Apoptosis Of Lens Epithelial Cells Research Articles

Application value of a nano-siRNA delivery system in treatment against senile cataracts by modulating apoptosis in lens epithelial cells

In this study, a novel nano-siRNA delivery system loaded with anti-ESR1 siRNA was designed. In a biological study, the new system was evaluated for the treatment and care of senile cataracts. The system significantly upregulated the expression level of the anti-apoptotic protein BCL-2, while significantly inhibiting the expression level of the inhibitory apoptotic protein BAX, thereby regulating apoptosis in lens epithelial cells. The system can be developed as a drug for the treatment of senile cataracts.

Role of C/EBP Homologous Protein (CHOP) and Nupr1 Interaction in Endoplasmic Reticulum Stress-Induced Apoptosis of Lens Epithelial Cells.

Our study mainly analyzed the mechanism of C/EBP homologous protein (CHOP) and its interacting protein Nupr1 on endoplasmic reticulum stress (ERS) induced lens epithelial cells (LEC) apoptosis. Cell proliferation was detected by CCK-8. Apoptosis was detected by flow cytometry and TUNEL. Nupr1 expressionwas detected byRT-qPCR. The expressions of CHOP, Nupr1, apoptosis-related protein, and ERS-related protein were detected by Western blot. DCFH-DA probe was used to detect cell ROS. The SOD, GSH-PX, and MDA contents were detected by the kit. Co-IP was used to detect the interaction between CHOP and Nupr1. The morphology of the lens was detected by HE staining. The result shows thatTunicamycin (TU) can induce endoplasmic reticulum stress and apoptosis in LEC in a concentration-dependent manner. TU induction leads to the occurrence of CHOP nuclear translocation. Overexpression of CHOP can further enhance the inhibitory effect of TU on LEC proliferation and the promotion of apoptosis, while knockdown of CHOP has the opposite effect. CHOP and Nupr1 are interacting proteins, and knockdown of Nupr1 or addition of Nupr1 inhibitor ZZW-115 can reverse the effects of TU and overexpression of CHOP, respectively. It has been observed in animal experiments that treatment with oe-CHOP can further aggravate the pathological lesions of the rat lens, while ZZW-115 can reverse the effect of oe-CHOP to a certain extent and improve the lesions of the rat lens. Overall,CHOP interacts with Nupr1 to regulate apoptosis caused by ERS and mediate cataract progression in rats, and this study provides a new potential therapeutic target for the treatment of cataract.

Regulation of transforming growth factor β2-induced epithelial-mesenchymal transition and autophagy in lens epithelial cells by the miR-492/NPM1 axis.

A cataract is a clinically common blinding disease closely related to the ageing of the eye cells, which has become a major health killer in the elderly. Our research seeks to analyze the primary targets linked to the pathogenesis of cataracts during the ageing process. We performed bioinformatics analyses on the GSE101727 dataset to discover genes linked with ageing and cataracts. To explore the impacts of Nucleophosmin 1 (NPM1) on cell apoptosis, proliferation, as well as epithelial-mesenchymal transition (EMT) processes, in vitro tests such as western blotting, flow cytometry, and MTT were carried out. Additionally, the study incorporated transforming growth factor β2 (TGF-β2) to examine its function in cellular responses, chloroquine (CQ) to regulate autophagic flow, and H2O2 therapy to mimic oxidative stress. Our study discovered seven ageing-related genes, including NPM1, that had substantial relationships with cataracts. NPM1 overexpression was shown to boost cell proliferation and prevent apoptosis in SRA01/04 cells. Notably, NPM1 modulated the TGF-β signalling pathway, influencing cell proliferation and EMT processes. miR-429 was shown to be adversely regulating NPM1 and autophagy-related proteins, as demonstrated by changes in their expression in response to TGF-β2 treatment. Furthermore, NPM1 knockdown restored autophagy activity suppressed by miR-429 mimics, indicating a complex interaction of miR-429, NPM1, and TGF-β2 pathways in regulating autophagy and EMT. Lens epithelial cell proliferation and apoptosis were largely regulated by NPM1, as well as autophagy and EMT, which were significantly mediated by TGF-β2 and the miR-429/NPM1 axis. These results imply new possible targets for prognosis and therapy of cataracts.

