Thioredoxin Binding Protein-2 Regulates Autophagy of Human Lens Epithelial Cells under Oxidative Stress via Inhibition of Akt Phosphorylation

Oxidative stress plays an essential role in the development of age-related cataract. Thioredoxin binding protein-2 (TBP-2) is a negative regulator of thioredoxin (Trx), which deteriorates cellular antioxidant system. Our study focused on the autophagy-regulating effect of TBP-2 under oxidative stress in human lens epithelial cells (LECs). Human lens epithelial cells were used for cell culture and treatment. Lentiviral-based transfection system was used for overexpression of TBP-2. Cytotoxicity assay, western blot analysis, GFP/mCherry-fused LC3 plasmid, immunofluorescence, and transmission electronic microscopy were performed. The results showed that autophagic response of LECs with increased LC3-II, p62, and GFP/mCherry-LC3 puncta (P<0.01) was induced by oxidative stress. Overexpression of TBP-2 further strengthens this response and worsens the cell viability (P<0.01). Knockdown of TBP-2 attenuates the autophagic response and cell viability loss induced by oxidative stress. TBP-2 mainly regulates autophagy in the initiation stage, which is mTOR-independent and probably caused by the dephosphorylation of Akt under oxidative stress. These findings suggest a novel role of TBP-2 in human LECs under oxidative stress. Oxidative stress can cause cell injury and autophagy in LECs, and TBP-2 regulates this response. Hence, this study provides evidence regarding the role of TBP-2 in lens and the possible mechanism of cataract development.

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The role of Trx/TBP-2 in oxidative stress induced autophagy in human lens epithelial cells

10.21203/rs.2.17897/v1 ◽

2019 ◽

Author(s):

Jianghua Hu ◽

Lifang Shen ◽

Chengshou Zhang ◽

Ke Yao ◽

Yibo Yu

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Cellular Response ◽

Negative Regulator ◽

Cell Counting ◽

Human Lens ◽

Lens Epithelial Cells ◽

Mrna Levels ◽

Human Lens Epithelial Cells

Abstract Background: Oxidative stress plays an important role in age-related cataract development. The cellular antioxidant protein thioredoxin (Trx) and its negative regulator, thioredoxin binding protein-2 (TBP-2), maintain the intracellular redox balance upon oxidative stress. The aim of this study is to investigate role of Trx and TBP-2 in human lens epithelial cells (LECs) under oxidative stress. Methods: LECs were treated with 50 μM of H2O2 serum-free medium for different duration, and the mRNA and protein levels of Trx-1, Trx-2 and TBP-2 were measured by reverse transcription-polymerase chain reaction and Western blot. Trx-1 activity was evaluated by Thioredoxin Activity Fluorescent Assay. The subcellular localization of Trx-1, Trx-2 and TBP-2 was evaluated by cellular immunofluorescence. The cell viability was detected by Cell Counting Kit-8 (CCK-8) and the LC3-II protein level was detected to evaluate the autophagy level. The interaction between Trx-2 and TBP-2 was examined by Co-Immunoprecipitation (Co-IP). Results: The results showed that the mRNA levels of the Trx-1, Trx-2 and TBP-2 were kinetically changed after treatment with 50 μM of H2O2 for different duration. Exposure to H2O2 increased the expression of Trx-2 and TBP-2 but not Trx-1, while the exposure inhibited Trx-1 activity. TBP-2 was co-localized with Trx-1 and Trx-2, exposure to H2O2 enriched co-localization of TBP-2 to Trx-1 but not Trx-2, and increased the interaction between TBP-2 and Trx-1. Under normal circumstances, Trx-1 over-expression enhanced autophagic response and mainly regulates autophagy in the initiation. Conclusions: This study demonstrates differential roles of Trx-1 and Trx-2 in cellular response to oxidative stress, and oxidative stress increased Trx-1 interacting to TBP-2 and Trx-1 regulating autophagic response.

