The Role of lncRNA GAS5 in Oxidative Stress and Mitochondrial Dysfunction in Age-Related Cataracts: Insights From Transcriptome Profiling.

The Role of lncRNA GAS5 in Oxidative Stress and Mitochondrial Dysfunction in Age-Related Cataracts: Insights From Transcriptome Profiling - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Invest Ophthalmol Vis Sci . 2026 Jun 3;67(6):7. doi: 10.1167/iovs.67.6.7 The Role of lncRNA GAS5 in Oxidative Stress and Mitochondrial Dysfunction in Age-Related Cataracts: Insights From Transcriptome Profiling Deqian Kong 1 , Yue Liu 1 , Li Li 1 , Huajun Wang 1 , Ke Li 1 , Tingting Zhang 2 , Naisong Qi 2 , Guangying Zheng 1, ✉ Author information Article notes Copyright and License information 1 Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China 2 Department of Pharmacology, Henan Provincial Administration for Market Regulation. Zhengzhou, Henan, China * Correspondence: Guangying Zheng, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe East Rd., Erqi District, Zhengzhou 450000, China; zzzgyzzdx@163.com . ✉ Corresponding author. Accepted 2026 May 6; Received 2025 Oct 21; Collection date 2026 Jun. Copyright 2026 The Authors This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. PMC Copyright notice PMCID: PMC13236481  PMID: 42233724 Abstract Purpose This study aimed to investigate the role of lncRNA GAS5 and GADD45a in H₂O₂-induced oxidative stress and mitochondrial dysfunction in human lens epithelial cells (LECs), and to explore the underlying ceRNA mechanism involving miR-3666. Methods Human LECs were exposed to H₂O₂ to induce oxidative stress. The functional roles of lncRNA GAS5, miR-3666, and GADD45a were assessed using gene knockdown and overexpression experiments. Apoptosis, oxidative damage, and mitochondrial function were evaluated. In addition, anterior lens capsule tissues were collected from patients with age-related cataract and normal controls, and the expression levels of lncRNA GAS5 and GADD45a were measured to assess their correlation in clinical samples. Additionally, antisense oligonucleotides targeting lncRNA GAS5 were tested in a rat model of age-related cataract (ARC). Results lncRNA GAS5 was found to act as a competing endogenous RNA that sponges miR-3666, leading to upregulation of GADD45a expression. This lncRNA GAS5/GADD45a axis increased the susceptibility of LECs to oxidative damage and apoptotic cell death. A pronounced positive association between lncRNA GAS5 and GADD45a was identified in cortical and nuclear cataracts. Inhibition of lncRNA GAS5 with antisense oligonucleotides alleviated oxidative stress in an ARC rat model. Conclusion These results reveal a novel ceRNA regulatory pathway in which lncRNA GAS5 promotes oxidative stress and apoptosis in LECs through the miR-3666/GADD45a axis, identifying lncRNA GAS5 as a potential therapeutic target for ARC treatment. Keywords: lncRNA GAS5, age-related cataract, oxidative stress, GADD45a, antisense oligonucleotide, bioinformatics Age-related cataract (ARC) is currently the most prominent ophthalmic disease that causes vision loss and blindness in older adults. 1 Numerous studies have demonstrated that oxidative stress is a crucial mechanism underlying the occurrence and progression of ARC. 2 Based on this pathological mechanism, most current drug research focuses on increasing antioxidant levels, such as glutathione, vitamins C and E, 25-hydroxycholesterol, lanosterol, and their derivatives, to delay disease progression. 3 , 4 Additionally, advancements in lens regeneration have opened new avenues for cataract treatment. 5 Although these antioxidant interventions ameliorate oxidative damage to some extent, their clinical efficacy has not yet reached ideal levels, suggesting the presence of more complex and precise regulatory mechanisms underlying ARC pathogenesis. The highly conserved secondary structure of long non-coding RNA (lncRNAs) suggests that they play important biological roles in the regulation of processes such as the cell cycle, differentiation, and metabolism, which depend on their structure and genomic localization. 6 – 8 In addition, lncRNAs can interact with microRNAs (miRNAs), DNA, mRNAs, and proteins to perform critical biological functions within cells. 9 – 11 Several lncRNAs have been shown to regulate lens metabolism. Shen et al. 12 reported that the transcription level of lncRNA MIAT was upregulated in patients with ARC. The lncRNA TUG1 was also found to be upregulated in the anterior lens capsule of patients with ARC, 13 suggesting that lncRNAs are closely related to the development of ARC. Nucleic acid drugs such as antisense oligonucleotides (ASOs) are designed to target specific genes or gene products based on a patient's genomic profile and can enhance treatment efficacy. 14 , 15 ASOs bind to complementary sequences of target RNAs to form RNA-DNA hybrids. 16 This specific pairing enables the ASOs to exert diverse regulatory effects. Recent preclinical studies have demonstrated the therapeutic potential of ASOs in various ocular diseases, such as retinal degeneration and corneal dystrophy, 17 , 18 underscoring their promise for treating RNA-mediated disorders in the eye. Given the complexity of the ARC and the ongoing advances in lncRNA research, numerous unidentified lncRNAs are likely to be discovered and investigated. High-throughput transcriptome sequencing revealed that the lncRNAs GAS5 and GADD45a were significantly upregulated in oxidatively stressed lens epithelial cells (LECs) and anterior capsule tissues from patients with ARC. In cortical and nuclear cataracts, we further observed a pronounced positive correlation between lncRNA GAS5 and GADD45a expression. Mechanistic studies have demonstrated that lncRNA GAS5 exhibits competing endogenous RNA (ceRNA) activity, sequestering miR-3666, thereby relieving its suppression of GADD45a. The lncRNA GAS5/miR-3666/GADD45a axis aggravates oxidative stress, mitochondrial dysfunction, and apoptosis in LECs. Targeting the lncRNA GAS5 with ASOs effectively alleviated oxidative damage in rat lens. Collectively, these findings suggest that the lncRNA GAS5 promotes ARC progression via a ceRNA mechanism and may serve as a novel therapeutic target for cataract