IGF-1 rs6218 polymorphisms modulate the susceptibility to age-related cataract.

Single nucleotide polymorphisms (SNPs), as the most abundant form of DNA variation in the human genome, contribute to age-related cataracts (ARC) development. Apoptosis of lens epithelial cells (LECs) is closely related to ARC formation. Insulin-like growth factor 1 (IGF1) contributes to cell apoptosis regulation. Moreover, IGF1 was indicated to exhibit a close association with cataract formation. Afterward, an investigation was conducted to examine the correlation between polymorphisms in IGF1 and the susceptibility to ARC. The present investigation was a case-control study. Venous blood draws were collected from the participants for DNA genotyping. Lens capsule samples were collected to detect mRNA and apoptosis. TaqMan RT-PCR was used to detect IGF1 polymorphism genotypes and qRT PCR was used to detect IGF1 mRNA levels in LECs. LEC apoptosis was evaluated through flow cytometry. The chi-square test was used to compare differences between ARCs and controls of each SNP. We found that the G allele frequency in the IGF1-rs6218 was higher in the ARCs than in the controls. Furthermore, it was observed that the rs6218 GG genotype exhibited a positive correlation to elevated levels of IGF1 mRNA in LECs. The IGF1 mRNA in the LECs and the apoptosis of LECs in nuclear type of ARCs (ARNC) was higher than the controls. The susceptibility to ARC was related to IGF1-rs6218 polymorphism, and this polymorphism is associated with IGF1 expression at the mRNA level. Moreover, apoptosis in LECs of ARNCs was found to be increased.

The Hsa_circ_0105558/miR-182-5p/ATF6 Cascade Affects H2O2-Triggered Oxidative Damage and Apoptosis of Human Lens Epithelial Cells.

Age-related cataract (ARC) is the prevalent cause of useful vision loss. Circular RNAs are related to ARC pathogenesis partly through their competing endogenous RNA (ceRNA) activity. Herein, we defined the action of hsa_circ_0105558 in hydrogen peroxide (H2O2)-driven apoptosis and oxidative damage in human lens epithelial SRA01/04 cells. Hsa_circ_0105558, microRNA (miR)-182-5p and activating transcription factor 6 (ATF6) were evaluated by a qRT-PCR or immunoblotting method. The hsa_circ_0105558/miR-182-5p and miR-182-5p/ATF6 relationships were predicted by bioinformatics analysis and confirmed by dual-luciferase reporter assay. Reactive oxygen species level, glutathione peroxidase level, superoxide dismutase activity, and malondialdehyde level were measured using the matched assay kits. Hsa_circ_0105558 was upregulated in human ARC lens and H2O2-exposed SRA01/04 cells. Suppression of hsa_circ_0105558 attenuated H2O2-driven SRA01/04 cell apoptosis and oxidative damage. Hsa_circ_0105558 targeted miR-182-5p, and reduced miR-182-5p expression reversed the influence of hsa_circ_0105558 depletion on H2O2-driven oxidative damage and apoptosis. ATF6 was a target of miR-182-5p, and miR-182-5p-driven downregulation of ATF6 regulated cell oxidative damage and apoptosis under H2O2 insult. Moreover, hsa_circ_0105558 functioned as a ceRNA to post-transcriptionally control ATF6 expression through miR-182-5p competition. Our study demonstrates that hsa_circ_0105558 modulates SRA01/04 cell oxidative damage and apoptosis under H2O2 insult through the miR-182-5p/ATF6 cascade.

Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca2+-dependent AMPK/mTOR pathway.

