Let-7c-3p Regulates Mechanisms Of Autophagy By Targeting ATG3 In Lens Epithelial Cells

Objective:Oxidative stress is an important factor during age-related cataract formation.Apoptosis and autophagy induced by oxidative stress have been reported as key factor in age-related cataract.In our research,we investigated the role of let-7c-3p in the regulation of autophagy and apoptosis during the formation of age-related cataract.Globally,cataract is a prior cause of blindness and aging is the leading contributor of the cataracts.Ultraviolet and oxidative stress are seen as key factors in cataract formation.A meaningful relationship has been revealed between age-related cataract and oxidative stress.Oxidative stress refers to the damage of exogenous beyond the antioxidant capacity of cells,which affects the signal transduction system of cells or subsequently damages macromolecules such as nucleic acids,proteins and lipids.Oxidative stress plays a critical role in regulating normal physiological functions associated with cell cycle,migration,and cell death.In previous study,oxidative stress has also been declared to induce apoptosis and autophagy in lens epithelial cells.There are many studies on the potential mechanism of new targets in the treatment of age-related cataract based on autophagy and apoptosis.Though there are some anti-oxidative agents can decreased H₂O₂-treated human lens epithelial cells damage,such as Ma et al.shows HO-1 can protects human lens epithelial cells from H2O2-induced oxidant stress by upregulated antioxidant enzyme activity,reducing ROS generation,and thus inhibiting caspase family-dependent apoptosis.Autophagy is a highly conserved process involving protein,lipids and organelles degradation,which resulting in influencing nutrition recycle.When LC3-II is measured as an autophagy related marker,the increase of LC3-II/LC3-I ratio represents the active autophagy activity.Almost all cells exist basal autophagy to maintain cellular homeostasis.But when under stress conditions,such as hypoxia and starvation,autophagy will be switched on to protect cells from these stress.Dysregulation of autophagy has been reported to be related to various diseases.In addition,sometimes the role of autophagy in disease progression is dual.Recent study has found autophagy can act a very important role in keeping the transparency of lenses.Moreover,autophagy can also participate in regulating pathophysiologic process in hereditary cataract and age-related cataract.It has been found autophagy is scheduled to be constitutively activated in lens epithelial cells during the period of fiber cell differentiation.It is very likely that autophagy is important for protecting lens cells from oxidative stress,which is one of the main causes of age-related cataract.A growing number of studies have indicated autophagy could be controlled by microRNAs?miRNAs?.MiRNAs are a number of small non-coding RNAs with 20?24 nucleotides that inhibit gene expression,such as mRNA degradation and translational inhibition,by binding the 3'UTR region of mRNA.MiRNAs have been recorded to regulate cell proliferation,metastasis,apoptosis and autophagy.Recently,the role of miRNAs in age-related cataract has been noted.The Bcl-2 protein locates in endoplasm momentum and mitochondrial membranes and it can inhibit the release of apoptosis inducing factors to prevent cell apoptosis.In apoptosis,Bax protein activates the cascade of reactions by releasing cytochrome c from the mitochondria that helps in successive activation of caspases and ultimately leads to cell death.Let-7b was noted to induce apoptosis of LECs through targeting leucine-rich repeat containing G protein-coupled receptor 4.The miRNA let-7c has been tried and found down-regulated in cataract.In mammals,let-7 is known as the keeper of differentiation,and its abnormal regulation and expression have been associated with disease progression.Let-7miRNA family has proven to inhibit the cellular reprogramming process.Reprogramming well-differentiated cells into induced persistent stem cells had a great significance on tissue repair and tumor occurrence.Recently,some findings suggest that microRNAs have a role in age-related cataracts.A local let-7 microRNA increase may represent a risk factor in the