treatment. Material and Methods Cell Culture and Transfection In this study, we used HLE-B3 cells from the BeNa Culture Collection and cultured them in high-glucose Dulbecco's modified Eagle's medium (10-013-CV; Corning Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum (10099-141, Gibco, USA) and 1% penicillin-streptomycin (CA0075; Beijing Leagene Biotechnology, Beijing, China). The cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Subsequent experiments were performed as follows: lentiviral particles for lncRNA GAS5 overexpression and knockdown, their corresponding controls, and GADD45a overexpression and knockdown plasmids were constructed by Genechem Corporation (Shanghai, China). LECs were treated with lentiviral particles carrying either lncRNA GAS5 overexpression or knockdown constructs and treated with 2 µg/mL puromycin to select successfully infected cells. Cells were transfected with GADD45a overexpression or shRNA knockdown plasmids using lipofectamine 8000 (C0533; Beyotime Institute of Biotechnology, Jiangsu, China). The cells were also transfected with miR-3666 mimics, inhibitors, or their respective negative controls using lipofectamine 2000 (11668-019; Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's protocol. The IBSBIO company (Shanghai, China) was responsible for the synthesis of all the plasmids, and their sequences are listed in Supplementary Table S1 . Clinical Samples Seventy patients (aged 60–90 years, including both sexes) with ARC, primarily featuring nuclear cataract opacity, were enrolled in the experimental anterior lens capsule group. Their lens opacity ranged from Grade III to V according to the Emery classification. The anterior capsules of the control group were obtained from 22 individuals (aged 10–50 years, of any sex) who were either corneal transplant donors or had undergone eye enucleation. Individuals with complex cataracts (e.g., owing to high myopia, glaucoma, uveitis, or ocular trauma) or systemic diseases (e.g., hypertension or diabetes) were excluded. Approval was obtained from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No. 2021-KY-1192-002), and all human procedures adhered to the Declaration of Helsinki. The clinical samples were processed as follows: specimens for PCR were lightly rinsed with sterile PBS and cryopreserved in liquid nitrogen; those for immunohistochemistry were rinsed with sterile PBS and fixed in ocular tissue fixative. Cell Viability Assay Cells were seeded into 96-well plates at a density of approximately 5 × 10 3 cells per well. Different concentrations of H 2 O 2 were then added, and the cells were treated for 24 hours. Subsequently, the CCK-8 working reagent (HY-K0301; MedChemExpress, Monmouth Junction, NJ, USA) was placed in each well, followed by an incubation period of 60 minutes in the dark. Absorbance was measured at a wavelength of 450 nm using a microplate reader (Molecular Devices, San Jose, CA, USA). Hoechst 33342 Staining Cells were treated with different concentrations of H₂O₂ and incubated for 24 hours. After treatment, the supernatant was discarded, and the cells were washed with PBS. The cell culture medium containing Hoechst 33342 (HY-15559; MedChemExpress) was added to each well and incubated in the dark. The nuclear morphology of LECs was observed under a fluorescence microscope (Zeiss, Oberkochen, Germany). FISH Assay After treatment with 200 µM H₂O₂, all procedures were performed according to the manufacturer's instructions (Bes1002, Bersin Biotechnology, Guangzhou, China). Nuclei were subsequently stained by dropwise addition of culture medium containing Hoechst 33342 at room temperature in the dark.The stained cells were then observed under a fluorescence microscope (Molecular Devices). RNA Extraction and Real-Time QRT-PCR Total RNA was extracted from tissues or cells using TRIzol reagent (10296010; Invitrogen, Carlsbad, CA, USA), and its concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Reverse transcription was carried out using the HiScript II Q RT SuperMix kit (R233; Nanjing Novizan Medical Technology Co., Ltd., Nanjing, China), followed by quantitative PCR using the AceQ qPCR SYBR Green kit (Q111; Nanjing Novizan Medical Technology Co., Ltd. ). Each 20 µL reaction system contained 10 µL SYBR Green Master Mix, 0.4 µL each of forward and reverse primers (10 µM), 0.4 µL 50 × ROX Reference Dye 1, 2 µL cDNA template, and 6.8 µL RNase-free water. Relative gene expression levels between groups were calculated using the 2 −ΔΔCT method. The sequences of all the primers have been documented in Supplementary Table S2 . RNA Sequencing (RNA-Seq) To identify differentially expressed lncRNAs under oxidative stress, LECs were treated with PBS or 200 µM H₂O₂. To further characterize lncRNA GAS5-associated transcriptional alterations, two RNA-seq comparisons were subsequently performed: normal vs. lncRNA GAS5 knockdown NC and lncRNA GAS5 knockdown NC versus lncRNA GAS5 knockdown. Total RNA was extracted using TRIzol reagent. All RNA samples that met the following criteria: concentration ≥ 100 ng/µL, total amount ≥ 3 µg, RNA integrity number ≥ 7.0, and rRNA was removed using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The mRNA was fragmented, and sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit (Illumina). All samples were sequenced on the Illumina NovaSeq 6000 platform. Bioinformatic Prediction of MiRNA The potential interactions between miRNAs and both lncRNA GAS5 and GADD45a were predicted using the following bioinformatic databases: Starbase ( http://starbase.sysu.edu.cn/ ), miRDB ( http://mirdb.org/ ), miRcode ( http://mircode.org/ ), and RNAhybird ( http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ ). StarBase predicts both miRNA-mRNA and miRNA-lncRNA interactions. MiRDB is designed for miRNA–mRNA target prediction and does not support miRNA-lncRNA analysis. miRcode annotates miRNA binding sites within non-coding RNAs and mRNA, including lncRNAs. RNAhybrid allows prediction of potential miRNA binding sites by using the FASTA sequence of the lncRNA as input. MiRNA–lncRNA interactions for lncRNA GAS5 were predicted using RNAhybird, whereas miRNA–mRNA interactions for GADD45a were predicted using StarBase, miRDB, and