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Long Non-Coding RNA H19 Regulates Human Lens Epithelial Cells Function

Cellular Physiology and Biochemistry ◽

10.1159/000494003 ◽

2018 ◽

Vol 50 (1) ◽

pp. 246-260 ◽

Cited By ~ 6

Author(s):

Xin Liu ◽

Chang Liu ◽

Kun Shan ◽

Shujie Zhang ◽

Yi Lu ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Differentially Expressed ◽

Human Lens ◽

Lens Epithelial Cells ◽

Double Staining ◽

Human Lens Epithelial Cells ◽

And Migration ◽

Proliferation And Migration

Background/Aims: Age-related cataract (ARC) remains the leading cause of visual impairment among the elderly population. Long non-coding RNAs (lncRNAs) have emerged as potential regulators in many ocular diseases. However, the role of lncRNAs in nuclear ARC, a subtype of ARC, requires further elucidation. Methods: LncRNA sequencing was performed to identify differentially expressed lncRNAs between the capsules of transparent and nuclear ARC lenses. Expression validation was confirmed by qRT-PCR. MTT assay, Calcein-AM and propidium iodide double staining, Rhodamine 123 and Hoechst double staining, EdU and transwell assay were used to determine the role of H19 or miR-675 in the viability, apoptosis, proliferation and migration of primary cultured human lens epithelial cells (HLECs). Bioinformatics and luciferase reporter assays were used to identify the binding target of miR-675. Results: Sixty-three lncRNAs are differentially expressed between the capsules of transparent and nuclear ARC lenses. One top abundantly expressed lncRNA, H19, is significantly up-regulated in the nuclear ARC lens capsules and positively associated with nuclear ARC grade. H19 knockdown accelerates apoptosis development and reduces the proliferation and migration of HLECs upon oxidative stress. H19 is the precursor of miR-675, and a reduction of H19 inhibits miR-675 expression. miR-675 regulates CRYAA expression by targeting the binding site within the 3’UTR. Moreover, miR-675 increases the proliferation and migration while decreasing the apoptosis of HLECs upon oxidative stress. Conclusion: H19 regulates HLECs function through miR-675-mediated CRYAA expression. This finding would provide a novel insight into the pathogenesis of nuclear ARC.

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Role of Aldehyde Dehydrogenase Isozymes in the Defense of Rat Lens and Human Lens Epithelial Cells against Oxidative Stress

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.04-0120 ◽

2005 ◽

Vol 46 (1) ◽

pp. 259 ◽

Cited By ~ 49

Author(s):

Sanjeev Choudhary ◽

Tianlin Xiao ◽

Leoncio A. Vergara ◽

Sanjay Srivastava ◽

David Nees ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Aldehyde Dehydrogenase ◽

Human Lens ◽

Lens Epithelial Cells ◽

Human Lens Epithelial Cells

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Metformin alleviates oxidative stress‐induced senescence of human lens epithelial cells via AMPK activation and autophagic flux restoration

Journal of Cellular and Molecular Medicine ◽

10.1111/jcmm.16797 ◽

2021 ◽

Author(s):

Mengmeng Chen ◽

Chunmei Zhang ◽

Nan Zhou ◽

Xu Wang ◽

Dongmei Su ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Human Lens ◽

Lens Epithelial Cells ◽

Autophagic Flux ◽

Ampk Activation ◽

Human Lens Epithelial Cells

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Mitochondria-Targeted Antioxidant Peptide SS31 Protects Cultured Human Lens Epithelial Cells against Oxidative Stress

Current Eye Research ◽

10.3109/02713683.2014.959607 ◽

2014 ◽

Vol 40 (8) ◽

pp. 822-829 ◽

Cited By ~ 13

Author(s):

Meng Cai ◽

Jing Li ◽

Shaofen Lin ◽

Xiaoyun Chen ◽

Juan Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Human Lens ◽

Lens Epithelial Cells ◽

Antioxidant Peptide ◽

Human Lens Epithelial Cells

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

BioMed Research International ◽

10.1155/2020/6069390 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Ting Li ◽

Yanhong Huang ◽

Wenkai Zhou ◽

Qichang Yan

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Western Blot ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Age Related ◽

Spot Number ◽

Gene Assay

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.

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