SP1 promotes high glucose-induced lens epithelial cell viability, migration and epithelial-mesenchymal transition via regulating FGF7 and PI3K/AKT pathway.

BACKGROUND: Diabetic cataract (DC) is a common complication of diabetes and its etiology and progression are multi-factorial. In this study, the roles of specific protein 1 (SP1) and fibroblast growth factor 7 (FGF7) in DC development were explored. METHODS: DC cell model was established by treating SRA01/04 cells with high glucose (HG). MTT assay was conducted to evaluate cell viability. Transwell assay and wound-healing assay were performed to assess cell migration and invasion. Western blot assay and qRT-PCR assay were conducted to measure the expression of N-cadherin, E-cadherin, Collagen I, Fibronectin, SP1 and FGF7 expression. CHIP assay and dual-luciferase reporter assay were conducted to analyze the combination between FGF7 and SP1. RESULTS: FGF7 was upregulated in DC patients and HG-induced SRA01/04 cells. HG treatment promoted SRA01/04 cell viability, migration, invasion and epithelial-mesenchymal transition (EMT), while FGF7 knockdown abated the effects. Transcription factor SP1 activated the transcription level of FGF7 and SP1 overexpression aggravated HG-induced SRA01/04 cell injury. SP1 silencing repressed HG-induced SRA01/04 cell viability, migration, invasion and EMT, but these effects were ameliorated by upregulating FGF7. Additionally, SP1 knockdown inhibited the PI3K/AKT pathway by regulating the transcription level of FGF7. CONCLUSION: Transcription factor SP1 activated the transcription level of FGF7 and the PI3K/AKT pathway to regulate HG-induced SRA01/04 cell viability, migration, invasion and EMT.

Circ_0000099/miR-223-3p/CTGF Regulates the Growth, Metastasis, and EMT Processes in TGF-ß2-Stimulated Human Lens Epithelial Cells.

PURPOSE: Posterior capsule opacification (PCO) is the major complication of visual impairment after cataract surgery. Circular RNAs (circRNAs) are involved in the development of many diseases. The purpose of this study was to explore the role and molecular mechanism of circ_0000099 in PCO. METHODS: SRA01/04 cells were treated with TGF-ß2 to establish a PCO cell model. The expression of circ_0000099, miR-223-3p, and connective tissue growth factor (CTGF) mRNA was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot assay was used to analyze the protein expression. Cell proliferation, migration, and invasion were analyzed by (4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2 '-Deoxyuridine (EdU), transwell, and wound healing tests. The circ_0000099/miR-223-3p/CTGF relationship was verified by dual luciferase reporter gene and RNA binding protein immunoprecipitation (RIP) assays. RESULTS: TGF-ß2 treatment promoted SRA01/04 cell proliferation invasion, migration, and EMT. Circ_0000099 expression was increased in POC patients and TGF-ß2-treated SRA01/04 cells.Knockdown of circ_0000099 suppressed TGF-ß2-induced proliferation, invasion, migration, and EMT in SRA01/04 cells. miR-223-3p was identified as the target of circ_0000099, and miR-223-3p inhibitor might partly abolish the repression of circ_0000099 silencing on TGF-ß2-triggered SRA01/04 cell disorders. MiR-223-3p directly targeted CTGF. Knockdown of CTGF suppressed TGF-ß2-induced SRA01/04 cell injury. Circ_0000099 can regulate CTGF expression by targeting miR-223-3p. CONCLUSIONS: Circ_0000099 silencing might relieve TGF-2-induced SRA01/04 cell injury by the miR-223-3p/CTGF axis, providing new avenues for the prevention and treatment of PCO.

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.

Paired Box Gene 6 Regulates Heme Oxygenase-1 Expression and Mitigates Hydrogen Peroxide-Induced Oxidative Stress in Lens Epithelial Cells.

