The Role And Mechanism Of MiR-34a/SIRT1 Signaling Pathway During Exprimental Age-related Cataract Formation

Age-Related Cataract is a condition where crystalline lens of the eye in middle-age and old people becomes increasingly opaque.Alhough suegery management is an effective procedure,age-related cataracts remains a leading cause of blindness and low vision in the world.It is proveed that SIRT1 is a gene target of miR-34a which is involved in apoptosis、cell aging and cell metabolism processes.Despit the increased level of miR-34a expressions is related to cataract developmet recently found,the action mechanism remains unclear.To investigate the role of miR-34a/SIRTl expression played in the pathogenesis of senile cataract, we detect the expression level differences between transparent and cataractou lens in 2 or 18 month old rats.To further explore the underling mechanism of miR-34a/SIRT1,we estabish oxidativestress damage cell model by lens epithelial cells(SRA01/04 cells) treated with H2O2 and observe the expression levels of miR-34a/SIRT1 in SRA01/04 cells. Transfer miR-34a mimics and miR-34a inhibitor into the cells, wherafer survey the impact of the expression level changes of miR-34a on oxidative stress-induced SRA01/04 cell injury.Part 1 The effect of miR-34a/SIRTl expression on the pathogenesis of rat cataractObjective:To investigate the expression level changes of miR-34a/SIRTl and downstream signal molecules in cataract lens in rats.Methods:Lenses from SD rats were distributed into three groups-.transparent lens in 2 month old rats、transparent lens in 18 month and cataractou lens in 18 month. Transcription of miR-34a/SIRT1 and translation of SIRT1 protein.. p53 protein and p53 acetylated protein in the lenses was respectively detected by quantitative real time RCR and Western blot.Results:Real time PCR analysis showed that miR-34a expression was significantly up accompanied down-expression of SIRT1 mRNA in rat cataractous lenses (t=-12.60, P=0.0002;t=3.299,P=0.03).miR-34a expression in 18-month-old rat cataractous lenses was lower than 2-month-old clear lenses(t=9.609,P=0.0007).SIRTl mRNA expression was higher accordingly(t=-7.242,P=0.0019).Western blot analysis showed that SIRT1 protein expression was significantly down accompanied up-expression of p53 acetylated protein in rat cataractous lenses (t=8.924,P=0.0009;tr=-9.280,P=0.0008). SIRT1 protein expression in 18-month-old rat cataractous lenses was higher than 2-month-old clear lenses(t=-3.419,P=0.0345). p53 acetylated protein expression was higher accordingly(t=4.768,P=0.0089).Conclusions:Higher level expression of miR-34a was related to cataract in rats. It will work by regulating the decrease of SIRT1 protein and promoting the acetylation of P53.Part 2 The effect of miR-34a/SIRTl expression on oxidative stress-induced injury of SRA01/04 cellObjective:To observe the effect of miR-34a/SIRT1 expression on oxidative stress-induced injury of SRA01/04 cell.Methods:Oxidativestress damage cell model is estabish by lens epithelial cells(SRA01/04 cells) treated with H2O2 the relationship between miR-34a/SIRTl/P53 expressionand oxidative stress reaction is tested by Real time PCR.The impact of miR-34a mimics�'�miR-34a inhibitor on cellur proliferation of oxidative stress-induced SRA01/04 cell is detected by CCK-8.Results:H2O2 can result in SRA01/04 cell oxidative stress damage and the effect is dose-dependent.The survival rate of cell is approximately 50% at a concentration of 200μM H2O2.In SRA01/04 cell injured by oxidative stress,the expression of miR-34a significantly increase.However,the expression of SIRT1 and P53 mRNA significantly decreased.miR-34a up--regulation can promote SRA01/04 cell oxidative stres injury,while miR-34a down-regulation restrain injury.Conclusions:miR-34a involve in oxidative stress of lens epithelial cells by regulating the expression of SIRT1 gene.