To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells (LECs) under hyperosmotic stress. LECs were treated with hyperosmotic stress at the concentration of 270, 300, 400, 500, or 600 mOsm for 6, 12, 18, 24h in vitro. Polymerase chain reaction (PCR) was employed for the mRNA expression of autophagy-related genes, while Western blotting detected the targeted protein expression. The transfection of stub-RFP-sens-GFP-LC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux. Scanning electron microscopy was used to detect the existence of autolysosome. Short interfering RNA of autophagy-related gene (ATG) 7, transient receptor potential vanilloid (TRPV) 1 overexpression plasmid, related agonists and inhibitors were employed to their influence on autophagy related pathway. Flow cytometry was employed to test the apoptosis and intracellular Ca2+ level. Mitochondrial membrane potential was measured by JC-1 staining. The cell counting kit-8 assay was used to calculate the cellular viability. The wound healing assay was used to evaluate the wound closure rate. GraphPad 6.0 software was utilized to evaluate the data. The hyperosmotic stress activated autophagy in a pressure- and time-dependent manner in LECs. Beclin 1 protein expression and conversion of LC3B II to LC3B I increased, whereas sequestosome-1 (SQSTM1) protein expression decreased. Transient Ca2+ influx was stimulated caused by hyperosmotic stress, levels of mammalian target of rapamycin (mTOR) phosphorylation decreased, and the level of AMP-activated protein kinase (AMPK) phosphorylation increased in the early stage. Based on this evidence, autophagy activation through the Ca2+-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress. Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased. Inhibition of autophagy by ATG7 knockdown had similar results. TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress. A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

CircRNA HLCS regulates lens epithelial cell apoptosis via miR-338-3p/BPNT1 axis.

The purpose of this study was to investigate the effect of circ_HLCS on age-related cataract (ARC). Circ_HLCS, microRNA (miR)-338-3p, and bisphosphate 3'-nucleotidase 1 (BPNT1) were quantified by quantitative real-time polymerase chain reaction or western blot. Cell proliferation and cell viability were assessed by the 5-Ethynyl-2'-deoxyuridinr and cell counting kit-8 assays. Cell apoptosis was detected by flow cytometry. Targeted correlations among circ_HLCS, miR-338-3p, and BPNT1 were verified by the dual-luciferase reporter and RNA pull-down assays. circ_HLCS was diminished in ARC tissues and UV-treated SRA01/04 cells. Elevated content of circ_HLCS undermined UV-induced cell proliferation inhibition and apoptosis. Mechanistically, circ_HLCS directly targeted miR-338-3p, and circ_HLCS regulated BPNT1 expression through miR-338-3p. Furthermore, reduction of miR-338-3p ameliorated UV-induced SRA01/04 cell damage by increasing BPNT1 expression. Taken together, these data suggested that circ_HLCS inhibited apoptosis of UV-treated SRA01/04 cells by miR-338-3p/BPNT1 axis. Therefore, circ_HLCS might be a potential therapeutic target for ARC.

Methyltransferase METTL3-mediated maturation of miR-4654 facilitates high glucose-induced apoptosis and oxidative stress in lens epithelial cells via decreasing SOD2.

N6-methyladenosine (m6 A) modification has been reported to have roles in modulating the development of diabetic cataract (DC). Methyltransferase-like 3 (METTL3) is a critical m6 A methyltransferase involving in m6 A modification activation. Here, we aimed to explore the action and mechanism of METTL3-mediated maturation of miR-4654 in DC progression. Human lens epithelial cells (HLECs) were exposed to high glucose (HG) to imitate DC condition invitro. Levels of genes and proteins were tested via qRT-PCR and western blotting assays. The proliferation and apoptosis of HLECs were evaluated by cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays, respectively. Oxidative stress was analyzed by detecting the contents of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). The binding of miR-4654 and SOD2 was confirmed by dual-luciferase reporter assay. The m6 A-RNA immunoprecipitation (MeRIP) assay detected the m6 A modification profile. Thereafter, we found that miR-4654 expression was elevated in DC samples and HG-induced HLECs. MiR-4654 knockdown reversed HG-mediated apoptosis and oxidative stress in HLECs. Mechanistically, miR-4654 directly targeted SOD2, silencing of SOD2 abolished the protective effects of miR-4654 knockdown on HLECs under HG condition. In addition, METTL3 induced miR-4654 maturation through promoting pri-miR-4654 m6 A modification, thereby increasing miR-4654 content in HLECs. METTL3 was highly expressed in DC samples and HG-induced HLECs, METTL3 deficiency protected HLECs against HG-mediated apoptotic and oxidative injury via down-regulating miR-4654. In all, METTL3 induced miR-4654 maturation in a m6 A-dependent manner, which was then reduced SOD2 expression, thus promoting apoptosis and oxidative stress in HLECs, suggesting a novel path for DC therapy.

