High-expression of ROCK1 modulates the apoptosis of lens epithelial cells in age-related cataracts by targeting p53 gene

Abstract Background: Age-related cataract (ARC) is a serious visual impairment disease, and its pathogenesis is unclear. This article aims to investigate the role of ROCK1 in the apoptosis of lens epithelial cells (LECs) in age-related cataracts. Methods: We collect anterior capsule samples from normal people, patients with age-related cataracts, young mice and naturally aging cataract mice. The Oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with H2O2. MTT, Hoechst 33342, and TUNEL assay were performed to explore proliferation and apoptosis. HE assay was used to observe cell morphology. The gene and protein expression were assessed by quantitative real-time PCR, western blot, immunofluorescence, and immunohistochemical staining.Result: The results from the clinic and mice experiments showed that the numbers of lens epithelial cells from cataract individuals were less than the control individuals. In vitro, the apoptotic cells were increased in lens epithelial cells under H2O2 treatment. The ROCK1 protein level increased in the lens epithelial cells from age-related cataract patients and the old mice, respectively. Meanwhile, the up-regulation of the ROCK1 gene was associated with H2O2-induced HLE-B3 cells apoptosis. MTT and apoptosis assay showed ROCK1 was necessary in mediating H2O2-induced lens epithelial cells apoptosis through ROCK1 over-expression and knockdown experiment, respectively. Further investigation showed that p53 protein levels had been increased during ROCK1-mediated apoptosis in response to H2O2. Besides, ROCK1 phosphorylated p53 at ser15 to up-regulate its protein level. Conclusions: This study established the novel association of ROCK1/p53 signaling with lens epithelial cells apoptosis and age-related cataract genesis.

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High-expression of ROCK1 modulates the apoptosis of lens epithelial cells in age-related cataracts by targeting p53 gene

Molecular Medicine ◽

10.1186/s10020-020-00251-6 ◽

2020 ◽

Vol 26 (1) ◽

Author(s):

Shanshan Hu ◽

Dongmei Su ◽

Lei Sun ◽

Zhongying Wang ◽

Lina Guan ◽

...

Keyword(s):

Epithelial Cells ◽

Protein Level ◽

P53 Protein ◽

P53 Gene ◽

Lens Epithelial Cells ◽

Protein Levels ◽

P53 Signaling ◽

Age Related ◽

Gene And Protein Expression

Abstract Background Age-related cataract (ARC) is a serious visual impairment disease, and its pathogenesis is unclear. This article aims to investigate the role of ROCK1 in the apoptosis of lens epithelial cells (LECs) in age-related cataracts. Methods We collect anterior capsule samples from normal people, patients with age-related cataracts, young mice and naturally aging cataract mice. The oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with H2O2. MTT, Hoechst 33342, and TUNEL assay were performed to explore proliferation and apoptosis. HE assay was used to observe cell morphology. The gene and protein expression were assessed by quantitative real-time PCR, western blot, immunofluorescence, and immunohistochemical staining. Result The results from the clinic and mice experiments showed that the numbers of lens epithelial cells from cataract individuals were less than the control individuals. In vitro, the apoptotic cells were increased in lens epithelial cells under H2O2 treatment. The ROCK1 protein level increased in the lens epithelial cells from age-related cataract patients and the old mice, respectively. Meanwhile, the up-regulation of the ROCK1 gene was associated with H2O2-induced HLE-B3 cells apoptosis. MTT and apoptosis assay showed ROCK1 was necessary in mediating H2O2-induced lens epithelial cells apoptosis through ROCK1 over-expression and knockdown experiment, respectively. Further investigation showed that p53 protein levels had been increased during ROCK1-mediated apoptosis in response to H2O2. Besides, ROCK1 phosphorylated p53 at ser15 to up-regulate its protein level. Conclusions This study established the novel association of ROCK1/p53 signaling with lens epithelial cells apoptosis and age-related cataract genesis.

