HLE-B3 Cells Research Articles

The Effects of Dexamethasone on Human Lens Epithelial Cells and the Analysis of Related Pathways with Transcriptome Sequencing.

The goal of this study was to investigate the effects of dexamethasone on human lens epithelial cells (HLECs) and the potential mechanisms. HLECs (HLE-B3) were cultured in vitro to assess the effects of dexamethasone on cell size at different concentrations. Immunofluorescence staining was used to detect specific protein expression in HLE-B3 cells. The cell size was observed using phase-contrast microscopy, and the length and area were quantitatively measured with ImageJ software for statistical analysis. Flow cytometry was used to verify these outcomes. The means of three groups were statistically analyzed using one-way analysis of variance, whereas the means of two groups were statistically analyzed with the parametric Student's t-test. Additionally, high-throughput transcriptome sequencing was performed to compare messenger RNA (mRNA) expression levels between different concentrations of dexamethasone treatment groups and the control group, to identify potential signaling pathways. Subsequently, we performed quantitative Polymerase Chain Reaction (qPCR), immunofluorescence staining, and molecular docking experiments on the key differentially expressed genes. Dexamethasone affected the size of HLE-B3 cells. Both 0.25 and 0.5 μmol/L dexamethasone increased cell length and area, exhibiting no significant difference between the two treatment groups. Flow cytometry showed that dexamethasone increased cell size and granularity, with 0.25 μmol/L dexamethasone leading to larger cell areas and higher intracellular granularity. High-throughput transcriptome sequencing revealed significant upregulation of lysophosphatidic acid receptor 1 (LPAR1) and the pathways related to the glucocorticoid (GC) receptor. Certain concentrations of dexamethasone impact the morphology and biological functions of HLECs. As a subtype of G protein-coupled receptors, LPAR1 on the cell membrane may interact with dexamethasone, affecting cell size and inhibiting autophagy via the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. These discoveries offer crucial biological insights into how dexamethasone influences the morphology and function of HLECs and the pathogenesis of GC-induced cataracts, offering potential molecular targets for future therapeutic strategies.

Effects of MMP2 and its inhibitor TIMP2 on DNA damage, apoptosis and senescence of human lens epithelial cells induced by oxidative stress.

Oxidative stress-induced lens epithelial cells (LECs) death plays a pivotal role in pathogenesis of age-related cataract (ARC), causing significant visual impairment. Apoptosis of porcine granulosa cells mediated by MMP2 is linked to DNA damage. The current study aimed to investigate the potential mechanism of MMP2 in DNA damage, apoptosis and senescence of lens epithelial cells caused by oxidative stress. HLE-B3 cells were treated with different doses of H2O2 for 24h, and CCK-8 was used to detect cell viability. Furthermore, western blotting was used to detect the expressions of MMP2, Bcl2, Bax, cleaved caspase3, γ-H2AX, p16, p21, and TIMP2. DCFH-DA staining was used to assess ROS levels. Moreover, EdU staining was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis. Then, 15A3 immunofluorescence staining and γ-H2AX staining were used to detect DNA damage. In addition, SA-β-gal staining was used to observe cell senescence. The present findings suggest that oxidative stress triggers damage to LECs viability and elevates the expression of MMP2. Furthermore, MMP2 interference attenuates H2O2-induced active damage, apoptosis, DNA damage, and cellular senescence in LECs. Additionally, TIMP2 expression is down-regulated in H2O2-induced LECs, which suppresses the expression of MMP2 induced by H2O2. These findings highlight the crucial role of MMP2 and TIMP2 in the modulation of oxidative stress-induced cellular responses in LECs. Collectively, TIMP2 alleviates H2O2-induced lens epithelial cell viability damage, apoptosis, DNA damage and cell senescence in LECs by inhibiting MMP2.

Mitofusin 2 inhibits high glucose-induced apoptosis of human lens epithelial cells via modulating mitochondrial function and autophagy.