Inhibition of miR-29a-3p Alleviates Apoptosis of Lens Epithelial Cells via Upregulation of CAND1

Purpose Accumulated evidence has shown that microRNAs (miRNAs) are closely related to the pathogenesis and progression of senile cataracts. Here we investigate the effect of miR-29a-3p in cataractogenesis and determined the potential molecular mechanism involved. Methods In this study, we constructed a selenite cataract model in rats and obtained the miRNAs related to cataracts by whole transcriptome sequencing. To investigate the effect and mechanism of miR-29a-3p on cataracts, we performed several in vivo and in vitro experiments, including CCK8 assay, flow cytometry, luciferase reporter assay, Edu assay, and western blot analysis. Result Sequencing data showed downregulation of miR-29a-3p in rats with selenite cataracts. Down-regulation of miR-29a-3p could promote lens epithelial cells (SRA01/04) proliferation and inhibit cell apoptosis, and miR-29a-3p silence could inhibit the development of cataracts. Additionally, CAND1 was a direct target gene for miR-29a-3p. Conclusion These data demonstrate that miR-29a-3p inhibits apoptosis of lens epithelial cells by regulating CAND1, which may be a potential target for senile cataracts.

JAM-C Is Important for Lens Epithelial Cell Proliferation and Lens Fiber Maturation in Murine Lens Development.

The underlying mechanism of congenital cataracts caused by deficiency or mutation of junctional adhesion molecule C (JAM-C) gene remains unclear. Our study aims to elucidate the abnormal developmental process in Jamc-/- lenses and reveal the genes related to lens development that JAM-C may regulate. Jamc knockout (Jamc-/-) mouse embryos and pups were generated for in vivo studies. Four key developmental stages from embryonic day (E) 12.5 to postnatal day (P) 0.5 were selected for the following experiments. Hematoxylin and eosin staining was used for histological analysis. The 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and TUNEL staining were performed to label lens epithelial cell (LEC) proliferation and apoptosis, respectively. Immunofluorescence and Western blot were used to analyze the markers of lens epithelium, cell cycle exit, and lens fiber differentiation. JAM-C was expressed throughout the process of lens development. Deletion of Jamc resulted in decreased lens size and disorganized lens fibers, which arose from E16.5 and aggravated gradually. The LECs of Jamc-/- lenses showed decreased quantity and proliferation, accompanied with reduction of key transcription factor, FOXE3. The fibers in Jamc-/- lenses were disorganized. Moreover, Jamc-deficient lens fibers showed significantly altered distribution patterns of Cx46 and Cx50. The marker of fiber homeostasis, γ-crystallin, was also decreased in the inner cortex and core fibers of Jamc-/- lenses. Deletion of JAM-C exhibits malfunction of LEC proliferation and fiber maturation during murine lens development, which may be related to the downregulation of FOXE3 expression and abnormal localization patterns of Cx46 and Cx50.