formation of age-related cataracts.The aim of our study is to research the differentially expressed let-7c in age-related cataract whether can influence autophagy,a fundamental degradation process in LECs,which can improve our understanding of cataract.In the current study,we explored the function of let-7c-3p in the regulation of autophagy in LECs during age-related cataract formation.We detected the expression of let-7c-3p in with age-related cataract tissues and LEC cells treated with hydrogen peroxide.The effect of let-7c-3p on autophagy and apoptosis was evaluated under oxidative stress.Moreover,we investigated molecular mechanisms of let-7c-3p regulating autophagy in LECs.We confirmed that let-7c-3p regulates autophagy by targeting ATG3 under oxidative stress in lens epithelial cells.Methods:1.According to the classification system of lens opacity II?LOCSII?,40samples of anterior capsular epithelial tissue were collected from patients with age-related cataract undergoing phacoemulsification in the fourth affiliated hospital of China medical university.All samples were divided into two groups,>65 group and?65 group.The study was approved by the hospital ethics committee.The expressions of let-7c-3p and ATG3 in tissue samples were detected by real-time quantitative polymerase chain reaction.Using the same operation method,the data obtained from three independent experiments are analyzed using the formula RQ=2-??Ct.Western blot was used to detect the expression of ATG3 protein in tissue samples.2.Human lens epithelial cell line?SRA01/04?was used to establish an in vitro culture model of oxidative stress in human lens epithelial cells.SRA01/04 cells were cultured with different concentrations of H2O2.H₂O₂ concentrations were 0?M,25?M,50?M,100?M and 150?M,respectively,and were treated in the incubator at 37?,5%CO₂ and full saturated humidity for 24 hours.The effects of H2O2 stimulation at different concentrations on SRA01/04 cell line viability were compared and detected by cck-8 method.SRA01/04 cells were treated with 50?M H₂O₂ at working concentration for 24h,and apoptosis was analyzed by FACS Calibur flow cytometry?BD Bioscience?.The expressions of let-7c-3p and ATG3 in SRA01/04 and their differences with those of the oxidative stress group were detected by real-time quantitative polymerase chain reaction and Western blot.3.The expression of let-7c-3p was regulated by transfection with a let-7c-3p mimic and inhibitor in the oxidative stress model,so as to regulate apoptosis and autophagy.The expressions of let-7c-3p,Bcl-2,Bax3,LC3B-II,LC3B-I were detected by real-time quantitative polymerase chain reaction and Western blot.The cell apoptosis rate of SRA01/04 was detected by flow cytometry.Immunofluorescence microscopy was used to detect the expression difference of LC3B-II in SRA01/04.4.Target genes that might play a role in let-7c-3p in human lens epithelial cells were predicted by targetscan,and the expression changes of the target genes at the protein level were predicted after the regulation of let-7c-3p expression by Western blot detection.The target gene siRNA was constructed,and the expression of the target gene in human lens epithelial cells and the effect of the target gene on the expression of LC3B-II and LC3B-I were detected by Western blot.The relationship between let-7c-3p and predictive target genes was detected by double luciferase reporter technique.SPSS17.0 software was used for statistical analysis.All experiments were repeated for 3 times,and the data were expressed as mean±standard deviation?mean±SD?.ANOVA analysis of variance and independent sample t test were used to compare the significance of the data,and P<0.05 was considered statistically significant.Results:1.Real-time PCR analysis of let-7c-3p expression showed that the level of let-7c-3p in anterior capsules of age-related cataract patients?age>65 years?was significantly lower than that in anterior capsules of age-related cataract patients?age?65years?.And we found that the SOD expression level was higher in the age?65 years group than the age>65 years group.Real-time PCR was then used to detect let-7c-3p expression.We demonstrated that the level of let-7c-3p expression in lens epithelial cells under oxidative stress was significantly lower than