miRcode. The overlapping miRNAs predicted to target both lncRNA GAS5 and GADD45a were identified as high-confidence candidates for subsequent validation. TUNEL Staining The identification of apoptotic LECs were detected using a one-step TUNEL apoptosis assay kit (C1086; Beyotime Institute of Biotechnology), and the nuclei were counterstained with Hoechst 33342. Apoptotic LECs were subsequently quantified under a fluorescence microscope. Immunohistochemistry All tissue samples were fixed in immunostaining fixative, embedded in paraffin, and sectioned into 4 µm serial slices. Sections were baked at 65°C for one to two hours. Endogenous peroxidase activity was blocked using 0.3% H₂O₂. Antigen retrieval was performed using a pressure cooker for one minute. The sections were then incubated overnight at 4°C with the primary antibodies. A biotin-conjugated secondary antibody was added dropwise, and the mixture was incubated for 60 minutes. Horseradish peroxidase–conjugated streptavidin was then added. DAB was used for chromogenic development, followed by hematoxylin counterstaining. Grayscale intensity was measured using Adobe Photoshop software to calculate relative expression levels. Detection of Reactive Oxygen Species (ROS) and Mitochondrial Membrane Potential (MMP) The intracellular ROS and MMP levels were detected using O08 (BB-46052; Beijing Best Bio Technical Co., Ltd., Beijing, China) and TMRE (C2001S; Beyotime Institute of Biotechnology) fluorescent probes, respectively, follow the manufacturer's directions. Hoechst 33342 was used for nuclear staining. Fluorescence images were captured using a fluorescence microscope. Assessment of Intracellular SOD and Malondialdehyde (MDA) The cells were processed according to the instructions provided by the SOD activity assay kit (A001-3; Nanjing Jiancheng, China). MDA levels were determined using an MDA assay kit (A003-1, Nanjing Jiancheng, China) according to the manufacturer's instructions. Reaction solutions were thoroughly mixed with the corresponding cell samples. The protein concentration in each group was calculated via a BCA protein quantification kit (P0012; Beyotime Institute of Biotechnology). The absorbance for the SOD, MDA, and BCA assays was measured at 450 nm, 532 nm, and 562 nm, respectively, using a microplate reader (Model iMark; Bio-Rad Laboratories, Ann Arbor, MI, USA). Quantification of Intracellular Glutathione (GSH) Standard samples were prepared according to the manufacturer's instructions provided by the GSH assay kit (BC1175; Solarbio Life Science, Beijing, China), and standard curves were constructed. The absorbance of the samples was measured, and the concentration of GSH was calculated using the provided formula. Dual-Luciferase Reporter Assay The Starbase, TargetScan, miRDB, miRcode, and RNAbird databases were used to predict miRNAs that potentially interact with both lncRNA GAS5 and GADD45a. The binding sites of miR-3666 in the 3′-UTR of GADD45a and lncRNA GAS5 were predicted by IBSBIO Company (Shanghai, China). Wild-type and mutant sequences of the lncRNAs GAS5 and GADD45a were cloned into pmiRGLO vectors. The plasmids were co-transfected into cells, along with either miR-3666 mimics or an miRNA negative control. Luciferase activity was determined using a Dual-Luciferase Reporter Gene Assay Kit (11402ES80; Yeasen, Shanghai, China). Western Blot Assay Cells were lysed on ice using a RIPA lysis buffer (PS0013; Beijing Leagene Biotechnology). After determining the protein concentration, lysates were separated by 10% or 15% SDS-PAGE rapid gel preparation kit (PE009 or PE035; Shanxi Zhonghui Hecai Biomedical Technology Co., Ltd., Shanxi, China) and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA), which were blocked with 5% skimmed milk for one hour. The membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with the secondary antibodies for one hour at room temperature. Signal detection was performed using an ECL reagent kit (BioSharp, Hefei, China), and band intensities were quantified using Adobe Photoshop software. The following primary antibodies were used: P53 (AF0865, 1:1500; Affinity Biosciences, Changzhou, China), Bax (AF0120, 1:1000; Affinity Biosciences), cleaved caspase-3 (9664, 1:1000; Cell Signaling Technology, Danvers, MA, USA), GADD45a (ab180768, 1:1000 for WB; Abcam, Cambridge, MA, USA), GADD45a (13747-1-AP, 1:400 for IHC; Proteintech, Rosemont, IL, USA), SOD1 (NBP2-24915, 1:500; Novus Biologicals, Littleton, CO, USA), MDA (NBP2-59366, 1:1000; Novus Biologicals), and Drp1 (DF7037, 1:1000; Affinity Biosciences), and Fis1 (DF12005, 1:1000; Affinity Biosciences). Rats and ASOs Injection All animal procedures were approved by the Department of Pharmacology, Henan Provincial Institute of Drug and Medical Device Inspection (approval number: HNYJ-20220921) and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Pregnant Sprague-Dawley rats ( n = 6) were purchased from Charles River company (Beijing, China), and ASO-GAS5 was synthesized by IBSBIO company (Shanghai, China). Eight-day-old rats were injected subcutaneously with sodium selenite to induce lens opacity characterized predominantly by oxidative stress-related nuclear cataract. Rats that developed Grade IV lens opacity were selected and randomly categorized into three groups: (i) Control group ( n = 10): Rats with completely clear lens were injected with ddH₂O in the anterior chamber; (ii) ASO-NC group ( n = 10): Rats with Grade IV lens opacity were injected with ASO-NC GAS5 in the anterior chamber; (iii) ASO injection group (n = 10): Rats with Grade IV were injected with ASO-GAS5 in the anterior chamber. All rats were housed in standard SPF cages under a 12/12-hour light/dark cycle. More than five punctures of the anterior chamber may lead to significant corneal edge erosion. We limited the number of injections to five. A total of 1 µL of ASO was injected into the anterior chamber every five days, for a total of five injections. Transmission Electron Microscopy Lens anterior capsule tissues containing lens epithelial cells were carefully dissected and cut into approximately 1 mm³ pieces, followed by fixation in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) at 4°C overnight and