Aging of the human eye lens: Epigenetic landscape and therapeutic targets in age‑related cataracts (Review)

Introduction

Cataracts

Cataracts, characterized by progressive opacification of the ocular lens, are the leading cause of visual impairment and blindness worldwide (1). Cataracts can be classified into age-related cataracts (ARCs), congenital cataracts and secondary cataracts caused by other reasons (2). ARCs, also known as senile cataracts, constitute the most prevalent cataract subtype, with typical onset after 50-60 years of age (3). Currently, no clinically proven interventions exist to prevent ARC progression. Surgery remains the gold-standard therapeutic approach, achieving rapid visual rehabilitation in most cases (4). Despite surgical efficacy, ARCs persist as a critical global health challenge due to high patient volume, substantial surgical costs and potential postoperative complications (5,6). Therefore, continuous and dedicated efforts are required to develop improved therapeutic strategies for ARC prevention and treatment.

Oxidative stress

ARCs develop through the long-term interplay of multiple pathogenic factors, with complex and heterogeneous underlying mechanisms. To date, multiple risk factors, including aging, diabetes, genetic predisposition, oxidative stress and ultraviolet (UV) radiation exposure, have been implicated in ARC pathogenesis (3). The current understanding of ARC pathogenesis strongly implicates reactive oxygen species (ROS) accumulation as a key driver, with numerous studies demonstrating the pathogenic roles of superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) generated during aerobic metabolism (7-9). Excessive ROS induce oxidative stress by chemically modifying and damaging critical cellular components (10). Oxidative stress, in turn, causes modifications in lens proteins (including enzymes, crystallins and other chaperones), thereby reducing their solubility and stability, and ultimately promoting crystallin aggregation (11). These alterations induce light scattering within the lens, progressively leading to lens opacity and subsequent visual acuity impairment (12). Oxidative stress also directly damages lens epithelial cells (LECs), which are critical for maintaining lens transparency and metabolic homeostasis, through the peroxidation of proteins, lipids and DNA, while concurrently activating signalling pathways and transcription factors (11,13-17). As the most metabolically active components of the lens, LECs possess a sophisticated antioxidant defence system to mitigate oxidative stress (18,19). However, when ROS levels exceed the antioxidant capacity of LECs, oxidative damage to the LECs triggers apoptosis, an early event in ARC pathogenesis (20,21).

Epigenetic modifications

Epigenetics refers to alterations in gene expression without changes to the DNA sequence in response to environmental, developmental and nutritional factors (22,23). These alterations are reversible and subject to dynamic regulation, leading to heritable changes in gene expression and cellular phenotypes (24). Extensive research has demonstrated that epigenetic mechanisms function as pivotal regulators of disease pathogenesis, influencing initiation, progression, prevention and treatment (25,26). The emergence of aberrant epigenetic changes is associated with the pathogenesis of various diseases, including cancer, diabetes, Alzheimer's disease and other age-related disorders (27-30). Epigenetic modifications not only underlie the molecular mechanisms driving disease development but also serve as promising biomarkers for clinical diagnosis and enable precision-targeted therapeutic interventions (31,32). Epigenetic modifications primarily encompass DNA methylation, histone modifications, RNA modifications, non-coding RNAs (ncRNAs) and chromatin remodelling. A substantial body of research has established the critical involvement of epigenetic processes in gene expression regulation, DNA damage repair, and cell cycle control and aging, highlighting their fundamental importance in molecular biology and genetics (33-35).

Breakthroughs in epigenetic research related to ocular diseases have only emerged within the past decade. Epigenetic modifications have grown into a leading frontier in biomedical research, with accumulating studies revealing their regulatory impact on several ocular diseases such as cataracts, glaucoma, diabetic retinopathy and age-related macular degeneration (36-39). Epigenetic research can deepen the understanding of the molecular mechanisms of ocular diseases and open novel avenues for the development of epigenome-targeted treatments. The present review summarizes current advances in epigenetic research on ARCs, discussing the implications of these research advances for future investigations and the therapeutic potential of epigenetic interventions in ARCs.

