Cataract is a common ophthalmic disease that is characterized by partial or total loss of lens transparency (1). If left untreated, a cataract can lead to loss of vision and eye function. A decrease of light transmission to the retina is the main cause of vision loss during aging, especially in patients with age-related cataracts (ARCs) (2,3). At present, no treatment to prevent the formation of cataracts exists. Although surgical extirpation and intraocular lens implantation are effective methods for treating cataracts, they are associated with a huge financial burden for patients, as well as serious postoperative complications, such as vitreous prolapse and corneal abrasions (4). Apoptosis of human lens epithelial cells (HLECs) has long been considered to be the cytological basis of non-congenital cataracts (5). Therefore, determining the pathogenesis of cataract is crucial to reducing the incidence this disease.
Usually, there is a homeostasis of free radicals and antioxidants present in the aqueous humor and lens. An imbalance in these molecules leads to the occurrence of oxidative stress (OS) (6). OS mediated by reactive oxygen species (ROS) disrupts the integrity and function of cells by attacking lens proteins, which is the main contributor to cataract formation (7,8). Increased ROS production or decreased levels of antioxidants exacerbate OS (9), alter the cellular environment, and trigger HLEC apoptosis, which is considered to be an early event of cataract development (10). Hydrogen peroxide (H2O2) is a non-free radical derived from the ROS family that can easily penetrate the lipid membrane and produce toxicity in the lens (11). Previous studies have shown that ROS production is stimulated by H2O2-induced epithelial cell injury and protein degradation, similar to human cataract damage (12). Therefore, H2O2 has been widely used to induce apoptosis of HLECs in order to simulate cataract formation in cell models (13).
Long non-coding RNAs (lncRNAs) are a class of transcripts exceeding 200 nucleotides in length that have little or no protein coding function (14). LncRNAs regulate gene expression at the transcriptional and posttranscriptional levels (15), and their abnormal expression in diseases is a topic that has been extensively researched. Abnormal expression of lncRNAs, such as lncRNA taurine up-regulated 1 (TUG1), has been found to play a role in the development of cataracts (16). Recent studies have reported that lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) has a protective effect on cells, and a lack of NEAT1 has been observed to increase the susceptibility to stress-induced cell death (17). Furthermore, the up-regulation of NEAT1 can help to alleviate doxorubicin-induced cardiac injury (18). To our knowledge, no report to date has explored the effect of NEAT1 on H2O2-stimulated HLECs. In this study, we studied the preventive role of NEAT1 in H2O2-treated HLECs (SRA01/04), and attempted to further elucidate the molecular mechanisms involved in this protective effect. We present the following article in accordance with the MDAR reporting checklist (available at http://dx.doi.org/10.21037/atm-20-7365).
Clinical pathology specimens
Between May 2018 and March 2020, tissue samples from 45 patients with ARC were obtained from the Affiliated Hospital of Nantong University, and all patients signed a written informed consent before taking part. The ARC cohort included 15 cases of age-related cortical cataract (ARCC), 15 cases of age-related nuclear cataract (ARNC), and 15 cases of age-related posterior subcapsular cataract (ARPC). The patients in this cohort were aged from 50 to 60 years and had no history of eye damage. A control group comprising 10 cases (aged 36–60 years old, with no known ocular or systemic diseases) who underwent anterior retinal vitrectomy was also enrolled. The 45 ARC patients were screened using the lens opacity classification system III (LOCS III) as the criteria (19). All procedures were approved by the ethics committee of Nantong University (No. NT20180513) and carried out in accordance with the Declaration of Helsinki (2013 version).
HLECs SRA01/04 were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China). The SRA01/04 cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, USA) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sigma, St. Louis, USA) in a humidified incubator at 37 °C with 5% CO2.
Cell viability assay
SRA01/04 cells were cultured in 96-well plates at a density of 1×106 cells/well, as previously described (20). The cells were exposed to H2O2 (Sigma) at different concentrations (0, 50, 100, 150, 200, 250, 300, 350, 400, or 500 µM) for 24 hours. Subsequently, 50 mL MTT reagent (Abcam, Cambridge, UK) was used to culture cells at 37 °C for 3 hours. Then, culture plates with 150 mL MTT solvent added were shaken on an orbital shaker for 15 minutes. A microplate reader (Bio-Rad, California, USA) was used to measure the optical density at 590 nm.