PURPOSE: This study aimed to investigate the regulation of heme oxygenase-1 (HO-1) by paired box gene 6 (Pax6) and their roles in hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in lens epithelial cells (LECs) (SRA01/04, HLE-B3). METHODS: Lens anterior capsule membranes of mice of different ages were obtained to compare differences in the expression of Pax6 and HO-1 using Western blotting. Pax6-overexpressing plasmid and small interfering RNA were designed to overexpress and silence Pax6, respectively. Cobalt protoporphyrin (CoPP) was used to promote the expression of HO-1. Oxidative damage in LECs was induced by treatment with H2O2 (400 µM) for 24 h. Cell viability was measured using the Cell Counting Kit-8 assay. Intracellular reactive oxygen species (ROS) were detected using flow cytometry and immunofluorescence. Superoxide dismutase (SOD) level was measured using SOD Assay Kit and apoptotic cells were quantified using annexin V-fluorescein isothiocyanate/propidium iodide staining. RESULTS: Pax6 and HO-1 expression levels showed an age-dependent decrease in LECs of mouse. Overexpressing Pax6 upregulated HO-1 expression level. Silencing Pax6 downregulated the HO-1 expression level, resulting in increased generation of ROS, reduced SOD activity, decreased cell viability, and increased apoptotic cells of LECs under H2O2-induced oxidative stress. Overexpressing Pax6 and CoPP both mitigates H2O2-induced oxidative stress by increasing the expression of HO-1 of LECs. CONCLUSION: Pax6 and HO-1 expression levels showed an age-dependent decrease in LECs in mouse anterior capsules. Pax6 could regulate the expression of HO-1 in LECs. The decrease of Pax6 weakened the antioxidant ability of LECs under H2O2-induced oxidative stress by downregulating HO-1, which may be a potential mechanism for the formation of age-related cataract.

EphA2 inhibits SRA01/04 cells apoptosis by suppressing autophagy via activating PI3K/Akt/mTOR pathway.

This study attempted to determine the effect of EphA2 on H2O2-treated lens epithelial cells (SRA01/04) and the underlying mechanisms. MTT assay and flow cytometry were performed to assess cell viability and cell apoptosis. Western blot was carried out to examine the levels of proteins associated with apoptosis and autophagy. Our results revealed that EphA2 significantly elevated the reduced cell viability, and inhibited the increased cell apoptosis in H2O2-treated SRA01/04 cells, along with the significant up-regulated Bcl-2 and down-regulated Cleaved-caspase-3 and Bax protein levels, but which were all abolished by Rapa (autophagy activator). We also found that EphA2 significantly suppressed cell autophagy in H2O2-treated SRA01/04 cells. Additionally, EphA2 significantly up-regulated the protein levels of p-Akt and p-mTOR in H2O2-treated SRA01/04 cells, and the inhibition of Akt by MK-2206 and inhibition of mTOR by Rapa both obviously reversed EphA2-mediated the inhibition of autophagy in H2O2-treated SRA01/04 cells. In summary, these data demonstrated that EphA2 inhibited the apoptosis of SRA01/04 cells by inhibiting autophagy via activating PI3K/Akt/mTOR pathway.

Quercetin Inhibits AQP1 Translocation in High-Glucose-Cultured SRA01/04 Cells Through PI3K/Akt/mTOR Pathway.