post-fixation in 1% osmium tetroxide. Samples were dehydrated through a graded ethanol series, embedded in epoxy resin, and sectioned into ultrathin sections (60–90 nm), which were stained with uranyl acetate and lead citrate. Ultrastructural images of lens epithelial cells were acquired using a JEM-1400-FLASH transmission electron microscope (JEOL, Tokyo, Japan). Statistical Analysis GraphPad Prism (v.9.5.0) was used to analyze the data. All data are presented as the mean ± SD. Student's t -test was used to compare two groups. One-way ANOVA was used to compare multiple groups, and the least significant difference test was used for post-hoc comparisons. Significance was defined as P < 0.05. Results H 2 O 2 Co-Culture Reduces LEC Viability and Induces Apoptosis H₂O₂ induces oxidative toxicity by generating ROS that penetrate cell membranes and cause oxidative damage. Exposure to increasing concentrations of H₂O₂ led to progressively greater damage to the LECs ( Fig. 1 A). At 200 µM, cell viability declined to 50.39% ± 1.58%, indicating a clear concentration-dependent effect. Hoechst 33342 staining revealed uniformly light-blue nuclei under normal conditions, whereas higher H₂O₂ concentrations resulted in increased punctate blue fluorescence, indicating chromatin condensation and nuclear fragmentation ( Fig. 1 B). TUNEL staining showed that H 2 O 2 exposure increased the number of TUNEL-positive apoptotic cells ( Fig. 1 C). Apoptosis increased in a concentration-dependent manner, consistent with the CCK-8 assay findings. Based on these results, 200 µM H₂O₂ for 24 hours was selected for subsequent experiments. Figure 1. Open in a new tab Identification of oxidative damage-related lncRNAs and the localization of lncRNA GAS5. (A) Effect of H 2 O 2 (0, 100, 200, 300, 400 and 500 µM) on cell viability as determined by the CCK-8 assay. (B) Hoechst 33342 staining and TUNEL staining. (C) Statistics of TUNEL-positive cells. (D) Volcano plot showing the differential expression of lncRNAs between the control and H 2 O 2 (Drug group). (E) Visualization of clustering analysis of differentially expressed lncRNAs using a heatmap. (F) Between-group correlation analysis. (G, H) The Gene Set Enrichment Analysis (GSEA) indicates that GADD45a is enriched in both apoptosis- and senescence-related pathways. (I) The mRNA expression of lncRNA GAS5/TPM4/STAT2/ AC087721.2 /DUXAP10 in LECs. (J) The mRNA expression of lncRNA GAS5/TPM4 in tissues. (K) Localization of lncRNA GAS5 in chromosomes. (L) Localization of lncRNA GAS5 in cells using FISH assay. Identification of Differentially Expressed lncRNAs by RNA-Seq Based on |log₂Fold Change| > 1 and P adj < 0.05, 162 differentially expressed lncRNAs were identified after H₂O₂ treatment, including 94 upregulated and 68 downregulated lncRNAs ( Fig. 1 D). These were visualized using the pheatmap package in R software ( Fig. 1 E). Intergroup correlation is shown in Figure 1 F. GSEA analyses identified GADD45a, a core gene in the apoptosis and senescence pathways, as a potential target regulated by lncRNA GAS5 ( Figs. 1 G, 1 H) and the core gene lists for both pathways are presented in Supplementary Table S3 . The qRT-PCR validation of the top five upregulated lncRNAs confirmed consistent expression trends for lncRNA GAS5-204 (ENST00000454068) and lncRNA TPM4-209 (ENST00000588483). However, lncRNA TPM4-209 did not show significant differential expression in anterior lens capsule tissues. In contrast, lncRNA GAS5-204 showed high expression in the anterior lens capsule tissue and was therefore selected for further analysis ( Figs. 1 I, 1 J). The positional information of lncRNA GAS5 is shown in Figure 1 K. FISH analysis revealed the predominant cytoplasmic localization of lncRNA GAS5 after H₂O₂ treatment, suggesting a post-transcriptional regulatory role ( Fig. 1 L). lncRNA GAS5 Positively Regulates GADD45a Expression and Is Associated With ARC The qRT-PCR confirmed successful overexpression and knockdown of lncRNA GAS5 in LECs. lncRNA GAS5 expression was significantly upregulated in the overexpression group ( Fig. 2 A) and was effectively silenced by sh-GAS5-2, which was selected for subsequent experiments ( Fig. 2 B). Both qRT-PCR and western blot analyses showed that GADD45a expression was significantly upregulated after lncRNA GAS5 overexpression and was downregulated upon lncRNA GAS5 knockdown ( Figs. 2 C–E), indicating that lncRNA GAS5 positively regulates. The qRT-PCR analysis showed that lncRNA GAS5 was upregulated in cortical, nuclear, and mature white cataracts but remained low in posterior subcapsular cataracts and the control group ( Fig. 2 F). Immunohistochemistry revealed elevated GADD45a expression in cortical and nuclear cataracts, with weaker expression in posterior subcapsular cataracts and control anterior lens capsule tissues ( n = 6 per group; Figs. 2 G, 2 H). The lncRNA GAS5 and GADD45a exhibited concordant expression patterns. Further correlation analysis ( n = 10 per group for cortical, nuclear, posterior subcapsular, and mature white cataracts) revealed that lncRNA GAS5 expression was significantly positively correlated with GADD45a expression in cortical and nuclear cataracts, whereas this correlation was markedly attenuated in mature white cataracts. Moreover, lncRNA GAS5 expression was positively correlated with age across these three cataract types ( Figs. 2 I–L). Figure 2. Open in a new tab Regulation of GADD45a expression by lncRNA GAS5. (A) The qRT-PCR detection of the effect of lncRNA GAS5 overexpression. (B) The qRT-PCR detection of the knockdown efficiency of sh-GAS5. (C) The qRT-PCR analysis of GADD45a mRNA levels after transfection with lncRNA GAS5 overexpression or knockdown lentivirus. (D, E) WB analysis of GADD45a protein levels after transfection with lncRNA GAS5 overexpression or knockdown lentivirus and quantification of protein bands. (F) The qRT-PCR detection of lncRNA GAS5 expression in different cataract types. (G, H) Immunohistochemical detection of high GADD45a expression in anterior capsule tissues from ARC patients and quantification. (I) Correlation analysis of lncRNA GAS5 and GADD45a in cortical cataract. (J) Correlation analysis of lncRNA GAS5 and GADD45a in nuclear cataract. (K) Correlation analysis of lncRNA GAS5 and GADD45a in mature white cataract. (L) Correlation analysis of lncRNA GAS5 and age. Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. lncRNA GAS5 Regulates GADD45a Expression via MiR-3666 Predicting the potential interaction between miRNAs and both lncRNA GAS5 and GADD45a can be achieved by using databases such as Starbase, miRDB, mircode, and RNAbird ( Fig. 3 A). Both miR-3666 and miR-4295 are capable of binding. Because the minimum free energy of miR-3666 binding to GADD45a is higher than that of miR-4295, greater minimum free energy indicates higher binding stability. We subsequently verified miR-3666 using a dual luciferase assay. A dual-luciferase reporter assay confirmed that miR-3666 targets both GADD45a and lncRNA GAS5. Luciferase activity was significantly decreased in the lncRNA GAS5-WT + miR-3666 mimic group, with no change observed in the lncRNA GAS5-Mut group ( Figs. 3 B, 3 C), confirming the interaction at the predicted binding site. Similarly, luciferase activity was significantly reduced in cells co-transfected with GADD45a-WT and miR-3666 mimics but remained unchanged in the GADD45a-Mut group ( Figs. 3 D, 3 E), indicating that GADD45a is a direct downstream target of miR-3666. Figure 3. Open in a new tab lncRNA GAS5 acts as a ceRNA to regulate the expression of miR-3666/GADD45a in HLE-B3 cells. (A) Predicted miRNAs potentially interacting with both lncRNA GAS5 and GADD45a. (B, C) Dual luciferase assays detecting the binding between lncRNA GAS5 and miR-3666. (D, E) Dual luciferase assays detecting the binding between GADD45a and miR-3666. (F) The qRT-PCR analysis of miR-3666 regulatory effects on GADD45a mRNA expression. (G, H) WB analysis of miR-3666 regulatory effects on GADD45a protein expression with quantification of protein bands. (I) The qRT-PCR analysis of lncRNA GAS5 and miR-3666 regulatory effects on GADD45a mRNA expression. (J, K) WB analysis of lncRNA GAS5 and miR-3666 regulatory effects on GADD45a protein expression with quantification of protein bands. Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. The qRT-PCR analysis revealed that GADD45a mRNA expression was significantly downregulated by the miR-3666 mimics and upregulated by the miR-3666 inhibitor ( Fig. 3 F), demonstrating a negative regulatory relationship. Consistent with this, Western blot analysis showed that the miR-3666 mimic significantly suppressed GADD45a protein levels, whereas the miR-3666 inhibitor further increased its expression ( Figs. 3 G, 3 H). These results indicate that miR-3666 directly suppresses GADD45a expression at both the transcriptional and translational levels. In addition, qRT-PCR results showed that lncRNA GAS5 overexpression increased GADD45a mRNA levels, whereas its knockdown led to a decrease. This effect was suppressed by the miR-3666 mimics and enhanced by the miR-3666 inhibitor ( Fig. 3 I). Western blot analysis showed consistent changes in GADD45a protein expression ( Figs. 3 J, 3 K), indicating that lncRNA GAS5 regulates GADD45a through miR-3666 in a ceRNA-dependent manner. lncRNA GAS5/GADD45a Promotes H 2 O 2 -Induced Apoptosis in LECs To investigate the role of lncRNA GAS5 in LECs apoptosis, TUNEL staining revealed a significant increase in TUNEL-positive cells in the lncRNA GAS5 overexpression group compared to the control. In contrast, knockdown of lncRNA GAS5 significantly reduced the number of apoptotic cells ( Figs. 4 A, 4 B). Western blot analysis further confirmed that lncRNA GAS5 overexpression elevated the levels of the proapoptotic proteins Bax, P53, GADD45a, and cleaved caspase-3, whereas lncRNA GAS5 knockdown led to reduced expression ( Figs. 4 C, 4 D). The qRT-PCR analysis shows that GADD45a expression was significantly upregulated in the overexpression group ( Fig. 4 E). The efficiency of GADD45a knockdown and overexpression was verified by qRT-PCR and Western blot analysis, with sh-GADD45a3 exhibiting the highest silencing efficiency and was thus selected for subsequent experiments ( Figs. 4 F–H). GADD45a overexpression led to a marked increase in TUNEL-positive cells, whereas its knockdown significantly reduced the number of apoptotic cells ( Figs. 4 I, 4 J). Additionally, cleaved caspase-3 expression was significantly elevated after GADD45a overexpression and suppressed by its knockdown ( Figs. 4 K, 4 L). These results indicate that both lncRNA GAS5 and GADD45a contribute to H₂O₂-induced apoptosis in LECs. Figure 4. Open in a new tab lncRNA GAS5/GADD45a enhances H₂O₂-triggered cell apoptosis. (A, B) TUNEL assay showing the effect of lncRNA GAS5 on apoptosis and statistics of TUNEL-positive cells. (C, D) WB analysis of lncRNA GAS5 effects on apoptosis-related proteins (Bax, cleaved caspase-3, P53 and GADD45) with quantification of protein band intensity. (E) The qRT-PCR identification of plasmids upregulating GADD45a mRNA. (F) The qRT-PCR identification of the most effective shRNA for GADD45a mRNA inhibition. (G, H) WB identification of the most effective shRNA for GADD45a protein inhibition with quantification of protein bands. (I, J) TUNEL assay showing the effect of GADD45a on apoptosis and statistics of TUNEL-positive cells. (K, L) WB analysis of GADD45a effects on cleaved caspase-3 with quantification of protein bands. Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. lncRNA GAS5/GADD45a Promotes Oxidative Stress and Mitochondrial Dysfunction Overexpression of lncRNA GAS5 significantly increased intracellular ROS levels and decreased MMP ( Figs. 5 A, 5 B). Additionally, WB assay showed lncRNA GAS5 overexpression reduced SOD1 expression and increased MDA levels ( Figs. 5 C, 5 D), accompanied by a reduction in GSH ( Fig. 5 E), indicating enhanced oxidative stress. Western blot analysis further demonstrated that lncRNA GAS5 overexpression upregulated mitochondrial fission proteins Drp1 and Fis1 and reduced the expression of phosphorylated Drp1 (p-Drp1) and Mfn1/2, whereas lncRNA GAS5 knockdown reversed these changes ( Figs. 5 F, 5 G). We performed two RNA-seq comparisons (normal vs. lncRNA GAS5 knockdown NC and lncRNA GAS5 knockdown NC vs. lncRNA GAS5 knockdown; Figs. 6 A, 6 B). Integrated differential expression analysis revealed approximately 14% overlap between the two datasets ( Fig. 6 C). KEGG enrichment analysis showed that lncRNA GAS5 knockdown attenuated senescence-associated pathways while enhancing mismatch repair and base excision repair ( Figs. 6 D, 6 E), suggesting that reduced lncRNA GAS5 expression reduced the enrichment of senescence-related pathways while increasing the enrichment of mismatch repair and base excision repair pathways. Figure 5. Open in a new tab lncRNA GAS5 promotes ROS accumulation and mitochondrial fragmentation. (A, B) Detection of ROS and MMP after transfection with lncRNA GAS5 overexpression or knockdown lentivirus and fluorescence intensity analysis. (C, D) WB analysis of lncRNA GAS5 regulation of antioxidant proteins (SOD1 and MDA) with quantification of protein bands. (E) Detection of GSH levels. (F, G) WB analysis and quantification of Drp1, p-Drp1, Fis1, and Mfn1/2 protein levels after lncRNA GAS5 overexpression or knockdown. Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Figure 6. Open in a new tab Transcriptome sequencing identifies reduced senescence and enhanced repair pathways following lncRNA GAS5 knockdown. (A) Volcano Plot: Normal vs. lncRNA GAS5 knockdown NC . (B) Volcano Plot: lncRNA GAS5 knockdown NC vs. lncRNA GAS5 knockdown. (C) Venn analysis of shared differentially expressed mRNAs. (D) KEGG pathway analysis of DEGs between Normal and lncRNA GAS5 knockdown NC groups. (E) KEGG pathway analysis of DEGs between lncRNA GAS5 knockdown NC versus lncRNA GAS5 knockdown groups. GADD45a overexpression led to a marked accumulation of ROS and a reduction in MMP ( Figs. 7 A, 7 B), along with decreased SOD1 protein expression ( Fig. 7 C), increased MDA levels ( Fig. 7 D), and reduced GSH level ( Fig. 7 E). Western blot analysis showed that Drp1, p-Drp1, Fis1 and Mfn1/2 expression was also elevated after GADD45a overexpression, whereas GADD45a knockdown suppressed their expression ( Figs. 7 F, 7 G). These findings suggest that both lncRNA GAS5 and GADD45a contribute to oxidative stress and promote mitochondrial dysfunction. Figure 7. Open in a new tab GADD45a drives oxidative stress and mitochondrial dysfunction. (A, B) Detection of ROS and MMP after transfection with GADD45a overexpression or knockdown plasmid and fluorescence intensity analysis. (C) Detection of SOD activity. (D) Measurement of MDA levels. (E) Detection of GSH levels. (F, G) WB analysis of Drp1, p-Drp1, Fis1, and Mfn1/2 protein levels after GADD45a overexpression or knockdown with quantification of protein bands. Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. ASO-GAS5 Alleviates Oxidative Damage in a Rat Model of Selenium-Induced Cataract After three subcutaneous injections of 2 mmol/L Na₂SeO₃ into the dorsal region, all rats developed varying degrees of lens opacity. Histological analysis and lens opacity assessment confirmed the successful establishment of the selenium-induced cataract model ( Figs. 8 A, 8 B). TEM revealed that lens epithelial cells primarily exhibited ultrastructural features of apoptosis at stages I–IV of selenite-induced cataract. At stages V-VI, the cell membrane became incomplete, vacuoles appeared in the cytoplasm, and cell death involved both necrosis and apoptosis. Therefore lenses at stage IV were selected for subsequent experiments ( Fig. 8 C). We evaluated the safety of ASO-GAS5. At one day after anterior chamber injection, no significant ciliary congestion was observed, showing no difference compared with the control group. OCT imaging revealed no corneal edema, and the anterior chamber remained clear ( Fig. 8 D). We decided to use ASO-GAS5 for the experiments because qRT-PCR showed that ASO-GAS5 2 worked best in the rat lens ( Fig. 8 E). Hematoxylin and eosin (H&E) staining revealed a significant decrease in vacuolization of the lens fibers after ASO-GAS5 injection. The anterior segment photograph reveals a decrease in lens opacity compared to previous results ( Fig. 8 F). ASO-GAS5 treatment significantly reduced GADD45a expression compared to the ASO-NC group via immunohistochemistry ( Figs. 9 A, 9 B), indicating that targeted intervention with lncRNA GAS5 expression led to significant downregulation of GADD45a levels. Furthermore, ASO-GAS5 treatment partially restored SOD activity ( Fig. 9 C). Furthermore, it reduced the expression of cleaved caspase-3 in rat lens, thereby decreasing apoptosis ( Figs. 9 D, 9 E). These findings suggest that silencing lncRNA GAS5 can effectively mitigate oxidative damage in selenium-induced cataracts. Figure 8. Open in a new tab Cataract model establishment and ASO-GAS5 injection safety evaluation. (A) Schematic diagram of oxidative stress model establishment and experimental procedures in rat lens. (B) H&E staining and anterior segment photography (grades I-VI) of rat lens after subcutaneous injection of 2 mmol/L Na₂SeO₃ solution. (C) TEM evaluation of LECs death modalities across lens opacification grades. (D) Ocular safety assessment of ASO-GAS5 injection. (E) Screening of ASOs for GAS5 knockdown. (F) H&E staining and anterior segment imaging of lens. Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Figure 9. Open in a new tab Assessment of the in vivo effects of ASO-GAS5 in the cataract model. (A, B) Immunohistochemical detection of GADD45a expression and quantification of GADD45a-positive area in LECs. (C) Detection of SOD activity. (D, E) WB analysis of cleaved caspase-3 after anterior chamber injection of ASO-GAS5 with quantification of protein band intensity. (F) Mechanistic Role of the lncRNA GAS5/miR-3666/GADD45a axis in lens epithelial cell dysfunction. Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Discussion ARC is the leading cause of blindness in the older population worldwide. 19 The stability of LECs plays a critical role in maintaining lens transparency, and oxidative damage to LECs is a key factor in cataract development. 