Let-7c-3p Regulates Autophagy under Oxidative Stress by Targeting ATG3 in Lens Epithelial Cells

Abstract

Background: Oxidative stress is an important factor during age-related cataract formation. Apoptosis and autophagy induced by oxidative stress have been reported as key factors 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. Material and Methods. Real-time PCR and western blot were employed to detect the expression of let-7c-3p in the tissues of age-related cataract. Human lens epithelial cells (LECs) were treated with H₂O₂ as an age-related cataract model. The extent of apoptosis was measured by flow cytometry and western blot. To detect autophagy, immunofluorescence was used to analyze the spot number of LC3, and western blot was used to detect the expression of LC3-II/I and ATG3. The molecular mechanisms of let-7c-3p regulating autophagy via ATG3 under oxidative stress were performed by a luciferase report gene assay and rescue experiment.

Results: Downregulation of let-7c-3p was found in the age-related cataract group aged >65 years relative to the age-related cataract group aged ≤65 years. Consistently, the expression of let-7c-3p was also lower under oxidative stress. The activities of LEC apoptosis and autophagy induced by oxidative stress were inhibited by let-7c-3p. By the bioinformatics database and the luciferase reporter assay, ATG3 was found to be a direct target of let-7c-3p. Let-7c-3p reduced the ATG3-mediated autophagy level, which was induced by oxidative stress in LECs.

Conclusion: Let-7c-3p inhibits autophagy by targeting ATG3 in LECs in age-related cataract.

Figure 1

The expressions of let-7c-3p were downregulated in the anterior lens capsules and in SRA01/04 cells under oxidative stress. (a) Let-7c-3p expression levels in samples with age > 65 years relative to samples with age ≤ 65 years were detected by real-time PCR. (b) Let-7c-3p expression levels in samples with age > 65 years relative to samples with age ≤ 65 years were detected by real-time PCR. (c) The cell viability in SRA01/04 cell treated with a series of H₂O₂ concentrations (0-150 μM). CCK8 assay was used to detect the levels of cell viability. (d) Let-7c-3p expression level in SRA01/04 cells under oxidative stress was detected by real-time PCR (^∗P < 0.05).

Figure 2

Let-7c-3p attenuated the apoptosis in SRA01/04 cells under oxidative stress. (a, b) The mRNA expression levels of let-7c-3p in SRA01/04 cells infected by let-7c-3p mimics, mimic controls, let-7c-3p inhibitors, or inhibitor controls were detected by real-time PCR. (c) Forty-eight hours after infection, downregulated let-7c-3p and control groups were treated with 50 μM H₂O₂ for 24 h. Flow cytometry was used to analyze apoptosis. (d) Western blot was used to analyze the expression level of Bcl-2 and Bax. The SRA01/04 cells were treated with 50 μM H₂O₂ for 24 h (^∗P < 0.05).

Figure 3

Let-7c-3p attenuated the autophagy in SRA01/04 cells under oxidative stress. (a) Western blot was used to analyze the expression level of LC3B II and LC3B I proteins in SRA01/04 cells infected by let-7c-3p mimics, let-7c-3p inhibitor, and mimic controls under oxidative stress. (b) The effect of enhanced let-7c-3p on LC3 puncta in SRA01/04 was explored by immunofluorescence. The SRA01/04 cells were treated with 50 μM H₂O₂ for 24 h (^∗P < 0.05).

Figure 4

ATG3 attenuated the autophagy in SRA01/04 cells under oxidative stress. (a, b) Western blot and real-time PCR showed the mRNA and protein expression level of ATG3 in SRA01/04 cells treated with 50 μM H₂O₂ for 0 h and 24 h. (c) Real-time PCR showed the transfection efficiency of ATG3. (d) Western blot was used to analyze the ratio of LC3B II and LC3B I proteins in SRA01/04 cells treated by si-ATG3 and NC group under oxidative stress. (e) The expression of ATG3 mRNA in cataract tissues. (f) The expression of ATG3 protein in cataract tissues was analyzed by western blot (^∗P < 0.05).

Figure 5

Let-7c-3p regulates autophagy by targeting ATG3 in SRA01/04 cells under oxidative stress. (a) Putative let-7c-3p binding site in the 3′UTR region of ATG3. (b, c) The expression levels of ATG3 mRNA and protein regulated by upregulated or downregulated let-7c-3p. Real-time PCR and western blot were used to detect the level of ATG3 mRNA and protein expression. ATG3 expression was downregulated when SRA01/04 cells were transfected with let-7c-3p mimics and upregulated with let-7c-3p inhibitors. (d) Relative luciferase level among wild-type ATG3-3′UTR, mutated ATG3-3′UTR, mimics-NC, let-7c-3p mimics, inhibitor-NC, and let-7c-3p inhibitor. LECs were transfected with let-7c-3p mimics, mimics-NC, inhibitor-NC, let-7c-3p inhibitor, wild-type 3′UTR, and mutated 3′UTR of ATG3. Luciferase activity was analyzed after 48 h. (e) Western blot was used to analyze the expression level of LC3B II and LC3 I proteins in SRA01/04 cells infected by mimic controls, let-7c-3p mimics, and let-7c-3p mimics with pcDNA3.1-ATG3 under oxidative stress. (f) The schematic of let-7c-3p-regulating autophagy by targeting ATG3 in SRA01/04 cells under oxidative stress (^∗P < 0.05).