γH2AX immunofluorescent staining
DNA damage was performed according to the methods previously described (21). H2O2-induced SRA01/04 cells grown on glass cover slides were fixed in 4% formaldehyde at 4 °C overnight, and then the membrane was permeated with phosphate-buffered saline (PBS) containing 0.2% Triton X-100 for 15 minutes. Next, the above cells were sealed with 5% bovine serum albumin (BSA) at room temperature for 1.5 hours, and then incubated with γ-H2A histone family member X (γ-H2AX) antibody (Abcam) overnight at 4 °C. Following that, they were stained with Alexa Fluor488-labeled (Thermo Fisher, Waltham, USA) secondary antibody for 1 hour at room temperature. Images were obtained with a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan).
Apoptosis of SRA01/04 cells was determined using an Annexin V-FITC/PI Apoptosis Detection Kit (Sigma). SRA01/04 cells were digested with trypsin (Beyotime) and collected. The cells were washed twice with ice-cold PBS and resuspended in 1× Binding Buffer (300 µL, Beyotime) according to the manufacturer’s instructions. Subsequently, the cells were double-stained with fluorescein isothiocyanate (FITC)-Annexin V (5 µL) and propidium iodide (PI, 5 µL) in the dark at room temperature for 15 minutes. Finally, a flow cytometer (Thermo Fisher) was used to analyze the fluorescence signals accompanied by light scattering.
Generation of OS
SRA01/04 cells were plated into a 6-well plate at a density of 2×105/well and incubated in a 5% CO2 incubator at 37 °C. When the cells reached 70–80% confluency, the upper culture medium was removed and the plate was washed with PBS. The cells were stained with 20 µM 2’,7’-dichlorofluorescin diacetate (DCFDA) solution from a DCFDA/H2DCFDA-Cellular ROS Assay Kit (ab113851, Abcam) for 30 minutes at 37 °C, according to the manufacturer’s instructions. Then, the living cells were imaged with a filter group suitable for FITC. Additionally, the content of malondialdehyde (MDA) and activity of glutathione (GSH) were examined using the corresponding assay kits (Beyotime, Haimen, China).
Low-expressed lncRNA NEAT1 was obtained in SRA01/04 cells using small interfering RNA (siRNA) against NEAT1 (5'-GAGCAATGACCCCGGTGACG-3') and a non-targeting siRNA as a normal control (NC, 5'-TAGATACCCCCAGGCCTACC-3'), which were synthesized by Sangon Biotech (Shanghai, China). SRA01/04 cells were transfected with si-NEAT1 or si-NC using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CA, USA), according to the manufacturer's instructions. After that, SRA01/04 cells were treated with H2O2 (300 µM).
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated from SRA01/04 cells and ARC tissues using TRIzol reagent (Invitrogen), and cDNA was synthesized using a PrimeScriptTM RT reagent kit (Takara, Otsu, Japan), according to the manufacturer's instructions. DNA was amplifiied through qRT-PCR using SYBR Green Mix Kit (Takara). The expression of NEAT1 was analyzed using the 2−ΔΔCt method (22), and was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers of NEAT1 and GAPDH used are listed below:
NEAT1: F 5'-CTTCCTCCCTTTAACTTATCCATTCAC-3',
GAPDH: F 5'-AGGTCGGTGTGAACGGATTTG-3',
5-Ethynyl-2'-deoxyuridine (EdU) assay
To evaluate the influence of NEAT1 on SRA01/04 cell proliferation, EdU staining (Invitrogen, Waltham, USA) was conducted. SRA01/04 cells were inoculated into a 6-well plate (1×104 cells/well) and incubated at 37 °C with 5% CO2 for 24 hours, then treated with 50 µM EdU labeling solution for 2 hours in the dark, according to the manufacturer’s instructions. After that, the cells were fixed in 4% paraformaldehyde for 15 minutes at 37 °C, and stained with 100 µL Hoechst 33342 (ApexBio, Houston, USA) at 37 °C for 10 minutes in the dark. Finally, images of EdU positive cells were captured with an inverted fluorescence microscope (Olympus, Tokyo, Japan).