20 Although many studies have revealed the importance of lncRNAs in the ARC, there is a lack of systematic screening focused on lncRNAs related to oxidative damage. In this study, we established an oxidative injury model in LECs using H₂O₂ and performed RNA-seq. Among the upregulated lncRNAs, GAS5 and TPM4 were identified as significantly differentially expressed, with lncRNA GAS5 being selected for further investigation due to its consistent upregulation in both H₂O₂-treated cells and anterior capsule tissues. Notably, the transcript of lncRNA GAS5 has not yet been studied in cataract. We found that lncRNA GAS5 and GADD45a were significantly upregulated in the anterior lens epithelial capsules of cortical, nuclear, and mature white cataracts, whereas their expression levels remained low in posterior subcapsular cataracts and normal lens. Further analysis showed that lncRNA GAS5 expression was significantly positively correlated with GADD45a in the anterior capsules of cortical and nuclear cataracts, whereas this correlation was markedly attenuated in mature white cataracts, suggesting that the regulatory relationship between the two changes during cataract progression. This difference may be closely related to distinct pathogenic stress environments among cataract subtypes. Epidemiological studies indicate that nuclear and cortical cataracts are more strongly associated with oxidative stress–related factors, such as smoking, ultraviolet exposure, and elevated ambient temperature, 21 – 23 whereas posterior subcapsular cataract appears less related to solar exposure, with high-dose folate intake reported as a potential risk factor. 24 In cortical and nuclear cataracts dominated by oxidative stress, lncRNA GAS5 may participate in age-related damage responses through regulation of GADD45a; in contrast, this regulatory axis is not prominently activated in posterior subcapsular cataracts. As the disease progresses to mature white cataracts, although lncRNA GAS5 expression remains age-associated, widespread epithelial damage and functional impairment may shift GADD45a activation toward a nonspecific global stress response, thereby reducing its dependence on lncRNA GAS5-mediated fine regulation and resulting in a weakened correlation between the two. Our study demonstrated that the overexpression of lncRNA GAS5 resulted in increased accumulation of ROS. Excessive ROS can lead to a reduction in the levels of intracellular antioxidants and MMP. 25 Notably, lncRNA GAS5 overexpression also leads to decreased expression of GSH, a critical intracellular antioxidant that plays a key role in maintaining redox balance and protecting cells from oxidative damage. 26 Consistently, RNA-seq analysis showed that lncRNA GAS5 knockdown reduced the enrichment of senescence-associated pathways while enhancing mismatch repair and base excision repair, suggesting that elevated lncRNA GAS5 promotes oxidative injury and senescence, whereas its suppression improves cellular repair capacity. Mitochondria are the primary targets of ROS and are highly dynamic organelles that constantly undergo fusion and fission. 27 , 28 These morphological changes are closely linked to the regulation of cell apoptosis. Subsequent analysis showed that overexpression of lncRNA GAS5 upregulated Drp1 and Fis1, which are key proteins involved in mitochondrial fission, indicating enhanced mitochondrial fragmentation. Interestingly, lncRNA GAS5 overexpression simultaneously increased total Drp1 protein levels while significantly reducing p-Drp1 at the inhibitory Ser637 site. Because Ser637 phosphorylation normally suppresses Drp1's fission activity, this dual regulation—elevated Drp1 expression combined with reduced inhibitory phosphorylation—synergistically enhances Drp1 mitochondrial translocation and fission capacity, thereby amplifying mitochondrial fragmentation. Drp1 is a key mediator of mitochondrial fission, and its recruitment to the outer mitochondrial membrane is facilitated by adaptor proteins such as Fis1. Studies have shown that Drp1 binds to the proapoptotic protein Bax to induce apoptosis. 29 Notably, several studies have reported that Drp1 and Fis1 act synergistically to promote mitochondrial fragmentation and trigger apoptosis. 30 Increased Drp1 expression exacerbates ROS production and depletes antioxidant enzymes such as GSH and SOD. 31 The resulting decrease in MMP leads to the opening of mitochondrial permeability transition pores, promoting the release of cytochrome c into the cytoplasm, upregulation of Bax, and the subsequent activation of cleaved caspase-3. 32 Our study showed that lncRNA GAS5 increased the expression of cleaved caspase-3, as confirmed by TUNEL assays, indicating enhanced cell apoptosis. Therefore we propose that the downregulation of lncRNA GAS5 inhibits excessive mitochondrial fission, reduces Drp1 and Fis1 expression, partially restores MMP, delays Bax activation, and alleviates oxidative stress-induced apoptosis. The GADD45 family (including GADD45a, GADD45b, and GADD45g) is involved in stress response signaling and has been implicated in aging, cardiovascular and autoimmune diseases. 33 – 35 GADD45a plays critical role in DNA repair, cell cycle regulation, and apoptosis. 36 Evidence has indicated that it contributes to age-related diseases. 37 Previous studies have shown that GADD45a impairs mitochondrial function in adipocytes, disrupts ATP synthesis in muscle tissue, and contributes to cellular aging. 38 Moreover, studies have demonstrated that GADD45a disrupts MMP in HepG2 cells, leading to mitochondrial dysfunction. 39 In this study, GADD45a overexpression increased ROS levels, decreased SOD activity, and reduced MMP. In addition, increased intracellular ROS production promotes excessive mitochondrial fission. 40 Upregulation of Drp1 and Fis1 facilitates mitochondrial fragmentation, thereby enhancing cleaved caspase-3 activation and triggering apoptosis. These findings suggested that GADD45a exacerbates oxidative damage by promoting ROS production and accumulation, thereby further aggravating mitochondrial dysfunction. A FISH assay confirmed the subcellular localization of lncRNA GAS5, showing predominant distribution in the cytoplasm, suggesting its potential involvement primarily in post-transcriptional regulatory processes. 