Epigenetic modifications in ARCs

Epigenetic modifications represent heritable changes in gene expression that occur independently of alterations in the underlying DNA sequence (40). Epigenetic modifications, including ncRNAs, DNA methylation, histone modifications and N6-methyladenosine (m6A) modification, are now widely acknowledged as key molecular drivers in the development of ARCs (41-43). A deeper understanding of these epigenetic mechanisms will elucidate disease pathogenesis and enable targeted epigenetic therapies for ARCs. The subsequent sections will first introduce key epigenetic alterations, including ncRNAs, DNA methylation, histone modifications and m6A modification, and then systematically examine their roles in ARC pathogenesis.

ncRNAs

The central dogma of molecular biology describes the flow of genetic information from DNA to RNA to protein (44,45). For decades, research on cataract pathogenesis has predominantly focused on protein-coding genes. However, advances in sequencing technologies have enabled the identification of numerous unique ncRNAs and confirmed their cellular presence. ncRNAs represent a class of RNA molecules that lack protein-coding sequences and are consequently not translated into polypeptides or proteins. This category includes microRNAs (miRNAs/miRs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), transfer RNA-derived small RNAs (tsRNAs) and small nuclear RNAs (46). Through specific interactions with DNA, RNA and proteins, ncRNAs precisely regulate nearly all biological pathways, including signal transduction (47), transcriptional and post-transcriptional regulation (48,49), translational repression (50), and translation initiation regulation (51) (Fig. 1). ncRNAs operate in both physiological and pathological contexts, exhibiting strong potential as novel molecular biomarkers and therapeutic targets (52-54). Consequently, ncRNAs serve a pivotal role in the pathogenesis and progression of ARCs, as extensively documented in several studies (55-57). The present review primarily focuses on reviewing advances in research on the four major ncRNAs (miRNAs, lncRNAs, circRNAs and tsRNAs) and their regulatory networks in ARCs.

Figure 1 Functional landscape of non-coding RNAs. miRNA, microRNA; lncRNA, long non-coding RNA; circRNA, circular RNA; tsRNA, transfer RNA-derived small RNA; piRNA, PIWI interacting RNA; ORF, open reading frame; RBP, RNA-binding protein; 3' UTR, 3' untranslated region; AGO, Argonaute; YB-1, Y-box binding protein 1.

miRNAs

miRNAs are the most extensively studied and best characterized small ncRNAs to date. miRNAs are small ncRNA molecules, typically 20-30 nucleotides in length, that serve critical roles in structural, enzymatic and regulatory processes across biological systems (58). Precursor miRNAs undergo a tightly regulated series of nuclear and cytoplasmic processing steps mediated by the endoribonucleases DROSHA and DICER, respectively, ultimately yielding mature miRNAs. The mature miRNAs are then incorporated into the RNA-induced silencing complex and regulate gene expression by binding to the 3' untranslated region (UTR) of target mRNAs, leading to either mRNA degradation or translational repression (58,59). miRNAs have also been reported to interact with other regions, such as the 5' UTR, coding sequence and gene promoters (60,61). Furthermore, accumulating evidence has demonstrated that miRNAs can post-transcriptionally activate gene expression in specific contexts, challenging the conventional paradigm of miRNA-mediated silencing and highlighting their functional versatility (62-64). Notably, miRNAs form intricate, highly interconnected regulatory networks, wherein individual miRNAs typically regulate multiple mRNA targets, and conversely, most mRNAs are subject to combinatorial regulation by multiple miRNAs (65). Diverse physiological and pathological processes, including apoptosis (66), autophagy (67), oxidative stress (68), senescence (69) and DNA repair (70), have been shown to influence the biological characteristics of LECs in ARC, as evidenced by both in vitro experiments and animal studies. miRNAs have been demonstrated to serve important roles in these processes, making it plausible that miRNAs are closely associated with cataract formation (Table SI).