Western blot (WB) analysis
Total protein from SRA01/04 cells was extracted using RIPA lysis buffer (Beyotime) and separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, after which the protein was transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The PVDF membranes were sealed in 5% nonfat milk at room temperature for 2 hours, and then incubated with the primary antibodies against B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, nuclear factor-kappa B (NF-κB) p65, Phospho-NF-κB p65, p38 mitogen-activated protein kinase (p38 MAPK), and Phospho-p38 MAPK at 4 °C overnight. On the second day, the appropriate secondary antibodies bound to horseradish peroxidase (Beyotime) were incubated at room temperature for 1 hour. Finally, the strips were exposed with electrochemiluminescence (ECL) reagent (Pierce, Rockford, USA), and quantitative analysis was performed using image laboratory software (Bio-Rad). All antibodies were obtained from Cell Signaling Technology (CST, Beverly, USA).
Data were expressed as mean ± standard deviation (SD) and analyzed with one-way analysis of variance (Significant differences between three or more groups) or Student’s t-test (significant differences between two groups). Statistical analyses were conducted with SPSS Statistics 19.0 (IBM, Armonk, USA) and Prism 7 (GraphPad, San Diego, USA) software. A P value of <0.05 was considered to show a statistically significant difference.
H2O2 inhibited the cell viability of HLECs
To examine the cytotoxic effects of H2O2 on HLECs, SRA01/04 cells were treated with different concentrations of H2O2 (from 0–500 µM) for 24 hours. As shown in Figure 1A, the viability of SRA01/04 cells was significantly reduced under treatment with 200, 250, 300, 350, 400, and 500 µM H2O2. Moreover, with 300 µM H2O2, cell viability was remarkably decreased at different time points (0, 24, and 48 h; Figure 1B). Therefore, H2O2 at a concentration of 300 µM was used for the subsequent experiments.
H2O2 induced HLEC oxidative injury
After stimulation with H2O2 (300 µM), the number of cells that stained positive for γ-H2AX was increased (Figure 2A) and p53 protein expression was remarkably up-regulated (Figure 2B,C) compared with the controls. As shown in Figure 2D,E, the percentage of apoptotic cells in the H2O2 group was dramatically higher than that in the control group. And H2O2 induced the up-regulation of Bax and down-regulation of cleaved caspase-3 and Bcl-2. Furthermore, the average fluorescence intensity of ROS and MDA content were increased after the exposure of SRA01/04 cells to H2O2. Similarly, H2O2 inhibited GSH activity in SRA01/04 cells (Figure 2F,G,H). Together, these results indicated that H2O2 induced HLEC oxidative injury.
NEAT1 was upregulated in ARC tissues and H2O2-treated HLECs
As shown in Figure 3A, qRT-PCR confirmed that NEAT1 expression in ARC tissues was increased compared to that in the control tissue samples. ARC subgroup analysis was performed to determine which subtype of ARC had the highest expression NEAT1. The expression of NEAT1 was found to be most significantly increased in the ARCC group (Figure 3B). Moreover, after treatment with 200 µM H2O2 for 24 hours, the expression of NEAT1 was significantly increased compared to the control group (Figure 3C).
NEAT1 knockdown improved H2O2-induced cell proliferation
qRT-PCR analysis (Figure 4A) showed that the expression of NEAT1 in SRA01/04 cells was increased by H2O2 treatment, while cells transfected with si-NEAT1 exhibited lower NEAT1 expression, which indicated high transfection efficiency. MTT detection found that H2O2 significantly reduced the cell viability of SRA01/04 cells at 0, 24, 48, and 72 hours, while a low expression of NEAT1 could partially attenuate the inhibitory effect of H2O2 on cell viability (Figure 4B). Furthermore, the data from the EdU assay demonstrated that while H2O2 triggered the inhibition of cell proliferation, si-NEAT1 transfection significantly weakened the inhibitory effect of H2O2 on cell proliferation (Figure 4C).