41 Mechanistically, lncRNA GAS5 may regulate GADD45a expression via a ceRNA mechanism. Bioinformatic analysis and dual-luciferase reporter assays in this study confirmed that miR-3666 binds to both lncRNA GAS5 and the 3'-UTR of GADD45a, suggesting that lncRNA GAS5 can function as a molecular sponge for miR-3666. Sequestration of miR-3666 relieved its inhibitory effect on GADD45a, thereby enhancing GADD45a expression. The lncRNA GAS5/miR-3666/GADD45a axis constitutes a regulatory pathway that enhances oxidative stress, disrupts mitochondrial dynamics, and promotes the apoptosis of LECs under oxidative damage. High levels of selenium induce ROS production, making cells highly susceptible to oxidative damage. 42 This was consistent with the criteria established for animal models. Therefore, selenium-induced cataracts are a valuable model of ARC. 43 , 44 ASOs designed to target specific genes or gene products based on genomic characteristics offer a promising strategy for improving therapeutic efficacy. 45 , 46 After the injection of ASO-GAS5, the expression of the stress-response protein GADD45a in the rat lens was significantly reduced, whereas the activity of the antioxidant enzyme SOD was significantly increased. SOD plays a critical role in neutralizing ROS and protecting lens from oxidative damage. 47 , 48 These results suggest that ASO-GAS5 alleviates oxidative damage in selenium-induced cataracts by downregulating GADD45a and upregulating SOD activity, thereby helping maintain the normal physiological function of LECs. We propose that the reduction in cortical fiber vacuolization results from improved LECs integrity. LECs apoptosis compromise Na⁺/K⁺-ATPase activity, leading to disturbed redox homeostasis and disrupted ionic–osmotic gradients, which in turn cause localized accumulation of endogenous water within peripheral cortical fibers and the appearance of vacuole-like spaces on H&E staining. ASO-GAS5 reduced LEC apoptosis and oxidative injury, preserved surface ionic pump function and redox balance, and thereby prevented secondary osmotic dysregulation in the peripheral cortex, markedly diminishing these vacuole-like changes. Nevertheless, despite the promising therapeutic effects of ASO targeting lncRNA GAS5 observed in our experimental models, several translational challenges must be acknowledged and addressed before clinical application. Potential issues such as off-target effects, suboptimal delivery efficiency, inadequate cellular uptake, and uncertain long-term safety remain inadequately explored and require thorough investigation in future studies. Moreover, although selenium exposure reliably induces oxidative injury, we selected lens at Grade IV opacity, in which LECs still undergo apoptosis as the predominant form of cell death, but with a severity far exceeding that of natural aging. This acute and intensified apoptotic pattern differs from the slow, mixed modes of epithelial cell injury that characterize human ARC, representing an inherent limitation of the selenite model. Future studies incorporating complementary chronic models will therefore be essential to better approximate the long-term biological context of human cataractogenesis. In summary, the steady-state environment of the lens is influenced by multiple factors, with ROS recognized as a major contributor. ROS is closely linked to the development of various ocular diseases, particularly age-related conditions. Therefore, greater attention should be given to maintaining redox balance, mitochondrial dysfunction, and the role of ROS in disease. The lncRNA GAS5/miR-3666/GADD45a axis may offer novel insights into the prevention and treatment of ARC by modulating ROS levels and mitochondrial function, as illustrated in Figure 9 F. Although the present study establishes the lncRNA GAS5–miR-3666–GADD45a axis as a key regulatory pathway in cataract-associated lens epithelial cells, its direct involvement in controlling apoptosis and oxidative injury has not yet been fully delineated. Future studies will therefore focus on systematically dissecting how this axis modulates oxidative stress signaling and apoptotic pathways, which will be essential for defining its mechanistic contribution to lens epithelial cell injury and for evaluating its potential as a therapeutic target in age-related cataract. Conclusions The lncRNA GAS5/miR-3666/GADD45a axis promotes oxidative stress and mitochondrial dysfunction in LECs, thereby contributing to ARC progression. Targeting GAS5 with ASOs alleviates oxidative damage and offers a potential therapeutic strategy for ARC. Supplementary Material Supplement 1 iovs-67-6-7_s001.pdf (79.1KB, pdf) Supplement 2 iovs-67-6-7_s002.pdf (79.1KB, pdf) Supplement 3 iovs-67-6-7_s003.pdf (166.3KB, pdf) Acknowledgments The authors thank the First Affiliated Hospital of Zhengzhou University and the Henan Provincial Administration for Market Regulation for their support. Supported by the National Natural Science Foundation of China [grant number 81970786]. Author Contributions Declaration: Guangying Zheng and Yue Liu conceived and designed the study. Deqian Kong conducted the experiments. Li Li, Ke Li, and Huanjun Wang analyzed the data. Tingting Zhang and Naisong Qi contributed to the data interpretation and manuscript preparation. All the authors have read and approved the final version of the manuscript. Data Availability Statements: The sequencing data generated in this study were deposited in the Gene Expression Omnibus (GEO) database under the accession number [ GSE298013 , GSE317222 ]. Disclosure: D. Kong , None; Y. Liu , None; L. Li , None; H. Wang , None; K. Li , None; T. Zhang , None; N. Qi , None; G. Zheng , None References 1. Cicinelli MV, Buchan JC, Nicholson M, Varadaraj V, Khanna RC. Cataracts. Lancet . 2023; 401(10374): 377–389. [ DOI ] [ PubMed ] [ Google Scholar ] 2. Świerczyńska M, Tronina A, Smędowski A.. 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Supplementary Materials Supplement 1 iovs-67-6-7_s001.pdf (79.1KB, pdf) Supplement 2 iovs-67-6-7_s002.pdf (79.1KB, pdf) Supplement 3 iovs-67-6-7_s003.pdf (166.3KB, pdf) Articles from Investigative Ophthalmology & Visual Science are provided here courtesy of Association for Research in Vision and Ophthalmology ACTIONS View on publisher site PDF (25.2 MB) Cite Collections Permalink RESOURCES Similar articles Cited by other articles Links to NCBI Databases Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

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