Studies have revealed alterations in miRNA expression profiles between transparent and cataractous lenses (71-73). For example, miRNA let-7b expression exhibits a positive association with patient age, and elevated miRNA let-7b expression is associated with higher nuclear (N), cortical (C) and posterior subcapsular (P) cataract scores according to the Lens Opacities Classification System III (74) in patients with ARCs (73). Similarly, miR-34a expression levels vary among patients of different ages, with moderate associations observed between high N, C and P cataract scores and high miR-34a levels (75). Elevated levels of miR-210-3p have been identified in the aqueous humour of patients with ARCs, and the elevation demonstrated strong diagnostic potential for ARCs while being associated with oxidative stress marker levels (76). miR-15a and miR16-1 are expressed at a higher level in the LECs of patients with cataracts compared with normal controls (77). Notably, ARCs can be classified into distinct subtypes based on the location of opacification, including cortical cataracts, nuclear cataracts (NCs), and posterior and anterior subcapsular cataracts (ASCs), while the remainder is classified as mixed cataracts, and differential expression of miRNAs has been observed among these subtypes (72). The expression levels of let-7a-5p, let-7d-5p, let-7g-5p and miR-23b-3p differ between patients with NC and ASC, as measured in lens epithelium samples (72). Thus, aberrant miRNA expression levels may not only act as risk factors in the formation and progression of ARC, but also serve as potential biomarkers for ARCs based on their differential expression patterns in different severity stages and subtypes of ARCs. However, no miRNAs are used for clinical diagnosis currently, requiring further validation in clinical practice.

miRNAs typically function by binding to target mRNAs and inhibiting translation (58,59). Thus, specific miRNAs indirectly contribute to cataract formation by regulating key factors involved in cellular processes such as apoptosis, proliferation and oxidative stress. For instance, miR-23b-3p promotes apoptosis and inhibits autophagy in LECs under oxidative stress by directly targeting and downregulating silent information regulator 1 (SIRT1), a critical mediator of oxidative stress resistance, thereby contributing to ARC pathogenesis (78). Similarly, miR-211 induces apoptosis and decreases cell viability by suppressing SIRT1 expression (79). E2F transcription factor 3 (E2F3), a member of the E2F transcription factor family, serves essential roles in regulating cell proliferation, apoptosis and differentiation (80,81). As a key cell cycle regulator, E2F3 promotes cell cycle re-entry in quiescent lens cells. However, aberrant cell cycle re-entry may trigger programmed cell death (82-84), and has been observed in LECs, which is closely linked to ARC pathogenesis (85). Experimental evidence has identified miR-34a (86), miR-15a (87), miR-221 (88), miR-630 and miR-378a-5p (89), as direct post-transcriptional suppressors of E2F3, collectively contributing to lens opacity progression. miR-34a, the expression of which is increased in the cataractous lens, triggers mitochondria-mediated apoptosis and oxidative stress by suppressing Notch2 (90). In addition, the upregulation of miR-34a together with the downregulation of hexokinase 1 (HK1) inhibits the proliferation and induces the apoptosis of LECs, thus accelerating the opacification of mouse lenses via the HK1/caspase 3 signalling pathway (91). Furthermore, Cao et al (67) demonstrated that miRNA let-7c-5p directly targeted excision repair cross-complementing rodent repair deficiency, complementation group 6 (ERCC6), and thus, inhibited its role in autophagy, a process necessary for maintaining lens transparency through degradation of misfolded proteins and damaged organelles. miR-378a is upregulated in human cataract tissues and inhibits LEC proliferation while promoting apoptosis by modulating the ROS/PI3K/AKT signalling pathway (92).

Emerging evidence has indicated that certain miRNAs function as cataract suppressors by preventing lens opacification (68,93,94). Given that ARC pathogenesis is associated with apoptotic cell death in the lens, extensive research has highlighted dysregulated apoptosis of LECs as a key pathogenic mechanism in cataract development (95-97). Bcl-2 family proteins are essential for the intrinsic apoptotic pathway (98). B-cell lymphoma-2-like-2 (BCL2L2), a Bcl-2 family member, is an important regulator of apoptosis (99). Zhang et al (93) identified BCL2L2 as a direct target of miR-133b, demonstrating that miR-133b-mediated downregulation of BCL2L2 expression led to apoptosis inhibition. While BCL2L2 promoted apoptosis, miR-133b functioned as an anti-apoptotic regulator by suppressing BCL2L2 expression (93). Likewise, miR-182, which is implicated in various ophthalmic disorders, including pterygium (100), glaucoma (101) and age-related macular degeneration (102), protects LECs against oxidative stress-induced apoptosis by regulating the nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (NOX4) and p38 MAPK signalling pathway (68). Furthermore, miR-125b is downregulated in ARC lens tissue, where it suppresses p53 mRNA expression and exerts anti-apoptotic effects on LECs (94). Although numerous miRNAs have been demonstrated to attenuate lens opacity progression, experimental validation by in vivo studies conducted in animals, such as rats or rabbits, remains limited. To the best of our knowledge, only three in vivo studies have been reported at present. Administration of antagomirs targeting miR-326 (103), miR-29a-3p (104) and miR-187 (105) independently inhibited cataract formation in animal models of ARC, suggesting their potential as non-surgical therapeutic candidates.