Diabetic cataract (DC) is a common ocular complication of diabetes. Mitofusin 2 (MFN2), a mitochondrial fusion protein, is involved in the pathogenesis of cataract and diabetic complications. However, its role and molecular mechanisms in DC remain unclear. DC models in rats were induced by intraperitoneal injection of streptozocin (STZ) for 12 weeks. We measured the body weight of rats, blood glucose concentrations, sorbitol dehydrogenase (SDH) activity and advanced glycation end products (AGE) content in the lenses of rats. MFN2 mRNA and protein expression levels in the lenses were detected by RT-qPCR and western blot assays. In vitro, human lens epithelial (HLE) B3 cells were treated for 48 h with 25 mM glucose (high glucose, HG) to induce cell damage. To determine the role of MFN2 in HG-induced cell damage, HLE-B3 cells were transfected with lentivirus loaded with MFN2 overexpression plasmid or short hairpin RNA (shRNA) to overexpress or knock down MFN2 expression, followed by HG exposure. Cell viability was assessed by CCK-8 assay. Flow cytometry was used to detect cell apoptosis and reactive oxygen species (ROS) level. JC-1 staining showed the changes in mitochondrial membrane potential (Δψm). The mediators related to apoptosis, mitochondrial damage, and autophagy were determined. STZ-administrated rats showed reduced body weight, increased blood glucose levels, elevated SDH activity and AGE content, suggesting successful establishment of the DC rat model. Interestingly, MFN2 expression was significantly downregulated in DC rat lens and HG-induced HLE-B3 cells. Further analysis showed that under HG conditions, MFN2 overexpression enhanced cell viability and inhibited apoptosis accompanied by decreased Bax, cleaved caspase-9 and increased Bcl-2 expression in HLE-B3 cells. MFN2 overexpression also suppressed the mitochondrial damage elicited by HG as manifested by reduced ROS production, recovered Δψm and increased mitochondrial cytochrome c (Cyto c) level. Moreover, MFN2 overexpression increased LC3BⅡ/LC3BⅠ ratio and Beclin-1 expression, but decreased p62 level, and blocked the phosphorylation of mTOR in HG-treated HLE-B3 cells. In contrast, MFN2 silencing exerted opposite effects. Presented findings indicate that MFN2 expression may be essential for preventing lens epithelial cell apoptosis during development of diabetic cataract.

Truncation mutations of CRYGD gene in congenital cataracts cause protein aggregation by disrupting the structural stability of γD-crystallin

Congenital cataracts, a prevalent cause of blindness in children, are associated with protein aggregation. γD-crystallin, essential for sustaining lens transparency, exists as a monomer and exhibits excellent structural stability. In our cohort, we identified a nonsense mutation (c.451_452insGACT, p.Y151X) in the CRYGD gene. To explore the effect of truncation mutations on the structure of γD-crystallin, we examined the Y151X and T160RfsX8 mutations, both located in the Greek key motif 4 at the cellular and protein level in this study. Both truncation mutations induced protein misfolding and resulted in the formation of insoluble aggregates when overexpressed in HLE B3 and HEK 293T cells. Moreover, heat, UV irradiation, and oxidative stress increased the proportion of aggregates of mutants in the cells. We next purified γD-crystallin to estimate its structural changes. Truncation mutations led to conformational disruption and a concomitant decrease in protein solubility. Molecular dynamics simulations further demonstrated that partial deletion of the conserved domain within the Greek key motif 4 markedly compromised the overall stability of the protein structure. Finally, co-expression of α-crystallins facilitated the proper folding of truncated mutants and mitigated protein aggregation. In summary, the structural integrity of the Greek key motif 4 in γD-crystallin is crucial for overall structural stability.

Association of CUL4B with the pathogenesis of age-related cataract.

