Protective Effect Of EphA2Gene Overexpression Against Oxidative Stress-mediated Human Lens Epithelial Cells Damage

Age-related cataract, also known as senile cataract, is a condition where cloudydeposits gradually accumulate on the crystalline lens of the eye in people aged50years and over. Despite advances in their surgical treatment, age-related cataractsremain a leading cause of visual impairment and blindness in the world.The lens epithelial cells play a key role in lens maintenance and defense.Oxidative stress occurs when the level of reactive oxygen species and other freeradicals exceeds the ability of a cell to respond through antioxidant defense systemsand ultimately leads to protein modification and degradation, DNA and mitochondrialdamage and eventual lens epithelial cells death.Eph-receptor tyrosine kinase-type A2(EphA2) is a member of the subclass A ofthe Eph subfamily. Located on the short arm of chromosome1at position36, thehuman EphA2gene encodes976amino acids, type-Ⅰ transmembrane protein with anextracellular NH2-terminal domain and a cytoplasmic COOH-terminal domain.Recently, the association of the various single nucleotide polymorphisms (SNPs) onthe EphA2gene with the risk for various subtypes of cataract has been assessed.However, the function of the EphA2gene on cataract is not well known.In the present study, in order to investigate the role of EphA2played in responding to oxidative stress，we detected the expression level of EphA2in human lensepithelial cells treated with multiple concentrations and time of H2O2as elements.With the method of constructing the eukaryotic plasmid which expressed humanEphA2gene and transfected into human lens epithelial cells, then observed the effectof EphA2plasmid on human lens epithelial cells and H2O2induced cell apoptosis. Tofurther investigate the relationship between level of EphA2and cataract, we examinedthe expression level of EphA2in the lenses of younger, older, and cataractous. Part1Expression of EphA2in human lens epithelial cells exposed tooxidative stressObjective: To investigate the expression level of EphA2in human lens epithelial cells(SRA01/04cells) exposed to oxidative stress.Methods: SRA01/04cells were cultured and treated with various concentrations ofH2O2(0μM,50μM,100μM,200μM, and300μM) for24hours. Cell viabilities weremonitored by Cell Counting Kit-8(CCK-8) assay. To established models of acuteoxidant stress in vitro, cultured SRA01/04cells were exposed to100μM and200μMhydrogen peroxide (H2O2) for24hours. Level of EphA2was detected by real timePCR and Western blot. Additionally, to established models of chronic oxidant stress invitro, SRA01/04cells were treated respectively with50μM and100μMhydrogen peroxide (H2O2) for15days and then analyzed the Level of EphA2by realtime PCR and Western blot.Results: The CCK-8assay showed that50μM H2O2did not significantly decreaseSRA01/04cells viability. However, from100μM to300μM of H2O2treatment,significantly decreased SRA01/04cells viability in a dose-dependent manner. TheSRA01/04cells were exposed to100μM and200μM hydrogen peroxide (H2O2) for24hours. Expression Level of EphA2mRNA and protein increased in H2O2-treatedSRA01/04cells. Additionally, The SRA01/04cells were exposed to50μM and100μM hydrogen peroxide (H2O2) for15days. Expression Level of EphA2mRNAand protein unregulated in SRA01/04cells treated with50μM H2O2anddownregulated in100μM H2O2-treated cells.Conclusions: The expression Level of EphA2in SRA01/04cells was increased whenexposed to acute oxidative stress. In the models of cell senescence to chronic oxidantstress, H2O2could promote expression of EphA2in early stage of cell senescence,and suppress expression of EphA2with the degree of cell senescence increased. Part2Construction of eukaryotic plasmid expressing human EphA2gene totransfect into human lens epithelial cellsObjective: To determine whether recombinant eukaryotic expression plasmidencoding the human EphA2gene can express in human lens epithelial cells.Methods: Pasmid pBABE-puro-EphA2was used as template for subsequent PCRamplifications of EphA2cDNA. EphA2cDNA was cloned into eukaryotic expressionplasmid (pIRES2-ZsGreen1) and identified by double restriction endnuclease (EcoRVand XhoI) and sequencing. EphA2plasmid was tranfected into SRA01/04cells withLipofectamine LTX. The transfection efficiency of EphA2plasmid in SRAS01/04cells