Single nucleotide polymorphisms (SNPs) within miRNA binding sites can affect the base pairing between miRNAs and their target mRNAs, thereby altering miRNA-mediated gene regulation (106). Oxidative DNA damage and impaired DNA repair capacity in LECs are strongly involved in ARC pathogenesis (107,108). Accumulating data have demonstrated that SNPs within miRNA binding sites of oxidative DNA damage repair genes may modulate ARC susceptibility (70,109,110). Zou et al (70) systematically analysed 10 miRNA-binding SNPs located in the 3' UTRs of seven oxidative damage-related genes. Their findings demonstrated that the C allele of XPC-rs2229090 increased susceptibility to the nuclear type of ARC (ARNC). Mechanistically, the C allele exhibited stronger binding affinity for hsa-miR-589-5p compared with the G allele, resulting in reduced XPC expression and impaired DNA repair capacity in LECs (70). Furthermore, Kang et al (109) demonstrated that the protective effect of Nei-like DNA glycosylase 2 (NEIL2)-rs4639T in ARCs may be mediated through maintenance of normal NEIL2 expression levels, potentially via disruption of the binding of rs4639T with hsa-miR-3912-5p. Additionally, the rs78378222 polymorphism in the p53 3' UTR contributes to ARC pathogenesis by regulating miR-125b-mediated apoptosis in LECs (110). Taken together, these findings suggest that SNP-mediated alterations in miRNA-dependent post-transcriptional regulation constitute a novel pathogenic mechanism in ARCs and reveal novel therapeutic targets.

In general, research on miRNA alterations in ARCs has reached a relatively mature stage, with comprehensive studies demonstrating the crucial roles of miRNAs in mediating LEC lesions in vitro and cataract progression in vivo (103-105). Exosomal miRNAs have become a research focus; however, their relationship with ARCs remains poorly understood. At present, only two studies have demonstrated the roles of exosomal miR-222-3p in ARC formation and miR-125a-3p in disease progression (66,111). Exosomes are 50-200-nm extracellular nanovesicles derived from multivesicular bodies in the cytoplasm and serve as an important medium of intercellular communication (112,113). Exosomes regulate receptor cells and tissues by transmitting effectors, including proteins, mRNAs or miRNAs (114). Various types of cells can secret miRNAs via exosomes, a process that protects miRNAs from degradation and ensures their function in target cells (115). Exosomes transport miRNAs to target cells without being degraded by RNAses, making exosomal miRNAs more stable and reliable biomarkers than their non-exosomal counterparts (116). Exosomal miRNAs are emerging as promising biomarkers for aging and age-related diseases (117-119). Furthermore, exosomes represent promising drug delivery vehicles due to their inherent advantages, including high biocompatibility, high biological barrier penetration and high circulatory stability (120). Therefore, investigating exosomal miRNAs could help to elucidate the pathogenesis of ARCs, facilitate biomarker identification and enable the development of innovative intervention strategies.

lncRNAs

Alongside short miRNAs, a wide array of longer transcripts, referred to as lncRNAs, has been increasingly characterized. lncRNAs are >200 bp long, and exhibit relatively low expression levels, poor stability and limited evolutionary conservation (121). Therefore, lncRNA research presents considerable challenges due to these inherent molecular characteristics, and is only recently undergoing a surge. Notably, similar to miRNAs, most lncRNAs are associated with human diseases; however, in contrast to their smaller counterparts, lncRNAs exhibit cell type-specific expression patterns and distinct subcellular localization (122). lncRNAs perform diverse regulatory functions, including acting as miRNA sponges, signal mediators, molecular decoys, chromatin modifiers, and regulators of gene expression and pre-mRNA splicing (123,124). Among these functions, their role as competing endogenous RNAs (ceRNAs) or miRNA sponges is particularly significant. The ceRNA hypothesis, first proposed by Salmena et al (125) in 2011, suggests that lncRNAs regulate gene expression post-transcriptionally by competitively binding to miRNAs via shared miRNA response elements, thereby preventing miRNA-mRNA interactions. Through diverse molecular mechanisms, lncRNAs regulate critical biological processes such as stem cell maintenance, cellular differentiation and chromatin remodelling, as well as transcription, splicing, translation, degradation and transport of mRNA (126,127). Previous studies have indicated that lncRNAs are highly active in ARCs, where they may either promote or suppress disease initiation and progression (Table SII) (128-130).