OBJECTIVE: The study aimed to investigate the effects of quercetin on Aquaporin 1 (AQP1) translocation in high glucose condition and made an attempt to clarify the underlying mechanisms and provide new ideas for the treatment of diabetic cataract (DC). METHODS: The human lens epithelial line SRA01/04 cells were divided into groups mentioned below: normal glucose, high glucose with a specific time (0 h, 2 h, 4 h, 8 h, 12 h, 24 h), high glucose plus the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, high glucose plus the mammalian target of rapamycin (mTOR) inhibitor rapamycin, and high glucose plus quercetin with different doses (2 µmol/L, 4 µmol/L and 8 µmol/L). The western blotting assay was used to detect the protein kinase B (Akt), phosphorylated protein kinase B (p-Akt), mammalian target of rapamycin (m- TOR), phosphorylated mammalian target of rapamycin (p-mTOR) and AQP1. Real-time polymerase chain reaction (RT-PCR) was used to detect the expression of AQP1. A Membrane and Cytosol Protein Extraction Kit was applied to separate membrane proteins. Immunofluorescence assay was performed to evaluate the expression and location of AQP1. The effect of quercetin on the expression of AQP1 and PI3K/Akt/mTOR signaling was detected. RESULTS: AQP1 protein was found to be significantly increased in 24 hour when exposed to high glucose condition (P<0.01). LY294002 and rapamycin inhibited PI3K/Akt/mTOR and AQP1 expression (P<0.01), preventing the change of AQP1 location in the SRA01/04 plasma membrane (P<0.01). This effect was further proved by immunofluorescence. In different doses of quercetin groups (2 µmol/L, 4 µmol/L and 8 µmol/L), the phosphorylation of mTOR and Akt were decreased and AQP1 in the membrane was changed compared with high glucose group (P<0.01). CONCLUSION: Quercetin significantly decreased the AQP1 elevation and prevented the change of AQP1 location through inhibiting the activation of the PI3K/Akt/mTOR signaling in high-glucose-- cultured SRA01/04 cells, which might have the preventable and inhibitory effects on the early development of diabetic cataract. The specific pathophysiological role of quercetin still needs to be verified.

Molecular characterization of the human lens epithelium-derived cell line SRA01/04.

Cataract-associated gene discovery in human and animal models have informed on key aspects of human lens development, homeostasis and pathology. Additionally, in vitro models such as the culture of permanent human lens epithelium-derived cell lines (LECs) have also been utilized to understand the molecular biology of lens cells. However, these resources remain uncharacterized, specifically regarding their global gene expression and suitability to model lens cell biology. Therefore, we sought to molecularly characterize gene expression in the human LEC, SRA01/04, which is commonly used in lens studies. We first performed short tandem repeat (STR) analysis and validated SRA01/04 LEC for its human origin, as recommended by the eye research community. Next, we used Illumina HumanHT-12 v3.0 Expression BeadChip arrays to gain insights into the global gene expression profile of SRA01/04. Comparative analysis of SRA01/04 microarray data was performed using other resources such as the lens expression database iSyTE (integrated Systems Tool for Eye gene discovery), the cataract gene database Cat-Map and the published lens literature. This analysis showed that SRA01/04 significantly expresses >40% of the top iSyTE lens-enriched genes (313 out of 749) across different developmental stages. Further, SRA01/04 also significantly expresses ~53% (168 out of 318) of cataract-associated genes in Cat-Map. We also performed comparative gene expression analysis between SRA01/04 cells and the previously validated mouse LEC 21EM15. To gain insight into whether SRA01/04 reflects epithelial or fiber cell characteristics, we compared its gene expression profile to previously reported differentially expressed genes in isolated mouse lens epithelial and fiber cells. This analysis suggests that SRA01/04 has reduced expression of several fiber cell-enriched genes. In agreement with these findings, cell culture analysis demonstrates that SRA01/04 has reduced potential to initiate spontaneous lentoid body formation compared to 21EM15 cells. Next, to independently validate SRA01/04 microarray gene expression, we subjected several candidate genes to RT-PCR and RT-qPCR assays. This analysis demonstrates that SRA01/04 supports expression of many key genes associated with lens development and cataract, including CRYAB, CRYBB2, CRYGS, DKK3, EPHA2, ETV5, GJA1, HSPB1, INPPL1, ITGB1, PAX6, PVRL3, SFRP1, SPARC, TDRD7, and VIM, among others, and therefore can be relevant for understanding the mechanistic basis of these factors. At the same time, SRA01/04 cells do not exhibit robust expression of several genes known to be important to lens biology and cataract such as ALDH1A1, COL4A6, CP, CRYBA4, FOXE3, HMX1, HSF4, MAF, MEIS1, PITX3, PRX, SIX3, and TRPM3, among many others. Therefore, the present study offers a rich transcript-level resource for case-by-case evaluation of the potential advantages and limitations of SRA01/04 cells prior to their use in downstream investigations. In sum, these data show that the human LEC, SRA01/04, exhibits lens epithelial cell-like character reflected in the expression of several lens-enriched and cataract-associated genes, and therefore can be considered as a useful in vitro resource when combined with in vivo studies to gain insight into specific aspects of human lens epithelial cells.