Age-related cataract (ARC) is the most common cause of visual impairment and blindness in older adults. However, the role of CUL4B in the ARC remains unclear. Therefore, we investigated CUL4B expression and its effects on apoptosis. CUL4B expression levels were detected by a quantitative real-time polymerase chain reaction from the anterior lens capsules of patients with ARC and HLE-B3 cells treated with different concentrations of H2O2. CUL4B expression was silenced by siRNA transfection to evaluate apoptosis. CUL4B and apoptotic proteins B cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), caspase-3, cleaved caspase-3, Bax, Bak, and Bid were assessed using western blot analysis. Apoptosis was monitored using the TUNEL assay. CUL4B expression was downregulated in the anterior lens capsules (P < 0.0001) and H2O2-treated HLE-B3 cells (P = 0.0405). CUL4B protein levels were significantly lower in 100µmol/L (P = 0.0012) and 200µmol/L (P = 0.0041) H2O2-treated HLE-B3 cells than in the untreated cells. CUL4B expression was significantly knocked down at the mRNA (P = 0.0043) and protein levels (P = 0.0002) in HLE-B3 cells. Bcl-2 (P = 0.0199), Mcl-1 (P = 0.0042), and caspase-3 (P = 0.0142) were significantly downregulated, whereas cleaved caspase-3 (P = 0.0089) and Bak (P = 0.009) were significantly upregulated in the knockdown group. The TUNEL assay showed a greater induction of apoptosis. CUL4B downregulation promotes the apoptosis of lens epithelial cells. Our study may help in understanding the role of CUL4B in ARC pathogenesis.

TRPM3-mediated inhibition of active Na-K transport by the ocular lens epithelium

Purpose: The TRPM3 channel is selectively expressed in the lens epithelium, a monolayer at the anterior lens surface, but undetectable in fiber cells that make up the lens bulk. A point mutation in TRPM3 gene (substitution of isoleucine by methionine at codon 65; I65M) causes early onset autosomal dominant cataract in humans and in mice. From studies on transgenic mice, it is apparent the I65M TRPM3 mutation disrupts lens Na-K homeostasis. However, the normal role of lens TRPM3 in the lens is currently unknown. Here, we explore the lens response to TRPM3 activation. Methods: Studies were carried out using intact mouse lenses, cultured mouse lens epithelial cells and a human lens epithelium cell line (HLEB3). Active Na-K transport was studied by using rubidium (Rb) as a tracer for potassium entry. Rb was detected by atomic absorption spectrophotometry. Intracellular calcium was measured in nonconfluent single cells by Fura-2 ratiometric imaging. Results: Rb uptake was measured over a period of 10 min in intact mouse lenses exposed to a TRPM3 agonist CIM0216 (10 μM). CIM0216 reduced the rate of Rb uptake by ~20% (Ctrl 6.4±0.4 vs CIM 4.5±0.4 mmoles/kg lens dry weight/10 min, n=7, p&lt;.01). The concentration dependence of the response to CIM0216 (1 to 50 μM) was examined in primary cultured mouse lens epithelium and Rb uptake was reduced by CIM0216 concentrations &gt; 10 μM. The TRPM3 agonist reduced Rb uptake to a similar extent (~20%) in mouse lens epithelium, HLEB3 cells and HLEB3 cells that had been stably transfected with the I65M mutant TRPM3. However, the mutant cells responded to CIM0216 at a 10-fold lower concentration than WT mouse and HLEB3 cells. The Rb uptake response to CIM0216 was significantly inhibited by a TRPM3 antagonist, primidone (50 μM), and was abolished when cells were bathed in calcium-free solution. To study calcium entry, Fura2-loaded mouse lens epithelial cells were exposed to the TRPM3 agonist pregnenolone (100 μM). Pregnenolone caused an immediate increase in cytoplasmic calcium that was inhibited by primidone (Peak calcium concentration Ctrl 201±37 vs primidone 137±8 nM, n=5, p&lt;.001). In cultures lens epithelium, agents that increase cellular cAMP, including IBMX, forskolin and 8p-CPT-cAMP, all caused a marked reduction of Rb uptake. Conclusions: TRPM3 activation causes a significant reduction in the rate of active Na-K transport and the response is associated with calcium entry. Because most lens Rb uptake is ouabain-sensitive, the response points to Na,K-ATPase inhibition. Studies are under way to test whether the Na-K transport response to TRPM3 activation involves cAMP signaling. This work was supported by NIH grants R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanosensor channels of the lens and ciliary body: patch clamp studies