In 2017, Zhang et al (131) collected anterior capsular samples from both healthy controls and patients with ARCs and conducted high-throughput sequencing to detect differentially expressed lncRNAs, identifying 7,041 candidate lncRNAs with differential expression. To investigate the comprehensive profile of ncRNAs and their interaction network in cataractogenesis, a study conducted in 2022 employed autophagy-related gene 7 (ATG7)-knockout LECs to inhibit autophagy under oxidative stress conditions, and performed detailed analyses of RNA sequencing data to characterize mRNA and lncRNA expression profiles in LECs exposed to oxidative stress. The authors identified 263 upregulated and 336 downregulated differential lncRNAs between wild-type and ATG7-deficient LECs treated with H2O2, among which 24 lncRNAs showed potential interactions with 10 cataract-related miRNAs (132). Collectively, these studies establish that lncRNAs and their associated ceRNA networks (lncRNA-miRNA-mRNA axis) are heavily involved in ARC pathogenesis (131,132).

Emerging evidence has established the pivotal role of numerous lncRNAs as pathogenic drivers in ARC pathogenesis. As aforementioned, lncRNAs can act as ceRNAs or endogenous miRNA sponges, effectively sequestering miRNAs and attenuating their post-transcriptional repression of target mRNAs (125). Building upon this theoretical framework, Jin et al (133) experimentally validated miR-214 as a direct target of lncKCNQ1OT1. Their findings demonstrated that lncKCNQ1OT1 knockdown upregulated miR-214 expression and subsequently downregulated the expression of its downstream effector caspase-1, indicating the pathogenic role of the lncKCNQ1OT1/miR-214/caspase-1 axis in cataract formation (133). Furthermore, lncKCNQ1OT1 functions as a molecular sponge for miR-223-3p, which directly targets BCL2L2. Through the lncKCNQ1OT1/miR-223-3p/BCL2L2 axis, lncKCNQ1OT1 promotes apoptosis and pyroptosis, while exacerbating oxidative damage in H2O2-treated LECs (134). Another study demonstrated that lncKCNQ1OT1 downregulation protected LECs from oxidative stress-induced damage via the miR-124-3p/BCL-2-like 11 pathway (135). In addition to lncKCNQ1OT1, TUG1 is another well-characterized lncRNA that exacerbates the pathogenesis of ARC, and has been shown to promote apoptosis in H2O2 or UV irradiation-treated LECs through three distinct molecular pathways: TUG1/miR-196a-5p (136), TUG1/miR-421/caspase-3 (20) and TUG1/miR-29b/second mitochondria-derived activator of caspases (137). Furthermore, research has identified multiple novel pathogenic lncRNAs, including NEAT1 (129,138), MEG3 (139,140) and OIP5-AS1 (141), with additional candidates expected to emerge as research progresses.

By contrast, other studies have indicated that certain lncRNAs exert protective effects to prevent ARC progression. For example, lncRNA phospholipase C δ3 (PLCD3)-OT1 may act as a ceRNA to promote PLCD3 expression by sponging miR-224-5p, thus promoting cell proliferation and viability, and inhibiting apoptosis, under oxidative stress conditions (142). Similarly, lncRNA NONHSAT143692.2 modulates 8-oxoguanine DNA glycosylase (OGG1) expression in LECs by competitively binding to miR-4728-5p, thereby protecting LECs from cell apoptosis and oxidative damage (130). Furthermore, Cheng et al (143) revealed that lncRNA H19 suppressed apoptosis, mitigated oxidative damage, and promoted proliferation and viability in UV irradiation-treated LECs through the functional interplay of lncRNA H19, miR-29a and thymine DNA glycosylase.