NEAT1 knockdown suppressed H2O2-induced apoptosis
To verify the effect of NEAT1 on H2O2-induced apoptosis of SRA01/04 cells, flow cytometry and western blot assay were performed. Annexin V FITC/PI double staining revealed that si-NEAT1 transfection markedly decreased the apoptosis rate of SRA01/04 cells, compared with the H2O2 group (Figure 5A). Also, western blot revealed that H2O2 treatment up-regulated the expression levels of cleaved caspase-3 and Bax, and down-regulated Bcl-2 expression. However, these changes were reversed by transfection with si-NEAT1 (Figure 5B).
NEAT1 knockdown alleviated H2O2-induced OS
The antioxidant defense system-related processes of SRA01/04 cells, such as ROS production, MDA, and GSH activity, was also examined. The average fluorescence intensity of intracellular ROS production was determined using a DCFHDA method (Figure 6A). After H2O2 treatment, the average fluorescence intensity of ROS in SRA01/04 cells was increased, while ROS content was significantly decreased after transfection with si-NEAT1. Compared to the control group, GSH activity (Figure 6B) was decreased and MDA content (Figure 6C) was increased in SRA01/04 cells after exposure to H2O2, and transfection with si-NEAT1 reversed these effects (Figure 6B,C).
NEAT1 protected against H2O2 stimuli through the NF-кB p65/p38 MAPK pathways
To explore the molecular mechanism of the protective effect of NEAT1 against H2O2 stimulation, we assessed the effects of NEAT1 on the expression of p65 and p38 MAPK, which play an important role in the regulation of intracellular metabolism (23). Our data revealed that stimulation with H2O2 resulted in a significant increase in p65 and p38 MAPK expression. As expected, the increases in p65 and p38 MAPK expression were significantly reduced by si-NEAT1 (Figure 7A). To further confirm the inhibitory effect of NEAT1, SRA01/04 cells exposed to H2O2 were treated with p65 (PDTC) or p-38 MAPK (LY2228820) inhibitors. Compared to those transfected with si-NEAT1 alone, H2O2-treated SRA01/04 cells transfected with si-NEAT1 combined with PDTC or LY2228820 exhibited much higher cell viability (Figure 7B) and GSH activity (Figure 7C), and a much lower MDA content (Figure 7D).
Cataract is a multifactorial ophthalmic disease characterized by opacity or a loss of transparency in the normally clear lens of the eye (1). Surgical intervention and replacement of the lens is the only clinical treatment for cataract patients. Therefore, the study of cataracts should focus on the exploration of new therapeutic targets. Apoptosis of lens epithelial cells is universally acknowledged to be closely related to the formation of cataracts, and OS induced by ROS has been considered as a major contributor to apoptosis of lens epithelial cells (12,24). In this study, H2O2 was used to construct a cell model of oxidative damage in HLECs. We found that H2O2 significantly suppressed cell viability in a dose- and time-dependent manner, and promoted OS and apoptosis of SRA01/04 cells, which was consistent with previous reports (25). Additionally, H2O2 treatment was observed to significantly increase the expression of NEAT1 in HLECs. Also, pathological examination showed that NEAT1 expression was up-regulated in ARCC, ARNC and, ARPC patient specimens compared to the control group.
Apoptosis of HLECs is a common cellular basis for non-congenital cataracts (5,10). Under multiple stresses, including H2O2 stimulation, lower levels of p53 proteins are activated through subsequent cell cycle arrest or apoptosis pathways (26). In this study, we found that H2O2 reduced cell viability of HLECs in a concentration- and time-dependent manner (Figure 1A,B), suggesting that H2O2 is cytotoxic to HLECs. To quantitatively assess the potential protective effect of NEAT1 for H2O2-induced cell death, we detected the percentage of apoptotic SRA01/04 cells by performing a V/PI double-staining assay. A significant increase was observed in the total number of apoptotic cells after exposure to 300 µM H2O2 (Figure 5). Compared with H2O2 alone, a low expression of NEAT1 led to an obvious decrease in the number of apoptotic cells. Caspases are recognized as the main promoters and executors of apoptosis (27). Caspase-3 is a key effector of apoptosis, and activated caspase-3 is involved in the mitochondrial-mediated pathway. Therefore, we examined the expression levels of cleaved caspase-3, Bax, and Bcl-2 through western blot (Figure 5B). Notably, si-NEAT1 significantly inhibited the down-regulation of Bcl-2, and up-regulation of cleaved caspase-3 and Bax. Overall, these results suggested that a low expression of NEAT1 protected against H2O2-induced HLEC apoptosis.