Epithelia from the lens and the ciliary body possess mechanosensors, including Piezo1 and transient receptor potential (TRP) channels, that may partake in the regulation of lens and aqueous humor volume. Although these mechanosensor channels commonly allow calcium entry, the outcomes of elevated intracellular calcium vary with the type of channel activated. For instance, separate activation of TRPV4 and TRPV1 increases Na-K-ATPase and Na/K/2Cl cotransporter activities, respectively. To further understand the role of mechanosensitivity in lens and ciliary body, we are currently using patch clamp techniques to characterize the response of primary cultured cells to chemical agonists and antagonists, as well as direct mechanical stimulation. Previously, we found that in mouse lens epithelial cells (mLEC), TRPV4 activation by agonist GSK1016790A (GSK) is followed by disappearance of a fast, transient K+-like outward current (Ipeak), decrease of a steady state outward current (Iss), fast depolarization of resting membrane potential (RMP) values from near -50 mV to near 0 mV, increase of Cx43 hemichannel-like events, and increase of overall membrane conductance ( gm) at holding potential (HP) = -50mV. More recently, we found that in mLEC, shear stress (streaming of external solution from a pipette tip over the cell membrane) reduced Ipeak but did not cause large RMP changes. Porcine non-pigmented epithelial cells (pNPE) in normal conditions display no Ipeak, small Iss, small RMP near -20 mV and spontaneous Cx43 hemichannel-like event activity. Nevertheless, GSK caused further depolarization of pNPE cells, and while no gm changes were seen when GSK was applied while holding cells at -50 mV, gm did increase when GSK exposure occurred during repeated ±80 mV pulses. Preliminary experiments show that activation of TRPM3 by its agonist pregnenolone causes a minimal hyperpolarization in mLEC. In comparison, in a line of human LEC (HLE B3) expressing TRPM3 channels, and which appear generally depolarized, pregnenolone caused a proportionally larger shift toward more negative RMP values; more intriguing, GSK has no measurable effect on the gm of HLE B3 cells. Taken together, and apart from considerations of cell- and species-specific differences, the data suggest that mechanosensor channels have different and/or opposite effects on the regulation of membrane ion channel activity, and thus on cell function. R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cataract-causing mutations S78F and S78P of γD-crystallin decrease protein conformational stability and drive aggregation

Congenital cataract is the leading cause of childhood blindness, which primarily results from genetic factors. γD-crystallin is the most abundant γ-crystallin and is essential for maintaining lens transparency and refractivity. Numerous mutations in γD-crystallin have been reported with unclear pathogenic mechanism. Two different cataract-causing mutations Ser78Phe and Ser78Pro in γD-crystallin were previously identified at the same conserved Ser78 residue. In this work, firstly, we purified the mutants and characterized for the structural change using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and size-exclusion chromatography (SEC). Both mutants were prone to form insoluble precipitates when expressed in Escherichia coli strain BL21 (DE3) cells. Compared with wild-type (WT), both mutations caused structural disruption, increased hydrophobic exposure, decreased solubility, and reduced thermal stability. Next, we investigated the aggregation of the mutants at the cellular level. Overexpression the mutants in HLE-B3 and HEK 293T cells could induce aggresome formations. The environmental stresses (including heat, ultraviolet irradiation and oxidative stress) promoted the formation of aggregates. Moreover, the intracellular S78F and S78P aggregates could be reversed by lanosterol. Molecular dynamic simulation indicated that both mutations disrupted the structural integrity of Greek-key motif 2. Hence, our results reveal the vital role of conserved Ser78 in maintaining the structural stability, which can offer new insights into the mechanism of cataract formation.

