The Role Of ILK In The Changes Of Cell Biological Behaviors Induced By High Glucose In Human Lens Epithelial Cells

BackgroundIntegrin-linked kinase(ILK) is a serine-threonine kinase.It mediates signals of extracellular matrix(ECM) and growth factors into intro-cellular through its binding to integrin subunits ofβ1 andβ3.It is a target of PI3-K,and adjusts the activity of PKB/AKT and GSK-3β,thus regulates numerous processes including cell proliferation,survival,cell cycles,differentiation, apoptosis,migration and tumor vasculogenesis.The onset age of diabetic cataract is somewhat younger compared with age-related cataract,and the postoperative complications like posterior capsule opacification(PCO) are more severe.Studies in the pathogenesis of lens fibrotic disease,such as PCO and anterior subcapsular cataract(ASC) have revealed that integrins are involved in the cell proliferation,apoptosis,and epithelial-to-mesenchymal transition (EMT) in the lens epithelial cells(LECs).Since ILK is an essential down-stream signal molecule of transmembrane integrins,it may play an important role in the bilateral signal transduction.It is reported that ILK was upregulated in human mesangial cells and podocyte cells cultured in high glucose.It induces EMT in renal tubular epithelial cells.These evidences suggest that ILK involves in the pathogenesis of diabetic nephrosis.However,it is still unknown in LECs whether ILK is involved in the cell proliferation,apoptosis,and EMT induced by high glucose.These changes may contribute to formation of diabetic cataract and PCO.ObjectiveTo investigate the effects of high glucose on cell proliferation,apoptosis, cell death and EMT in human LECs in vitro.To detect the changes of ILK expression induced by high glucose in the cells,and to determine its role in cell proliferation and EMT via RNA interference technology.MethodsHuman LECs line SRA01/04 cells were cultured in DMEM containing 2% serum and exposed to 5.5 mmol/L glucose as control or 30.5 mmol/L high glucose,respectively.After 24 hours,the expression of proliferating cell nuclear antigen(PCNA),α-smooth muscle actin(α-SMA),and fibronectin(FN) were detected by indirect immunofluorescence assay,reverse transcription polymerase chain reacation(RT-PCR),and flow cytometry.Cell apoptosis or cell death was detected by flow cytometry with PI and AnnexinⅤstaining after treatment with 5.5,30.5,55.5,80.5 and 105.5mmol/L glucose for 24h,respectively.After 24 hours of treatment with high glucose,the expression of ILK was detected by indirect immunofluorescence staining.ILK mRNA was also analyzed by RT-PCR at 0,6,and 24 hours after treatment with high glucose.SRA01/04 cells were transfected with a selected small-interfering RNA targeting for ILK gene. Six hours after transfection,the cells were exposed to normal or high concentration of glucose for 24 hours.The expression of ILK,PCNA,α-SMA and FN at mRNA level were detected by RT-PCR to evaluate the cell proliferation and EMT. ResultsIn the cells with the treatment of high glucose for 24 hours,the fluorescence intensities of ILK,PCNA,α-SMA and FN were enhanced.The mRNA levels of ILK,PCNA,α-SMA and FN increased by 2.23,2.00,1.74 and 1.78 folds compared with control group,respectively(P＜0.05).The percentages of PCNA andα-SMA positive cells detected by flow cytometry were 55.8%and 59.7%, respectively,which was higher than the control group(P＜0.05).After 24h of treatment with five different concentrations of glucose,the percentages of apoptotic cells were no different(P=0.227,F=1.489).The percentages of dead cells in 30.5 and 55.5mmol/L glucose groups were no significant differences compared with 5.5 mmol/L control group.In 80.5 and 105.5mmol/L glucose groups,the percentages of dead cells were increased compared with control group(P＜0.01,P＜0.001).The percentages of the dead cells in 55.5,80.5 and 105.5mmol/L glucose groups were increased gradually.The interfering efficiency of the selected ILK siRNA was about 70%.After transfected with ILK siRNA,the expression of ILK mRNA was reduced to 30%of that in normal control cells.In the transfected cells of high glucose group,the expression ILK mRNA was reduced to 21%of that in untransfected cells,and PCNA,α-SMA, and FN mRNA was 29%,33%and 39%of those in untransfected cells, respectively(P＜0.01).ConclusionsHigh glucose stimulates cell proliferation and EMT but apoptosis in the lens epithelial cells.The percentage of the dead cells was gradually increased with high concentrations of glucose.The expression of ILK was upregulated by high glucose in human LECs.ILKsiRNA suppresses cell proliferation and EMT induced by high glucose.