Epigallocatechin gallate delays age-related cataract development via the RASSF2/AKT pathway

Age-related cataract (ARC) is a common eye disease, the main cause of which is oxidative stress-mediated apoptosis of lens epithelial cells (LECs). Epigallocatechin gallate (EGCG) is the most potent antioxidant in green tea. Our results demonstrated that EGCG could effectively reduce apoptosis of LECs and retard lens clouding in aged mice. By comparing transcriptome sequencing results of three groups of mice (young control, untreated aged, and EGCG-treated) and screening using GO and KEGG analyses, we selected RASSF2 as the effector gene of EGCG for mechanistic exploration. We verified that the differential expression of RASSF2 was associated with the occurrence of ARC in clinical samples and mouse tissues by immunohistochemistry and western blotting, respectively. We showed that high RASSF2 expression plays a crucial role in the oxidative induction of apoptosis in LECs, as revealed by overexpression and interference experiments. Further studies showed that RASSF2 mediates the inhibitory effect of EGCG on apoptosis and ARCogenesis in LECs by regulating AKT (Ser473) phosphorylation. In this study, we found for the first time the retarding effect of EGCG on lens clouding in mice and revealed the mechanism of action of RASSF2/AKT in it, which provides a theoretical basis for the targeted treatment of EGCG.

LncRNA TUG1 regulates Smac/DIABLO expression by competitively inhibiting miR-29b and modulates the apoptosis of lens epithelial cells in age-related cataracts.

As one of the early discovered long non-coding RNAs (lncRNA), taurine upregulation gene 1 (TUG1) has been widely expressed in a variety of tumors. Moreover, it promotes cell proliferation, differentiation, apoptosis, and migration. However, our understanding of its importance in the pathogenesis of cataracts remains limited. This study aimed to explore the mechanism by which lncRNA TUG1 mediates lens epithelial cell apoptosis in age-related cataracts (ARC) by regulating the microRNAs (miR-29b)/second mitochondria-derived activator of caspases axis, and to identify more non-surgical strategies for cataract treatment. The messenger RNA expression levels of TUG1, miR-29b, and Smac were detected using quantitative real-time polymerase chain reaction in vivo and in vitro. The expression of the Smac protein was analyzed by Western blotting and immunofluorescence. Flow cytometry and cell counting kit-8 assays were used to detect the cell apoptosis and proliferation rates, respectively. The targeted regulatory relationship between lncRNA TUG1, miR-29b, and Smac was verified by viral vector construction, co-transfection, nuclear and cytoplasmic separation, luciferase reporter assays, and RNA immunoprecipitation. TUG1 and Smac were expressed at high levels in ARC and HLE-B3 cells treated with 200 μmol/L H2O2, whereas miR-29b expression was decreased. In vitro cell experiments confirmed that down-regulation of TUG1 could inhibit the apoptosis of lens epithelial cells. Mechanistically, Smac expression was negatively regulated by miR-29b. TUG1 competitively inhibited miR-29b expression and caused greater release of Smac. In addition, miR-29b partially reversed the effects of TUG1 on human lens epithelial cell line cells. lncRNA TUG1 increases Smac expression and promotes apoptosis of lens epithelial cells in ARC by competitively inhibiting miR-29b. This mechanism is the cytological basis for ARC formation. Based on these results, the lncRNA TUG1/miR29b/Smac axis may be a new molecular pathway that regulates ARC development.

Current Concepts of Molecular Mechanisms of Age-Related Changes in Eye Lens

Age-related cataract is the leading cause of vision loss in old people worldwide. According to the World Health Organization it accounts for 47.8 % of the total number of ocular pathologies in people over 50 years old. Despite the rapid development of cataract surgery technology, surgery remains a challenge due to its cost and the increasing number of patients. Literature review is devoted to current concepts of pathogenesis and molecular mechanisms of age-related changes in eye lens. There are the three main theories of cataractogenesis: oxidative stress; the impact of quinoid substances, which are formed due to the impairment of aromatic amino acid metabolism and the activation of aldo reductase enzyme with subsequent accumulation of sorbitol, reactive oxygen species (ROS) generation, dysfunction of Na+/K+ channels and calcium deregulation causing lens epithelial cells apoptosis. Theories of pathogenesis are linked and based on the development of age-related changes in protein metabolism (the majority of nuclear α-crystallins are insoluble), glucose metabolism (non-enzymatic glycosylation of proteins), lipid metabolism, enzyme activity and the loss of membrane potential of cells ( the increase of Na+ and Ca2+ level and the decrease of K+ level). Key element of all theories of age-related changes in eye lens is the aggregation of high molecular weight proteins covalent-bonded of disulfide linkages. Based on molecular mechanisms of cataractogenesis, the development of pathogenetically oriented medical methods of correction of the age-related changes in lens is carried on. This review provides information on results of experimental and clinical studies which demonstrate the anti-cataract effect of Pirenoxine 0, 005 %.