Metformin protects human lens epithelial cells from high glucose-induced senescence and autophagy inhibition by upregulating SIRT1.

The aim of this study is to explore whether metformin (MET) protects the human lens epithelial cells (HLECs) from high glucose-induced senescence and to identify the underlying mechanisms. A cellular senescence model was established by treating HLE-B3 cells with D-glucose and then intervened with MET. Concentrations of high glucose (HG) and MET were detected using CCK-8 and western blot. qRT-PCR, western blot, and senescence-associated β-galactosidase (SA-β-gal) were performed to verify the protective effect of MET on senescent HLE-B3 cells. Additionally, western blot and qRT-PCR were conducted to detect the effects of MET on autophagy-related markers p62 and LC3, as well as SIRT1. In vitro, we observed apparent senescence in human lens epithelial cells (HLECs) under high glucose conditions. This was characterized by increased senescence-associated genes p21 and p53. However, the addition of MET significantly reduced the occurrence of HLECs senescence. We also observed that high glucose inhibited both autophagy and SIRT1, which could be restored by MET. Moreover, we verified that the anti-senescence effect of MET was mediated by SIRT1 using SIRT1 activators and inhibitors. We have demonstrated that autophagy and SIRT1 activity are inhibited in HLE-B3 cells using the HG induced senescence model. Furthermore, our results showed that MET can delay senescence by activating SIRT1 and autophagy. These findings suggest that MET may be a promising candidate for alleviating cataract development and provide a direction for further investigation into the underlying molecular mechanisms.

GSK-3β-dependent Nrf2 antioxidant response modulates ferroptosis of lens epithelial cells in age-related cataract.

Oxidative stress-induced lens epithelial cells (LECs) death plays a pivotal role in age-related cataract (ARC) with severe visual impairment, in which ferroptosis is gradually receiving numerous attention resulting from lipid peroxide accumulation and reactive oxygen species (ROS) overproduction. However, the essential pathogenic factors and the targeted medical strategies still remain skeptical and indistinct. In this work, by transmission electron microscopy (TEM) analysis, the major pathological courses in the LECs of ARC patients have been identified as ferroptosis, which was manifested with remarkable mitochondrial alterations, and similar results were found in aged mice (24-month-old). Furthermore, the primary pathological processes in the NaIO3-induced mice and HLE-B3 cell model have also been verified to be ferroptosis with an irreplaceable function of Nrf2, proved by the increased sensitivity to ferroptosis when Nrf2 was blocked in Nrf2-KO mice and si-Nrf2-treated HLE-B3 cells. Importantly, it has been found that an increased expression of GSK-3β was indicated in low-Nrf2-expressed tissues and cells. Subsequently, the contributions of abnormal GSK-3β expression to NaIO3-induced mice and HLE-B3 cell model were further evaluated, inhibition of GSK-3β utilizing SB216763 significantly alleviated LECs ferroptosis with less iron accumulation and ROS generation, as well as reversed expression alterations of ferroptosis markers, including GPX4, SLC7A11, SLC40A1, FTH1 and TfR1, in vitro and in vivo. Collectively, our findings conclude that targeting GSK-3β/Nrf2 balance might be a promising therapeutic strategy to mitigate LECs ferroptosis and thus probably delay the pathogenesis and development of ARC.

Role of Chondroitin Sulfate Proteoglycan 5 in Steroid-Induced Cataract.