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High-Expression of ROCK1 Modulates the Apoptosis of Lens Epithelial Cells in Age-Related Cataracts by Targeting p53 Gene

10.21203/rs.3.rs-48570/v1 ◽

2020 ◽

Author(s):

Shanshan Hu ◽

Dongmei Su ◽

Lei Sun ◽

Zhongying Wang ◽

Lina Guan ◽

...

Keyword(s):

Epithelial Cells ◽

Protein Level ◽

P53 Protein ◽

P53 Gene ◽

Lens Epithelial Cells ◽

Protein Levels ◽

P53 Signaling ◽

Age Related ◽

Gene And Protein Expression

Abstract Background: Age-related cataract (ARC) is a serious visual impairment disease, and its pathogenesis is unclear. This article aims to investigate the role of ROCK1 in the apoptosis of lens epithelial cells (LECs) in age-related cataracts. Methods: We collect anterior capsule samples from normal people, patients with age-related cataracts, young mice and naturally aging cataract mice. The Oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with H2O2. MTT, Hoechst 33342, and TUNEL assay were performed to explore proliferation and apoptosis. HE assay was used to observe cell morphology. The gene and protein expression were assessed by quantitative real-time PCR, western blot, and immunohistochemical staining.Result: The results from the clinic and mice experiments showed that the numbers of lens epithelial cells from cataract individuals were less than the control individuals. In vitro, the apoptotic cells were increased in lens epithelial cells under H2O2 treatment. The ROCK1 protein level increased in the lens epithelial cells from age-related cataract patients and the old mice, respectively. Meanwhile, the up-regulation of the ROCK1 gene was associated with H2O2-induced HLE-B3 cells apoptosis. MTT and apoptosis assay showed ROCK1 was necessary in mediating H2O2-induced lens epithelial cells apoptosis through ROCK1 over-expression and knockdown experiment, respectively. Further investigation showed that p53 protein levels had been increased during ROCK1-mediated apoptosis in response to H2O2. Besides, ROCK1 phosphorylated p53 at ser15 to up-regulate its protein level. Conclusions: This study established the novel association of ROCK1/p53 signaling with lens epithelial cells apoptosis and age-related cataract genesis.

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MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

BMC Ophthalmology ◽

10.1186/s12886-020-01489-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Luciferase Reporter Gene ◽

Age Related ◽

Induced Apoptosis

Abstract Background MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear. Methods The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2′,7′-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis. Results We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p. Conclusions We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

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Age-related changes in fibre differentiation of rat lens epithelial cells in vitro

Experimental Eye Research ◽

10.1016/s0014-4835(88)80083-6 ◽

1988 ◽

Vol 46 (2) ◽

pp. 259-267 ◽

Cited By ~ 7

Author(s):

N.A. Richardson ◽

J.W. McAvoy

Keyword(s):

Epithelial Cells ◽

Lens Epithelial Cells ◽

Age Related ◽

Age Related Changes

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In vitro exposure of human lens epithelial cells to X-rays at varied dose-rates leads to protein-level changes relevant to cataractogenesis

International Journal of Radiation Biology ◽

10.1080/09553002.2020.1846819 ◽

2020 ◽

pp. 1-13

Author(s):

Vinita Chauhan ◽

Ngoc Q. Vuong ◽

Simran Bahia ◽

Nazila Nazemof ◽

Premkumari Kumarathasan

Keyword(s):

Epithelial Cells ◽

Protein Level ◽

Human Lens ◽

Lens Epithelial Cells ◽

X Rays ◽

Dose Rates ◽

Human Lens Epithelial Cells ◽

In Vitro Exposure

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MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

10.21203/rs.3.rs-16028/v3 ◽

2020 ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Luciferase Reporter Gene ◽

Age Related ◽

Induced Apoptosis

Abstract Background: MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear.Methods: The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2',7'-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis.Results: We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p.Conclusions: We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

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MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

10.21203/rs.3.rs-16028/v2 ◽

2020 ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Luciferase Reporter Gene ◽