Upregulation of EphA2 is associated with apoptosis in response to H2O2 and UV radiation-induced cataracts

In this article, we examine the role of erythropoietin-producing hepatocellular receptor A2 (EphA2) in the apoptosis of lens epithelial cells (LECs) in H2O2 and UV radiation-induced cataracts. We treated SRA01/04 cells with H2O2 or ultraviolet (UV) radiation to create a cataract cell model. We constructed a cataract lens model by exposing mice to UV radiation. We used CCK8 assays, Annexin V-FITC analysis, and immunohistochemical staining to explore proliferation and apoptosis of the cataract model. Thereafter, we used quantitative real-time PCR (qPCR) analysis, Western blot assays, and immunofluorescence to determine gene and protein expression levels. We also employed Crispr/Cas9 gene editing to create an EphA2 knockout in SRA01/04 cells. Results: H2O2 or UV radiation induced SRA01/04 cells showed EphA2 gene upregulation. CCK8 and apoptosis assays showed that EphA2 over-expression (OE) reduced epithelial cell apoptosis, but knockout of EphA2 induced it in response to H2O2 and UV radiation, respectively. Mutation of the EphA2 protein kinase domain (c.2003G > A, p. G668D) had a limited effect on cell apoptosis. In vivo, the EphA2 protein level increased in the lenses of UV-treated mice. Our results showed that EphA2 was upregulated in SRA01/04 cells in response to H2O2 and UV radiation. Mutation of the EphA2 protein kinase domain (c.2003G > A, p. G668D) had a limited effect on H2O2 and UV radiation-induced cell apoptosis. We confirmed this change with an experiment on UV-treated mice. The present study established a novel association between EphA2 and LEC apoptosis.

SUMO1-regulated DBC1 promotes p53-dependent stress-induced apoptosis of lens epithelial cells.

Deleted in breast cancer 1 (DBC1) was initially identified from a homozygously deleted region in human chromosome 8p21. It has been well established that DBC1 plays a dual role during cancer development. Depending on the physiological context, it can promote or inhibit tumorigenesis. Whether it plays a role in lens pathogenesis remains elusive. In the present study, we demonstrated that DBC1 is highly expressed in lens epithelial cells from different vertebrates and in retina pigment epithelial cells as well. Moreover, DBC1 is SUMOylated through SUMO1 conjugation at K591 residue in human and mouse lens epithelial cells. The SUMOylated DBC1 is localized in the nucleus and plays an essential role in promoting stress-induced apoptosis. Silence of DBC1 attenuates oxidative stress-induced apoptosis. In contrast, overexpression of DBC1 enhances oxidative stress-induced apoptosis, and this process depends on p53. Mechanistically, DBC1 interacts with p53 to regulate its phosphorylation status at multiple sites and the SUMOylation of DBC1 enhances its interaction with p53. Together, our results identify that DBC1 is an important regulator mediating stress-induced apoptosis in lens, and thus participates in control of lens cataractogenesis.

Ultraviolet B radiation induces oxidative stress and apoptosis in human lens epithelium cells by activating NF-κB signaling to down-regulate sodium vitamin C transporter 2 (SVCT2) expression