Although acting as miRNA sponges represents a well-established function of lncRNAs (144-146), their other regulatory functions are also implicated in cataract pathogenesis. lncRNAs are typically classified into five major classes according to their relative genomic locations: Antisense (AS), intergenic, overlapping, intronic and full lapping (147). Within these lncRNA classes, AS lncRNAs, which are reverse-strand complements of their endogenous protein-coding counterparts, are generally non-protein-coding and account for a substantial proportion of the entire long non-coding transcriptome (148-150). Previous studies have demonstrated that natural AS transcripts participate in diverse biological processes by regulating sense gene expression at multiple levels, including transcriptional regulation via gene promoter activation, and post-transcriptional regulation through mRNA stabilization and translational modulation (151,152). Glutathione peroxidase 3 (GPX3)-AS has been found to upregulate GPX3 expression at both the mRNA and protein levels, enhancing the activity of this ROS scavenger to inhibit LEC apoptosis, thereby revealing a potential therapeutic target for ARCs (153). Furthermore, lncRNAs can also serve as direct precursors for miRNA biogenesis (154). A representative example is lncRNA H19, which serves as the precursor for miR-675, where H19 downregulation leads to reduced miR-675 expression. lncRNA H19 modulates LEC function via miR-675-mediated regulation of crystallin αA (CRYAA) expression, offering novel insights into ARNC pathogenesis (155).

Overall, lncRNAs participate in ARC pathogenesis through multifaceted regulation of LEC biology, including oxidative stress responses and apoptotic pathways. Given their functional versatility and multi-target nature, the precise roles and regulatory networks of several lncRNAs in ARC pathogenesis have yet to be comprehensively characterized. Furthermore, although in vitro experiments offer valuable mechanistic insights, their translational relevance to whole organisms remains limited. To the best of our knowledge, no in vivo models investigating lncRNAs in ARCs have been reported at present. Animal models, which better recapitulate the complexity of physiological systems, are crucial for validating the observed in vitro effects.

circRNAs

circRNAs are single-stranded, covalently closed RNA molecules without 5' caps and 3' poly-A tails, and are usually produced through a back-splicing process (156,157). The absence of both 5' caps and 3' polyadenylated tails renders circRNAs resistant to exonuclease-mediated degradation, thus ensuring their stability and longevity in cellular processes (158). circRNAs possess distinctive properties and diverse functions that continue to be elucidated. Similar to lncRNAs, circRNAs can function as efficient miRNA sponges, representing one of their most well-characterized regulatory modes (159-161). Beyond their well-known role as miRNA sponges, studies have revealed that circRNAs can also interact with RNA-binding proteins (162), regulate RNA splicing and transcription (163,164), and undergo translation into functional peptides or proteins (165,166). circRNAs also participate in a wide range of biological processes, including fibrosis, cell apoptosis, proliferation, differentiation and angiogenesis (167). In recent years, their roles as vital regulators in multiple ocular diseases have attracted increasing attention (168-170). Similar to linear ncRNAs, circRNAs are implicated in cataract formation (Table SIII) (171-173). The circRNA/miRNA/mRNA regulatory network has advanced the understanding of the molecular basis of cataractogenesis.

Studies have characterized specific circRNAs that contribute to ARC pathogenesis. For example, hsa_circ_0105558 promotes apoptosis and aggravates the oxidative damage of LECs under H2O2 exposure through the miR-182-5p/activating transcription factor 6 axis (174). hsa_circ_0007905 upregulates eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) expression by competitively binding to miR-6749-3p, consequently enhancing LEC apoptosis while inhibiting LEC proliferation, resulting in ARC progression (175). Notably, hsa_circ_0007905 expression in LECs is upregulated through methyltransferase like 3 (METTL3)-mediated m6A modification (175). A comprehensive discussion of m6A modifications of circRNAs is presented in the m6A modification section. Notably, circMRE11A_013 (circMRE11A) acts primarily as a molecular scaffold that interacts with UBX domain-containing protein 1 rather than functioning as a miRNA sponge, and promotes excessive activation of ataxia-telangiectasia mutated kinase (ATM), ultimately inducing LEC cell cycle arrest and senescence through the ATM/p53/p21 signalling pathway (176). Furthermore, recombinant adeno-associated virus vector virions of circMRE11A have been injected into the mouse vitreous cavity, providing in vivo evidence that circMRE11A drives lens aging and opacification in mice (176). In addition to circMRE11A, circSTRBP (hsa_circ_0088,427) exerts its biological effects independently of miRNA sponging activity by interacting with insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), and enhances the stability and expression of NOX4 mRNA in LECs (171). circSTRBP exacerbates H2O2-induced oxidative stress and apoptosis in LECs by enhancing NOX4 mRNA stability through IGF2BP1 recruitment, providing novel perspectives for ARC intervention (171).