Steroid-induced cataracts (SIC) are defined as cataracts associated with the administration of corticosteroids. Long-term glucocorticoid treatment for inflammatory diseases reportedly increases the risk of SIC, and steroids can induce cataracts by disrupting ocular growth factor balance or homeostasis. In this study, we verified the effect of chondroitin sulfate proteoglycan 5 (CSPG5) using dexamethasone (dexa)-treated human lens epithelial (HLE-B3) cells and the lens epithelium from the anterior capsule of SIC patients obtained during cataract surgery. CSPG5 expression increased in the lens epithelium of SIC patients. The downregulation of CSPG5 suppressed the dexa-induced epithelial-mesenchymal transition (EMT)-related protein expression and motility in HLE-B3 cells. The disruption of the transcription factors EZH2 and B-Myb downregulated CSPG5, dexa-induced fibronectin expression, and cell migration in HLE-B3 cells, reaffirming that CSPG5 expression regulates EMT in lens epithelial cells. Taken together, these results indicate that the steroid-induced effects on lens epithelial cells are mediated via alterations in CSPG5 expression. Therefore, our study emphasizes the potential of CSPG5 as a therapeutic target for the prevention and treatment of SIC.

Characterization of a Novel Gja8 (Cx50) Mutation in a New Cataract Rat Model.

To describe a novel spontaneous cataract inbred strain isolated from large-scale breeding SD rats, identify the responsible gene mutation, and understand how this mutation affects lens function. Exome sequencing of 12 cataract-associated genes was performed in the affected and healthy relatives. Sequences of rat wild-type or mutant gap junction protein alpha 8 gene (Gja8) were transfected into cells. The expression level of protein was assayed by Western blot analysis. Subcellular localization of connexin 50 (Cx50) was analyzed in confocal fluorescent images. Wound-healing, 5-ethynyl-2'-deoxyuridine incorporation, and attachment assay were performed to characterize the cell migration, proliferation and adhesion. The abnormality was found to be inheritable in an autosomal semi-dominant pattern through different mating patterns. We found a G to T transversion at codon 655 in Gja8, leading to a substitution of valine by phenylalanine (p.V219F). Gja8V219F/+ heterozygotes expressed nuclear cataract while Gja8V219F/V219F homozygotes manifested microphthalmia in addition to cataract. Histology revealed fiber disorders and loss of organelle-free zone in the mutant lens. Cx50V219F altered its location in HeLa cells and inhibited the proliferation, migration and adhesion abilities of HLEB3 cells. The mutation also reduced the expression of focal adhesion kinase and its phosphorylation. The c.655G>T mutation (p.V219F) is a novel mutation in Gja8, inducing semi-dominant nuclear cataracts in a new spontaneous cataract rat model. The p.V219F mutation altered Cx50 distribution, inhibited lens epithelial cell proliferation, migration, and adhesion, and disrupted fiber cell differentiation. As a consequence, the nuclear cataract and small lens formed.

Expression of αA-crystallin (CRYAA) in vivo and in vitro models of age-related cataract and the effect of its silencing on HLEB3 cells.

To investigate the expression of αA-crystallin (CRYAA) in age-related cataract (ARC) models and its role in lens epithelial cells (LECs). We used Flow cytometry to detect the apoptosis and cell cycle in HLEB3 cells and Real-time fluorescence quantitative polymerase chain reaction to detect the expression of CRYAA mRNA in HLEB3 and in rabbit lens. The expression of CRYAA in HLEB3 cells and rabbit lenses as well as the proteins related to apoptosis and autophagy in transfected cells were detected by western blotting. The lens structure in rabbits was investigated using hematoxylin-eosin staining. Protein thermostability assay was performed to detect the thermal stability of rabbit lens proteins. CCK- 8 assay was used to detect the viability of transfected cells, and the transfection was recorded by fluorescence photography. Hydrogen peroxide can promote apoptosis and arrest the cell cycle in HLEB3 cells, and naphthalene can cause cataract formation and damage the structure of the lens in rabbits. Both ARC models can reduce the expression of CRYAA. The expression of CRYAA silencing increased apoptosis and autophagy in HLEB3 cells.

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.

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.