ABSTRACT Ultraviolet B (UVB) exposure is reported to cause cataract formation by inducing excessive reactive oxygen species (ROS) and apoptosis in human lens epithelial cells (HLECs). Sodium-dependent Vitamin C transports-2 (SVCT2) is a ascorbic acid (AsA) transporter for that can protect cells and tissues from oxidative stress. Here, we focus on the functional characterization and mechanism analysis of SVCT2 in UVB-treated HLECs. The results showed a significant reduction of SVCT2 expression in HLECs treated with UVB. SVCT2 abated apoptosis and Bax expression and increased Bcl-2 expression. Moreover, SVCT2 decreased ROS accumulation and MDA level, but increased the activities of antioxidant enzymes (SOD and GSH-PX). NF-κB inhibitor (PDTC) alleviated ROS production and apoptosis, and promoted SVCT2 expression in UVB-treated HLECs. Additionally, ROS inhibitor (NAC) suppressed oxidative stress, apoptosis, and induced SVCT2 expression in UVB-treated HLECs, while these effects were significantly abated due to the activation of NF-κB signaling. Furthermore, SVCT2 facilitated 14C-AsA absorption in UVB-treated HLECs. Together, our findings demonstrated that UVB exposure-induced ROS generation, which further activated NF-κB signaling to down-regulate SVCT2 expression in HLECs. Then, downregulated SVCT2 promoted ROS accumulation and induced apoptosis by decreasing AsA uptake. Our data reveal a novel NF-κB/SVCT2/AsA regulatory pathway and suggest the therapeutic potential of SVCT2 in UVB-induced cataract.

Long non-coding RNA nuclear paraspeckle assembly transcript 1 downregulation protects lens epithelial cells from oxidative stress-induced apoptosis by regulating the microRNA-124-3p/death-associated protein kinase 1 axis in age-related cataract.

Oxidative stress plays a significant role in cataract development. It causes the apoptosis of lens epithelial cells (LECs), resulting in lens opacification and accelerating cataract progression. Long non-coding RNAs (lncRNAs) and microRNAs have been linked to cataract development. Notably, lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in LEC apoptosis and cataract formation. However, the molecular mechanism by which NEAT1 causes age-related cataracts remains unknown. In this study, LECs (SRA01/04) were exposed to 200μM H2O2 to generate an in vitro cataract model. The apoptosis and viability of cells were determined using flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assays, respectively. Additionally, western blotting and quantitative polymerase chain reaction were used to determine the miRNA and lncRNA expression levels. When LECs were treated with hydrogen peroxide, lncRNA NEAT1 expression levels were significantly upregulated, which contributed to LEC apoptosis. Notably, lncRNA NEAT1 suppressed the expression of miR-124-3p, a critical regulator of apoptosis, whereas NEAT1 inhibition increased miR-124-3p expression and alleviated apoptosis. However, this effect was reversed when miR1243p expression was inhibited. Additionally, the miR1243p mimic effectively inhibited the death-associated protein kinase 1 (DAPK1) expression and apoptosis of LECs, while the DAPK1 mimic reversed these effects. In conclusion, our findings indicate that the lncRNA NEAT1/miR-124-3p/DAPK1 signaling loop is involved in the regulation of LEC apoptosis induced by oxidative stress, which can be exploited to develop potential treatment strategies for age-related cataracts.

MicroRNA-22-3p Regulates the Apoptosis of Lens Epithelial Cells Through Targeting KLF6 in Diabetic Cataracts.

The purpose of this study was to identify novel abnormally expressed microRNAs (miRNAs) and their downstream target in diabetic cataract (DC). General feature, fasting blood glucose, glycosylated hemoglobin, and type A1c (HbA1c) expression level of patients were collected. DC capsular tissues were obtained from patients and the lens cells (HLE-B3) exposed to different concentrations of glucose were used to simulate the model in vitro. Both mimic and inhibitor of miR-22-3p were transferred into HLE-B3 to up- and downregulate miR-22-3p expression, respectively. The cellular apoptosis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence. The downstream target gene of miR-22-3p was identified by dual luciferase reporter. In DC capsules and HLE-B3 under hyperglycemia, miR-22-3p showed a significant downward trend. The expression of BAX was upregulated and the BCL-2 was downregulated following high glucose. The expression of BAX was significantly down- or upregulated in HLE-B3 cells following transfection of mimic or inhibitor of miR-22-3p, respectively. Conversely, BCL-2 was significantly increased or decreased. Dual luciferase reporter assay showed that miR-22-3p directly targeted Krüppel Like Factor 6 (KLF6) to regulate cell apoptosis. In addition, the expression of KLF6 were significantly up- or downregulated following transfection of inhibitor or mimic of miR-22-3p. This study suggested that miR-22-3p could inhibit lens apoptosis by targeting KLF6 directly under high glucose condition. The miR-22-3p/KLF6 signal axis may provide novel insights into the pathogenesis of DC. Differential expression of miR-22-3p may account for the pathogenesis of DC and lead to a new therapeutic strategy for DC.

