promote Human Lens Epithelial Cells Apoptosis

PrefacePosterior capsule opacification(PCO) is a common complication of cataract surgery,which incidence is approximately 50%in adults and 100%in children.PCO develops in a significant proportion of patients to such an extant that a secondary vision loss,which consequently requires further corrective laser surgery.The proliferation,migration and abnormal differentiation of residual lens epithelial cells (LECs) and fiber cells in the capsular bag have been implicated in the pathogenesis of PCO.Growth factors such as TGF-beta(TGFβ) and FGF appear to play a key role in this process.At present PCO is treated by YAG capsulotomy,which carries a some risk of sight threatening complications such as cystoid macular edema or retinal detachment Several attempts have been made to find an appropriate therapeutic concept to significantly lower the rate of PCO,which include mechanical strategies by striving to remove all LECs during surgery,certain IOL materials and sharp-edged IOL designs. Different pharmacological studies have been conducted in vitro and in vivo,using immunotoxins,cytotoxins,and antimetabolic agents.Such approaches have shown promising results regarding the reduction of the PCO rate,but are limited in clinical application by the possible side effects affecting the surrounding normal structures inside the eye.Therefore,the development of an alternative therapy for preventing PCO is of critical importance.Apoptosis is the physiological,energy-dependent form of cell death.It is,in contrast to necrosis,controlled and,under certain conditions,even initiated by the cell itself.Apoptosis causes no inflammatory side effects.The programmed cell death is initiated either extracellularly by death ligands binding specifically to death receptors or cellular stress factors such as ionizing radiation,heat or hypoxia,thereby initiating an intracellular signalling cascade leading finally to the death of the cell.The IAPs(inhibitor of apoptosis proteins) and the Bcl-2 families are among the most important regulators of the cell suicide process.Bcl-2 and Bax are,respectively, anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family mainly involved in the regulation of the intrinsic apoptosis pathway.And Survivin is a recently identified member of the IAPs,with an intriguing bifunctional role:it facilitates cell cycle progression while counteracting a large variety of stimuli involved both in the extrinsic and in the intrinsic apoptotic pathways.Research on apoptosis,its intracellular signalling cascade has made apoptosis a central issue in the development of anti-proliferative chemotherapies.In turn,this information could provide new opportunities and targets for therapies to prevent PCO. In this study we focused on LECs proliferation inhibition strategy,reducing PCO by means of inducing Bax gene overexpression and silencing Survivin gene expression, characterizing the pathway of apoptosis in LECs.PartⅠSurvivin and Bax gene expressions in LECs by FGF or TGF-βstimuliPurpose To investigate the impact of FGF or TGF-βstimuli on Survivin and Bax gene expressions.Method Human Lens cell line(SRA01/04) was cultured in vitro and treated with different concentrations of TGF-βor FGF respectively.LECs proliferation was monitored by phase contrast microscopy,and determined using CCK-8 regent.Protein expressions of Survivin,Bcl-2 and Bax were observed by western blot analysis.And mRNA expressions of target gene were investigated by means of RT-PCR using specific primers.Results It was found that LEC proliferation was suppressed when exposed to the increasing concentration of TGF-β,whereas FGF enhanced proliferation. Proliferation suppression by TGF-βwas blocked by FGF.TGF-βcaused a significant down-regulated in expression of Survivin and Bcl-2,while increased Bax expression. FGF could up-regulate Survivin and Bcl-2 protein and mRNA expression while reduce Bax expression. Conclusion Growth factor TGF-βor FGF regulated the proliferation of LECs.Survivin,Bcl-2 and Bax,the members of the apoptosis regulatory gene,might play a role in these processes.The findings suggest that these genes are good candidates for blocking proliferation of LEC.PartⅡBax gene overexpression to induce LECs apoptosisPurpose To investigate the influence of Bax overexpression on the apoptosis of LECs.Methods The plasmid EGFP-N1-Bax,EGFP-N1 were transferred to LECs using lipofectamine 2000.48 hours after transfection,Bax gene mRNA expression was measured using RT-PCR and protein expression was determined by western blot analysis.The expression level of Bcl2 and Survivin gene was also examined using the same techniques.Flow cytometry was used to evaluate Bax transfection and the effect of Bax on apoptosis.Results EGFP-N1-Bax transfection rate was 45.47±1.30%evaluated by cytometry.48 hours after Bax gene transfection,LECs exhibited a significant increase in the expression level of Bax gene mRNA and protein,compared to cells transferred with plasmid EGFP-N1.The apoptosis rate in EGFP-N1-Bax group was approximately 23.98±1.88%.The Bax transfection group also showed a significant decrease in the mRNA expression of Survivin and Bcl-2.Conclusion After treatment with plasmid EGFP-N1-Bax,expression of Bax gene was greatly enhanced.Overexpression of Bax gene could trigger the apoptosis of LECs.PartⅢInducing apoptosis of LECs by Silencing Survivin gene expressionPurpose To investigate the role of silencing Survivin gene expression in apoptosis pathway and the response of LECs death induced by silencing Survivin and over expressing Bax.Methods Survivin siRNA was introduced into LECs using lipofectamine alone and combining with EGFP-N1-GFP.The transfection rates and apoptosis rates were assayed by flow cytometry.The change of mRNA and protein of the Survivin gene and Bax/Bcl-2 gene were detected by RT-PCR and Western blot,respectively.Results Survivin siRNA transfection rate was 56.58±2.67%evaluated by cytometry. LECs showed a significant apoptosis by 24.49±2.42%,33.28±2.69%when they transferred with Survivin siRNA,and combining with EGFP-N1-Bax.The Survivin mRNA and protein were knocked down in LECs.Combined with Bax transfection, the cell presented a decrease in apoptosis index Bcl-2/Bax and Survivin expression.Conclusion Small interfering RNA can exert a knockdown of Survivin gene expression in LECs,and induce apoptosis significantly.Combined with Bax transfection could enhanc cell apoptosis.Summary1.Survivin,Bcl-2 and Bax,the members of the apoptosis regulatory gene,might play a role in the process of LEC proliferation mediated by growth factor(FGF and TGF-β).2.Overexpression of Bax gene by transfection EGFP-N1-Bax could trigger the apoptosis of LEC.3.Small interfering RNA could exert a knockdown of Survivin gene expression in LECs,and effectively induce apoptosis.4.Combined Survivin gene inhibition with Bax overexpression may enhance LEC apoptosis.