Effect of senescence marker protein 30 on the proliferation and apoptosis of human lens epithelial cells SRA01/04.

AIM: To study the effect of senescence marker protein 30 (SMP30) on the proliferation and apoptosis of human lens epithelial cell (HLEC) SRA01/04. METHODS: SMP30 overexpression (OE) and knock down (KD) type cell lines were cultivated by using two groups regucalcin (RGN; SMP30) lentiviral vectors (LV-RGN, LV-RGN-RNAi) and the respective negative control virus infect SRA01/04 cells. Western blot and real-time quantitative polymerase chain reaction (q-PCR) analysis were used to determine RGN overexpression and knock down efficiency. We use cell counting kit-8 (CCK8) assay to measure cell viability and 5-bromodeoxyuridine (BrdU) assay to test cell proliferation. Cell cycle was measured by PI FACS assay and cell apoptosis was tested by Annexin V-APC assay through flow cytometry. We use Western blot to measure the content of caspase-3 in SRA01/04. RESULTS: We used PCR and Western blot techniques to determine the successful transfection of SMP30 OE and KD SRA01/04 cell lines. By CCK8, Brdu and PI FACS cell cycle assay, it was found that the SMP30 OE group promoted cell proliferation (P<0.05) compared with the control group, and the KD group inhibited cell proliferation (P<0.05). The results of Annexin V-APC signal staining detection indicated that compared with respective control group, the cell apoptosis rate was higher in KD group (P<0.05) but lower in OE group (P<0.01). The expression of caspase-3 was down-regulated in OE group through Western blot assay and up-regulated in KD group compared with respective control group. CONCLUSION: Proliferation of SRA01/04 was promoted by SMP30 OE and apoptosis was suppressed. Increasing the expression of SMP30 may protect HLEC SRA01/04 against apoptosis in cataract.

Heme oxygenase-1 (HO-1) protects human lens epithelial cells (SRA01/04) against hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis.

OBJECTIVES: This study aimed to investigate the protective role of heme oxygenase-1 (HO-1) in H2O2-induced oxidative stress and apoptosis in human lens epithelial cells (hLEC; SRA01/04). METHODS: SRA01/04 cells were exposed to a hydrogen peroxide (H2O2) concentration gradient and inducers of HO-1 such as cobalt protoporphyrin (CoPP) and zinc protoporphyrin (ZnPP), respectively. In addition, an RNA silencing experiment was conducted to investigate the HO-1 function in this study. A Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Western blot and ELISA were used to detect the level of HO-1 expression. In our study, hLECs were exposed to 400 µM hydrogen peroxide (H2O2) for 24 h with or without pretreatment with 10µΜ CoPP or 10µΜ ZnPP, respectively. Double immunofluorescence staining was used for cell identification and the qualitative expression of HO-1. Expression of HO-1 was monitored using Western blot and ELISA. Intracellular reactive oxygen species (ROS) were detected by flow cytometry analyses; commercial enzymatic kits were used to measure the levels of glutathione (GSH), as well as superoxide dismutase (SOD). The proportion of cell apoptosis was quantified by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The expression of caspase family (-8, -3) proteins was measured by Western blot analysis. RESULTS: HO-1 significantly restored the cell viability under H2O2 injury via reducing the generation of ROS and increasing the levels of SOD and GSH activity. Moreover, HO-1 also inhibited H2O2-induced caspase-8 and caspase-3 proteins, thus significantly reducing the apoptosis of SRA01/04. An RNA silencing experiment demonstrated the increased resistance of LECs to oxidative stress specifically for increased levels of HO-1. CONCLUSIONS: These findings suggested that HO-1 protects human lens epithelial cells from H2O2-induced oxidant stress by upregulating antioxidant enzyme activity, reducing ROS generation, and thus inhibiting caspase family-dependent apoptosis.