Emerging evidence has identified multiple protective circRNAs in ARC pathogenesis, with several candidates exhibiting potent antioxidant and anti-apoptotic effects in LECs. circRNA homeodomain interacting protein kinase 3 (circHIPK3) promotes viability and proliferation, inhibits apoptosis, and alleviates oxidative damage in H2O2-treated LECs via three distinct molecular pathways: circHIPK3/miR-221-3p/PI3K/AKT (177), circHIPK3/miR-193a/CRYAA (178) and circHIPK3/miR-495-3p/histone deacetylase 4 (HDAC4) (179). circ_0060,144 regulates HIPK3 expression by sponging miR-23b-3p, thereby influencing LEC homeostasis through regulation of proliferation, apoptosis and the oxidative stress response (180). UV radiation downregulates circRNA erythrocyte membrane protein band 4.1 expression, which subsequently reduces 3'(2'), 5'-bisphosphate nucleotidase 1 levels via miR-24-3p binding, thus inhibiting proliferation and promoting apoptosis in LECs (181). Furthermore, circRNA 06209 acts as a ceRNA by sequestering miR-6848-5p to neutralize its suppressive effect on arachidonate 15-lipoxygenase, ultimately promoting proliferation and inhibiting apoptosis in H2O2-treated LECs (182). Notably, injection of a circRNA_06209-overexpressing plasmid into a Na2SeO3-induced cataract model attenuated lens opacity while reducing insoluble protein aggregation and increasing soluble protein levels in the lens, demonstrating the anti-cataract efficacy of circRNA_06209 in both in vitro and in vivo settings (182).

Given that circRNAs exhibit high stability, evolutionary conservation across species and tissue specificity (183), investigating ARC-related circRNAs as potential biomarkers or therapeutic targets could open novel avenues for the diagnosis, prevention and treatment of ARCs. However, only a limited number of circRNAs in ARCs have well-characterized biological functions and mechanistic pathways. While circRNAs employ diverse molecular mechanisms and functions, current research on their roles in ARCs has largely focused on their function as miRNA sponges and their interactions with proteins (171,174,184-186), suggesting that novel functions or mechanisms remain to be identified.

tsRNAs

tsRNAs constitute a family of small ncRNAs produced by the cleavage of precursor or mature transfer RNAs (tRNAs) via specific endonucleases (187). tsRNAs are mainly classified into two types: tRNA halves and tRNA-derived fragments, based on the cleavage position of the precursor or mature tRNAs (188). Advances in high-throughput sequencing technologies have revealed that tsRNAs serve crucial roles in the regulation of gene expression, post-transcriptional modifications, apoptotic inhibition and protein translation (189-191), while their dysregulation contributes to various pathological processes in diseases such as cancer, cardiovascular diseases and Alzheimer's disease (192-194). Investigations have begun to reveal the crucial involvement of tsRNAs in ophthalmic diseases (195-197). Through integrated next-generation sequencing analysis, a study conducted in 2022 systematically established the first evidence of the changes in tsRNA profiles in Emory mice, a well-characterized model for human ARCs (198). A total of 422 differentially expressed tsRNAs were identified in 8-month-old mice (156 upregulated and 266 downregulated), with Gene Ontology (GO) analysis revealing that these target genes were predominantly enriched in processes including but not limited to camera-type eye development and sensory organ development (198). As tsRNA research remains in its infancy, further investigations focusing on the association between tsRNAs and ARCs, as well as their underlying molecular mechanisms, are needed to provide novel insights into cataractogenic processes and ARC disease management.

ncRNA regulatory networks

Understanding ncRNA functions in isolation becomes increasingly difficult due to intricate regulatory networks among ncRNAs. These networks involve multiple layers of interaction: ncRNAs frequently share common protein-coding mRNA targets; miRNAs functionally interact with other ncRNA species rather than operating in isolation; and lncRNAs and circRNAs, in turn, regulate the abundance of available miRNAs (199). ncRNA networks serve a pivotal role in ARC pathogenesis by modulating gene expression, oxidative stress responses and pathological processes in LECs, indicating potential targets for early diagnosis and therapeutic intervention (134,200).