was determined by fluorescence microscope after48-hour post transfection.Expression of EphA2mRNA and protein were analyzed by real time PCR andWestern blot at48hours after transfection.Results: The recombinant EphA2eukaryotic expression plasmid was successfullycloned. The transfection efficiency of EphA2plasmid in SRAS01/04cells wasapproximately reached to65%at48hours after transfection. There wasstronger expression of EphA2mRNA and protein in SRA01/04cells transfected withEphA2plasmid at48hours post transfection.Conclusions: The recombinant EphA2eukaryotic expression plasmid was expressedsuccessfully and effectively in SRA01/04cells.Part3Effects of EphA2gene overexpression on the proliferation，cell cycle，apoptosis and migration in human lens epithelial cellsObjective: To investigate the effect of EphA2gene overexpression on theproliferation，cell cycle，apoptosis and migration in human lens epithelial cells. Methods: SRA01/04cells were cultured and transfected with empty vector (Vector)or EphA2plasmids (EphA2OE), to obtain cells with a transient EphA2overexpression. After48hours of transfection, the cells were collected; Cellproliferation was monitored by Cell Counting Kit-8(CCK-8) assay. The apoptosisrate and cell cycle were detected by flow cytometric analysis. Cell migration wasstudied by scratch wound healing assay.Results: The CCK-8assay showed that the proliferation of EphA2OE-transfectedSRA01/04cells was significantly increased compared with that of theVector-transfected SRA01/04cells (P=0.0003). EphA2OE-transfected SRA01/04cells led to significant increased number of cells in S phase (P=0.008, t=-4.988).There was no significant difference in apoptotic rate between the EphA2OE cells andVector--transfected cells (P=0.486, t=0.776). The migration rate of EphA2-transfectedcells was significantly faster than Vector--transfected cells (P<0.001).Conclusion: Overexpression of EphA2could promote cellular proliferation,migration and increase cell cycle in SRA01/04cells, but it has no remarkable effecton the apoptosis of SRA01/04cells.Part4Overexpression of EphA2gene protects human lens epithelial cellsagainst oxidative stress-induced apoptosisObjective: To investigate the protective effect of EphA2gene overexpression onoxidative stress-induced apoptosis in human lens epithelial cells.Method: SRA01/04cells were cultured and transfected with empty vector (Vector)or EphA2plasmids (Vector OE) for48h, to obtain cells with a transient EphA2overexpression and then treated by H2O2(200μM) for24h. Cell viabilities weremonitored by Cell Counting Kit-8(CCK-8) assay. The apoptosis rate and ROSgeneration were detected by flow cytometric analysis. Expression levels of BCL-2,BAX and Caspase-3proteins were measured by western-blotting analysis. Results: Overexpression of EphA2in SRA01/04clearly increased cells viability andreduced H2O2-induced cell apoptosis and ROS accumulation; protected SRA01/04cells from H2O2induced oxidative damage, increased the expression levels of BCL-2,downregulated the expression levels of BAX and did not affect the expression levelsof Caspase-3.Conclusions: These findings suggested that EphA2overexpression protectedSRA01/04cells from H2O2induced oxidative damage, presumably by upregulatingthe expression levels of BCL-2and downregulating the expression levels of BAX.Part5The relationship between expression level of EphA2gene andcataract in ratObjective: To investigate the relationship between expression level of EphA2andcataract in rat.Methods: Thirty-six lenses from SD rats were allocated into three groups: clearyoung, old and cataractous lenses. Expression of EphA2in the lenses of young, old,and cataractous was detected by quantitative real time PCR and western blot.Results: Western blot analysis showed that EphA2protein in rat cataractous lenseswas lower compared to clear old lenses (P=0.0000006, t=10.952). The old lensesslightly higher in the level expression of EphA2protein compared to young lenses(P=0.001, t=-4.4562). Real time RT-PCR analysis showed that EphA2mRNAexpression in rat cataractous lenses was higher than clear old lenses (P=0.001,t=-4.518). However, EphA2mRNA expression in old lenses was lower than younglenses (P=0.000009, t=8.237).Conclusions: Lower level expression of EphA2was related to cataract in rats. Theexpression level of EphA2protein in rats’ lenses was affected by age.