Age Related ◽

Induced Apoptosis

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MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibit ing NOX4 and p38 MAPK signaling

10.21203/rs.3.rs-16028/v1 ◽

2020 ◽

Author(s):

Zhao-Na Li ◽

Ming-Xu Ge ◽

Zhong-Fang Yuan

Keyword(s):

Epithelial Cells ◽

P38 Mapk ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Human Lens ◽

Lens Epithelial Cells ◽

Luciferase Reporter Gene ◽

Age Related ◽

Induced Apoptosis

Abstract Background: MicroRNAs (miRNAs) are abnormally expressed in varying ocular diseases, including age-related cataract. However, the roles of miR-182-5p in age-related cataract progression remains unclear.Methods: The expression of miR-182-5p in HLE-B3 cells were detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2',7'-dichlorodihydrofluorescein diacetate, JC-1 kit, and Western bolt were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro . The target relationship of miR-182-5p and NOX4 was confirmed using dual-luciferase reporter gene analysis.Results: We found that miR-182-5p was significantly decreased by the H 2 O 2 exposure. Overexpression of miR-182-5p promoted cell proliferation, inhibited ROS production and apoptosis in H 2 O 2 -induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H 2 O 2 -treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effect of H 2 O 2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p.Conclusions: We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

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CircHIPK3 knockdown suppresses E2F3 expression to inhibit the viability and promote the apoptosis of lens epithelial cells in age-related cataract by sponging miR-499a-5p

Archives of Medical Science ◽

10.5114/aoms/138262 ◽

2021 ◽

Author(s):

Qichao Han ◽

Rong Zhang ◽

Lan Ma ◽

Li Shao ◽

Meiyan Feng

Keyword(s):

Epithelial Cells ◽

Cell Viability ◽

Circular Rna ◽

Cell Counting ◽

Luciferase Reporter ◽

Lens Epithelial Cells ◽

Protein Levels ◽

Age Related ◽

Anterior Lens Capsule ◽

E2f Transcription Factor

IntroductionCircular RNA (circRNA) is considered to be a vital regulator of disease progression, including age-related cataract (ARC). However, the molecular mechanism of circHIPK3 in ARC progression has not been fully elucidated.Material and methodsThe expression levels of circHIPK3, miR-499a-5p and E2F transcription factor 3 (E2F3) were measured by quantitative real-time PCR. Cell counting kit 8 assay and flow cytometry were performed to detect cell viability and apoptosis, respectively. Western blot analysis was employed to test the protein levels of apoptosis-related markers and E2F3. Biotin-labeled RNA pull-down assay was used to select miRNAs that could be targeted by circHIPK3, and dual-luciferase reporter assay was performed to confirm the interaction between miR-499a-5p and circHIPK3 or E2F3.ResultsCircHIPK3 is a stable circRNA that is significantly under-expressed in the anterior lens capsule tissues of ARC patients. Knockdown of circHIPK3 suppressed SRA01/04 cell viability and accelerated apoptosis. Moreover, miR-499a-5p could be targeted by circHIPK3, and its inhibitor reversed the effect of circHIPK3 silencing on cell viability and apoptosis. Furthermore, E2F3 was a target of miR-499a-5p, and its overexpression reversed the effect of miR-499a-5p on the viability and apoptosis of SRA01/04 cells. In addition, circHIPK3 positively regulated E2F3 expression by sponging miR-499a-5p.ConclusionsCircHIPK3 knockdown inhibited viability and enhanced apoptosis of lens epithelial cells to promote ARC progression by regulating miR-499a-5p/E2F3 axis.

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Platelet Microtubule Subunit Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657068 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1630-1633 ◽

Cited By ~ 3

Author(s):

A G Castle ◽

N Crawford

Keyword(s):

Epithelial Cells ◽

Gel Electrophoresis ◽

Blood Platelets ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Lens Epithelial Cells ◽

Molecular Weights ◽

Kinetics Of ◽

Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

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