OS is a loss of balance between oxidants and antioxidants due to increased free radical production or reduced free radical scavenging capacity (28). H2O2 is the main component of ROS. Stimulation by H2O2 destroys the lens’ natural antioxidant defense system, resulting in the decrease of SOD and GSH activity, and the increase of MDA content (29). We observed that H2O2 induced the production of intracellular ROS in HLECs, while si-NEAT1 pretreatment significantly inhibited ROS production of intracellular free radicals (Figure 6A). Meanwhile, we also found that, compared with the control group, GSH activity was significantly decreased in H2O2-treated HLECs, while MDA content was increased, suggesting that H2O2 induced the OS of HLECs. Transfection with si-NEAT1 significantly increased GSH activity and reduced MDA content (Figure 6B,C). These results indicated that NEAT1 knockdown had antioxidant activity in H2O2-treated HLECs (13,24,25).
To explore the molecular mechanism of NEAT1 on H2O2-induced HLEs, we assessed the potential role of NEAT1 on NF-κB p65 and p38 MAPK expression, which plays an important role in the regulation of intracellular metabolism and response to stress (30,31). NF-κB belongs to the family of evolutionarily conserved transcription factors, which regulate cell survival, apoptosis, and inflammatory response (32). Previous research has shown that H2O2 could activate NF-κB pathways in HLECs, activated p65 transferred from the cytoplasm to the nucleus (33). However, the inactivated p65 effectively attenuated the cytotoxicity of HLECs exposed to H2O2 (34). We found that the expression of phosphorylated p65 was significantly increased in H2O2-induced HLECs (Figure 7A). OS can specifically activate p38 MAPK signaling pathways under different conditions. Inactivation of p38 phosphorylation reduces H2O2-induced apoptosis and ROS production in HLECs (35). Our data showed that H2O2 treatment led to a significant increase in p38 phosphorylation. As expected, si-NEAT1 significantly interfered with this increase in p38 phosphorylation (Figure 7A). To further confirm the role of p65 and p38 MAPK signaling pathways, HLECs exposed to H2O2 being treated with p65 inhibitors (PDTC) or p38 inhibitors (LY2228820). Compared to those transfected with si-NEAT1 alone, SRA01/04 cells treated with si-NEAT1 in combination with either PDTC or LY2228820 showed higher cell viability (Figure 7B). Similarly, after treatment with si-NEAT1 combined with PDTC or LY2228820, more significant changes were observed in GSH activity (Figure 7C) and MDA level (Figure 7D). Taken together, these results demonstrated that NEAT1 exerts protective effects against H2O2-induced cell injury in HLECs through mediating p65 and p38 MAPK signaling pathways (36).
In conclusion, we constructed an in vitro model of oxidative damage by exposing HLECs (SRA01/04) to H2O2. The present study showed for the first time that NEAT1 knockdown could protect HLECs against H2O2-induced cell damage by inactivating the phosphorylation of p65 and p38 MAPK. Our study has provided a potential therapeutic target for the treatment of cataracts, but further in vivo studies are needed to fully elucidate NEAT1 functional role.
Reporting Checklist: The authors have completed the MDAR checklist. Available at http://dx.doi.org/10.21037/atm-20-7365
Data Sharing Statement: Available at http://dx.doi.org/10.21037/atm-20-7365
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-7365). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures were approved by the ethics committee of Nantong University (No. NT20180513) and carried out in accordance with the Declaration of Helsinki (2013 version). All patients signed a written informed consent before taking part.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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