The Role And Mechanism Of IGFBP5 In Apoptosis And Proliferation Of Human Lens Epithelial Cells,via Endoplasmic Reticulum Stress

Background:Cataracts are the world’s leading eye disease which result in avoidable blindness.As the population ages,the prevalence of age-related cataracts（ARC）will gradually increase.Endoplasmic reticulum stress（ERS）-mediated lens epithelial cell apoptosis plays an important role in ARC pathogenesis theories.The previous research showed that insulin like growth factor binding protein 5（IGFBP5）may be an important protein involved in the regulation of ERS in human lens epithelial cells.Up to now,studies have found that the expression of IGFBP5 in lens epithelial cells of ARC patients is apparently lower than that of transparent lenses,which uncovers that IGFBP5 is closely related to the occurrence and development of ARC.As an important part of insulin-like growth factor family,IGFBP5 is widely involved in the regulation of important life activities such as cell proliferation,differentiation and apoptosis.However,the studies related IGFBP5 have not been reported yet in the side of ARC pathogenesis,which need to be investigated.Objective:1.To observe the changes of IGFBP5 expression in human lens epithelial cells under oxidative stress.2.To study the regulation and mechanism of IGFBP5 on cell proliferation and apoptosis through endoplasmic reticulum stress in human lens epithelial cells.Methods:Part I:Firstly,appropriate concentration of H₂O₂ was screened to construct the human lens epithelial cell line（HLE-B3）oxidative stress cell model.Then,real-time fluorescence quantitative polymerase chain reaction（q RT-PCR）and Western blot were used to detect the expression changes of m RNA and protein of IGFBP5 under oxidative stress.PartⅡ:Lentivirus was used as transfection tool to construct and screen IGFBP5 overexpressed and down-regulated stable transfected cell lines,and IGFBP5gene function verification was performed after treating cells with appropriate concentration of H₂O₂.Cell proliferation ability was detected by Cell counting Kit-8（CCK-8）.The percentage of apoptosis was detected by Annexin V-PE/7-ADD double staining flow cytometry.q RT-PCR and Western blot were used to detect the m RNA and protein expression levels of IGFBP5,IGF-1 and endoplasmic Reticulum stress-related GRP78,CHOP and caspase-12.Results:Part I:The appropriate H₂O₂ treatment concentration is 155.3μmol/L.Under oxidative stress,the expression of IGFBP5 m RNA and protein in HLE-B3 was dramatically down-regulated.Part II:HLE-B3 cell lines were successfully constructed and screened that is IGFBP5 overexpression and interference expression stable transfection.After treating by H₂O₂,ERS of HLE-B3 cells were activated,showing that GRP78,CHOP,Caspase-12 m RNA and protein were up-regulated,and cell proliferation was decreased,and apoptosis rate was increased.Under oxidative stress,IGF-1 m RNA and protein expression in HLE-B3 cells were down-regulated simultaneously with IGFBP5.Among the cells treated by H₂O₂:in the group of IGFBP5 overexpression,IGF-1 m RNA and protein expressions were up-regulated.And with inhibited ERS,increasing cell proliferation rate,decreasing apoptosis rate,the expression of GRP78,CHOP,caspase-12 m RNA and protein were decreased which is compared with the control group.In the group of IGFBP5 interference expression,IGF-1 m RNA and protein expressions were down-regulated.And with increasing ERS,decreasing cell proliferation rate,increasing apoptosis rate,the expressions of GRP78,CHOP,caspase-12 m RNA and protein were up-regulated which is compared with the control group.Conclusions:1.IGFBP5 is closely related to the occurrence and development of ARC.2.Under oxidative stress,IGFBP5 overexpression can up-regulate IGF-1.By inhibiting ERS,the apoptosis of human lens epithelial cells was reduced and cell proliferation was promoted.However,IGFBP5 interference expression can inhibit the expression of IGF-1,promoting apoptosis of human lens epithelial cells and inhibiting cell proliferation by intensifying ERS.3.In human lens epithelial cells,IGFBP5 can interact with IGF-1 to regulate ERS and apoptosis and proliferation of associated cells.