FOXM1 promotes TGF-β2-induced injury of human lens epithelial cells by up regulating VEGFA expression.

To explore whether Fork head box protein M1 (FOXM1) is involved in TGF-β2-induced injury of human lens epithelial cells and its related mechanism. Human lens epithelium samples from cataract patients and healthy controls were collected. A cellular epithelial injury model was established by treating HLE-B3 cells with TGF-β2. QPCR, immunoblot assays were performed to detect the levels of FOXM1 in human cataract samples and the lens epithelial injury cell model. FOXM1 siRNA and pcDNA3.1-FOXM1 plasmids were transfected into the cells to knockdown and overexpress FOXM1, respectively. MTT and wound closure and transwell assays were performed to analyze cell proliferation and migration in HLE-B3 cells. Immunoblot assays were also conducted to detect the effects of FOXM1 on EMT, VEGFA and MAPK/ERK signaling. We found high expression of FOXM1 in lens tissues of cataract patients. Silencing of FOXM1 in TGF-β2-induced HLE-B3 cells suppressed cell proliferation, migration, and the EMT process. Mechanistically, we found that downregulation of FOXM1 inhibited the VEGFA/MAPK signaling pathway in TGF-β2-induced HLE-B3 cells. FOXM1 promoted TGF-β2-induced injury of human lens epithelial cells (hLECs) by promoting VEGFA expression. FOXM1 could be a potential drug target for the treatment of ocular diseases.

M echanism of lncRNA FOXD3 AS1 targeting miR 338 3p in human lens epithelial cell injury in diabetic cataract

Purpose: To explore the influence of lncRNA FOXD3-AS1 on high glucose (HG)-stimulated human lens epithelial cell injury in diabetic cataract. Methods: Lens epithelial cell HLEB3 were cultured in vitro and transfected with si-FOXD3-AS1, miR-338-3p mimic or miR-338-3p inhibitor, followed by HG (40 mmol/L) treatment. FOXD3-AS1, miR-338-3p, cleaved caspases3 and cleaved caspases9 expression were analyzed by RT-qPCR or western blot. SOD and CAT activities and MDA production in cells were determined using special kits. Cell apoptotic rate was quantified by flow cytometry. Regulatory relationship of FOXD3-AS1 and miR-338-3p was investigated by mechanism assay. Results: HG enhanced FOXD3-AS1 expression but repressed miR-338-3p (P&lt;0.05) in HLEB3 cells. FoxD3-AS1 depletion or miR-338-3p introduction promoted SOD and CAT activities, decreased MDA content, apoptosis rate, and cleaved caspases3 and cleaved caspases9 protein production in HG-induced HLEB3 cells (P&lt;0.05). FOXD3-AS1 could target and negatively regulate miR-338-3p. FOXD3-AS1-mediated influence on HG-induced HLEB3 cells was rescued by miR-338-3p inhibitors. Conclusion: FOXD3-AS1 silenced upregulated miR-338-3p expression to alleviate HG-induced oxidative stress and apoptosis of HLEB3 cells.

Plasmin-Induced Lens Epithelial Cells Detachment for the Reduction of Posterior Capsular Opacification.

We sought to evaluate the efficacy and safety of plasmin injection in the capsular bag during the cataract operation for the prevention of posterior capsule opacification. Thirty-seven anterior capsular flaps taken from phacoemulsification surgery were immersed in either 1 µg/mL plasmin (plasmin group, n = 27) or phosphate-buffered saline (control group, n = 10) for 2 minutes and photographed after fixation and nuclear staining to compare the numbers of residual lens epithelial cells. In the animal experiments, the plasmin solution was injected into the capsular bag and remained for 5 minutes during hydrodissection or after lens extraction. The degree of posterior capsular opacity of the rabbits at 2 months were photographed by slit lamp biomicroscopy. In HLE-B3 cell culture, the cell detachment rate, proliferation, and apoptosis after the plasmin digestion were analyzed. The residual lens epithelial cell numbers on the capsule after plasmin treatment were 168 ± 190.7/mm2 in the 1 µg/mL plasmin group, which was significantly lower than that of the control (1012 ± 798.8/mm2; P < 0.0001). In a rabbit model, the treatment of plasmin resulted in a significantly clearer posterior capsule compared with that of the control group at 2 months postoperatively. This study suggested that plasmin injection can induce effective lens epithelial cell detachment, which could be a promising adjunctive treatment to further improve the success rate in posterior capsule opacification prevention. Plasmin injection for lens epithelial cell detachment could significantly decrease the number of residual lens epithelial cells. This approach could be a promising treatment incorporating the current treatment approach to further improve the success rate in posterior capsule opacification prevention.