that of the normal group.This indicated that let-7c-3p was downregulated in SRA01/04 cells under oxidative stress.Let-7c-3p Attenuated the apoptosis in SRA01/04 Cells under Oxidative Stress.2.To explore the effect of let-7c-3p on apoptosis under oxidative stress,LECs were infected with let-7c-3p mimics and let-7c-3p inhibitors,respectively.The transfection efficiency was analyzed by real-time PCR.We observed that the apoptosis rate of SRA01/04 cells was induced by oxidative stress.The rate of apoptosis in SRA01/04 cells increased from6.04%to 22.50%.Meanwhile,the rate of LEC apoptosis decreased from 18.50%to7.70%when SRA01/04 cells were infected by let-7c-3p mimics compared to the negative control.And the rate of LEC apoptosis decreased from 20.40%to 26.02%when SRA01/04 cells were infected by let-7c-3p inhibitor compared to the negative control.To further confirm this result,we analyzed Bcl-2 and Bax protein expression and found that the results were consistent with flow cytometry.These results implied that let-7c-3p attenuated apoptosis under oxidative stress.3.Let-7c-3p Attenuated the Autophagy in SRA01/04 Cells under Oxidative Stress.As autophagy and apoptosis both participate in formation of cataract,we tried to investigate whether let-7c-3p could modulate autophagy.SRA01/04 cells were exposed to oxidative stress as an experiment group for24h and then treated with let-7c-3p mimics and let-7c-3p inhibitor.Under oxidative stress,we observed that the ratio of LC3B-II and LC3B-I proteins increased significantly in SRA01/04 cells,while the ratio decreased when LECs were transfected by let-7c-3p mimics compared with the control group.However,the let-7c-3p inhibitor could increase the ratio of LC3B II and LC3B I.To further investigate the effect of let-7c-3p on autophagy,an immunofluorescence assay was conducted.The result showed that let-7c-3p could suppress autophagy induced by H₂O₂.Thus,the findings suggested that let-7c-3p attenuated the level of autophagy in SRA01/04 cells under oxidative stress.4.ATG3Facilitated Autophagy in SRA01/04 Cells under Oxidative Stress.We conducted real-time PCR and western blot assays to detect the expression of ATG3 in SRA01/04 cells under oxidative stress.We found that ATG3 was upregulated in SRA01/04 cells under oxidative stress.ATG3 has been reported as a vital modulator of autophagy in mediating mitochondrial homeostasis.To confirm the effect of ATG3 in LECs,we performed the loss-of-function study.After transfection for 24h,the level of ATG3 was downregulated by si-ATG3.We found that the ratio of LC3B II and LC3B I proteins in the si-ATG3group was lower than that in the negative control.These findings revealed that ATG3facilitatedthe autophagy in SRA01/04 cells under oxidative stress.In addition,ATG3,an E2-like enzyme,is essential for vesicle elongation formation and plays a significant role in autophagy regulation.Then,we detected the expression level of ATG3 in cataract tissues.The results showed that ATG3 in patients aged>65 years was higher than that in the patients aged?65 years.5.Let-7c-3p Regulates Autophagy by Targeting ATG3 in SRA01/04 Cells under Oxidative Stress.The prediction by the TargetScan database indicated that ATG3 might be the target gene of let-7c-3p.To indicate the prediction result,we use real-time PCR and western blot to measure ATG3 expression levels after transfecting with let-7c-3p mimics and let-7c-3p inhibitors in SRA01/04 cells.The results showed that ATG3 mRNA and protein expression increased when let-7c-3p was downregulated,while the levels of ATG3 mRNA and protein expression decreased when let-7c-3p was upregulated.Consistently,the luciferase reporter assay was performed,showing that let-7c-3p could bind with ATG3 mRNA directly.To examine whether let-7c-3p could regulate autophagy via ATG3,we performed a rescue experiment.The results showed that ATG3 reversed the effect of let-7c-3p on attenuating the autophagy level.In conclusion,let-7c-3p could regulate autophagy by targeting ATG3 in SRA01/04cells under oxidative stress.Conclusion:This study aimed to investigate the effects of let-7c-3p in lens epithelial cells?LECs?in vitro and involved the process of autophagy and apoptosis.Let-7c-3p inhibits autophagy by targeting ATG3 in LECs in age-related cataract.