Ubiquitination of Ku70 by Parkin promotes apoptosis of lens epithelial cells.

Oxidative damage-triggered apoptosis in lens epithelial cells is considered as a main risk factor in the pathogenesis of age-related cataracts. Ku70 is a key factor in the DNA repair process of double-strand breaks. In the present study, we aimed to investigate the role of Ku70 and its related E3 ubiquitin ligase in lens epithelial cell apoptosis. The levels of Ku70 in the anterior lens capsules of human cataracts and Emory mice were lower compared to controls. H2 O2 treatment resulted in decreased expression of Ku70 through accelerating Ku70 ubiquitination. Parkin, an E3 ubiquitin ligase, could interact with Ku70 and promote the ubiquitination and degradation of this protein. In addition, ubiquitinated Ku70 was regulated by ubiquitin-proteasome, autophagy-lysosome and mitophagy pathways. Ectopic expression of Ku70 protected SRA01/04 cells from H2 O2 -induced apoptosis, whereas silencing Ku70 exhibited the opposite trend. Co-transfected with Parkin non-ubiquitinatable Ku70 mutant could maintain its anti-apoptosis ability, whereas wild-type Ku70 failed. Moreover, Ku70 might facilitate mitochondrial fusion by increasing the expression of Mitofusin 1/2. The present study revealed that Parkin-mediated Ku70 ubiquitination facilitated H2 O2 -induced lens epithelial cell apoptosis through alleviating mitochondrial fusion, which could provide potential targets for age-related cataract treatment.

Glutaredoxin 1 protects lens epithelial cells from epithelial-mesenchymal transition by preventing casein kinase 1α S-glutathionylation during posterior capsular opacification

Oxidative stress drives protein S-glutathionylation, which regulates the structure and function of target proteins and is implicated in the pathogenesis of many diseases. Glutaredoxin 1 (Grx1), a cytoplasmic deglutathionylating enzyme, maintains a reducing environment within the cell under various conditions by reversing S-glutathionylation. Grx1 performs a wide range of antioxidant activities in the lens and prevents protein-thiol mixed disulfide accumulation, reducing protein–protein aggregation, insolubilization, and apoptosis of lens epithelial cells. Oxidative stress is related to epithelial–mesenchymal transition (EMT) during posterior capsular opacification (PCO). However, whether Grx1-regulated protein S-glutathionylation plays an essential role in PCO remains unclear. In this study, we revealed that Grx1 expression was decreased in mice following cataract surgery. Furthermore, the absence of Grx1 elevated oxidative stress and protein S-glutathionylation and aggravated EMT in both in vitro and in vivo models. Concurrently, these results could be reversed by Grx1 overexpression. Notably, liquid chromatography–tandem mass spectrometry results showed that casein kinase 1α (CK1α) was susceptible to S-glutathionylation under oxidative stress, and CK1α S-glutathionylation (CK1α-SSG) was mediated at Cys249. The absence of Grx1 upregulated CK1α-SSG, subsequently decreasing the CK1α-induced phosphorylation of β-catenin at Ser45. The consequential downregulation of degradative β-catenin and upregulation of its nuclear translocation activated the Wnt/β-catenin signaling pathway and aggravated EMT. In conclusion, the downregulated expression of Grx1 in mice following cataract surgery aggravated EMT by upregulating the extent of CK1α-SSG. To the best of our knowledge, our study is the first to report how S-glutathionylation regulates CK1α activity under oxidative stress.