Vascular endothelial cell-derived exosomal miR-1246 facilitates posterior capsule opacification development by targeting GSK-3β in diabetes mellitus

Posterior capsule opacification (PCO) is a serious complication after cataract surgery. Diabetes could increase the occurrence of PCO, but the mechanism is still unclear. The purpose of this study is to investigate the role of small extracellular vesicles (sEVs) derived from diabetic aqueous humor in PCO process. Intraoperatively-derived aqueous humor sEVs from patients with diabetic related cataract (DRC) promoted the epithelial-mesenchymal transition (EMT) and metastasis of human lens epithelial cells (LECs). Via mouse PCO surgical model and DiI labeled fluorescence detection of sEVs, the sEVs derived from vascular endothelium were discovered directly contacting with LECs. Furthermore, we demonstrated that high-glucose-cultured human umbilical vein endothelial cells (HUVEC) -derived sEVs facilitated EMT process of HLE-B3 using co-culture model in vitro. miRNA-seq data and GEO datasets analysis revealed that miR-1246 was essential in EMT process with diabetes. The miR-1246 was highly expressed in diabetic aqueous humor sEVs and high-glucose-treated vascular-endothelial-cell-derived sEVs. Moreover, miR-1246 promoted the metastasis and EMT process of HLE-B3 cells by directly targeting GSK-3β. Inhibiting miR-1246 could negatively regulated EMT. This finding might serve as a potential therapy for diabetic PCO.

Aberrant expression of COL4A1 in age-related cataract and its effect on cell proliferation, apoptosis and gene expression changes

To evaluate the regulation of the aberrant expression of collagen type IV alpha 1 chain (COL4A1) in the development of age-related cataract (ARC). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis were employed to evaluate the expression of COL4A1 in ARC patients and healthy controls. The proliferation, apoptosis, cell cycle and epithelial-mesenchymal transition (EMT) of human lens epithelial cell (HLE-B3) were further analyzed under the condition of COL4A1 gene silence. Alteration of gene expression at mRNA level after knockdown COL4A1 were also evaluated by qRT-PCR on HLE-B3 cells. The aberrant expression of COL4A1 was identified a clinically associated with the ARC. Silencing of COL4A1 promoted the apoptosis and inhibited the proliferation of HLE-B3 by blocking the cell cycle. Moreover, COL4A1 gene silence didn't affect the cytoskeleton of HLE-B3 but down-regulated the Collagen type IV Alpha 2 Chain (COL4A2), paired box 6 (PAX6), procollagen-lysine 2-oxoglutarate 5-dioxygenases 1 (PLOD1) and procollagen-lysine 2-oxoglutarate 5-dioxygenases 2 (PLOD2) expression levels in HLE-B3 cells. Silencing the COL4A1 gene induced EMT of the HLE-B3 cells by promoting the transforming growth factor beta (TGF-β) expression. Silencing of COL4A1 induces S-phase arrest, also inhibits the proliferation and enhance HLE-B3 apoptosis and EMT, and down-regulates the expression of COL4A2, PAX6, PLOD1 and PLOD2. Thus, the expression alteration of COL4A1 may play a critical role in the pathogenesis of ARC.