In addition to the networks among different types of ncRNAs, such as the aforementioned ceRNAs, interactions also exist between the same type of ncRNAs. Emerging research across various diseases, including cancers and cardiovascular diseases, has revealed that certain miRNAs can be involved in the same biological pathways either as effectors or regulators (201). Krek et al (202) demonstrated that myotrophin (Mtpn) was a direct target of miR-124, let-7b and miR-375, as evidenced by small interfering RNA (siRNA)-mediated downregulation of Mtpn protein levels and suppression of luciferase activity in dual-luciferase reporter assay. Cotransfection of the luciferase reporter and a pool of siRNA duplexes designed to mimic the function of miR-124, let-7b and miR-375, resulted in normalized luciferase activity that was substantially less than the activity in any of the groups transfected with a single siRNA, revealing cooperative regulation of Mtpn by these miRNAs (202). Similarly, lncRNAs could interact with each other by co-regulating genes participating in the same or similar biological functions (203). Multiple lncRNAs, such as OIP5-AS1, TUG1 and NEAT1, have been implicated in the synergistic regulation of genes and pathways in cancer (204). Current research on ncRNAs in ARCs primarily focuses on their dysregulation in ARCs and the ceRNA mechanism (104,140,174,205). The cooperative interactions among ncRNAs remain poorly characterized and require further analyses and validation to achieve a comprehensive understanding of the cooperative behaviours of ncRNAs in ARC pathogenesis.

DNA methylation

DNA methylation, a fundamental epigenetic modification, stably modulates gene expression through heritable regulatory mechanisms, influencing diverse physiological processes and disease pathogenesis (206). In mammals and humans, DNA methylation critically participates in numerous biological processes, including genomic imprinting (207), X-chromosome inactivation (208), transcriptional regulation (209) and tumorigenesis (210). DNA methylation is a covalent chemical modification involving the selective addition of methyl groups to DNA molecules, catalysed by DNA methyltransferases (211). Although DNA methylation occurs at various sites, including the C5 position of cytosine (212), N6 position of adenine (213) and N7 position of guanine (214), 5-methylcytosine (m5C) remains the most prevalent and extensively studied modification in humans, representing the principal focus of DNA methylation research (215-217). The term 'DNA methylation' in general primarily refers to the methylation of the 5th carbon atom of cytosine within cytosine-phosphate-guanine (CpG) sites, yielding m5C (215,218).

CpG sites, consisting of cytosine-guanine dinucleotides, are distributed throughout the genome (219). These sites frequently cluster in CpG islands, genomic regions with a high density of CpG dinucleotides, which are predominantly located near gene promoters (220). CpG islands are critical regulatory elements for gene expression, as promoter DNA methylation can inhibit transcription factor binding or recruit methyl-CpG binding domain (MBD) proteins and HDACs, ultimately inducing gene silencing (221,222). Hypermethylation of CpG islands in promoter regions is strongly associated with transcriptional silencing, whereas unmethylated states are typically associated with active gene expression (108,223).

DNA methylation is catalysed and maintained by DNA methyltransferases (DNMTs) (224). DNMTs can be classified into two functional types: Maintenance DNA methyltransferase (DNMT1), which preserves the epigenetic inheritance of DNA methylation patterns during DNA replication, and de novo methyltransferases (including DNMT3A and DNMT3B), which establish new methylation patterns during development (225). During DNA methylation, cytosine protrudes from the DNA double helix into a cleft that can bind to the enzyme. Cytosine methyltransferase catalyses the transfer of an active methyl group from S-adenosylmethionine (SAM) to the C5 position of cytosine, forming m5C (218). This modification alters gene expression without changing the DNA sequence and is one of the most important epigenetic regulatory mechanisms (226).

Previous studies in ophthalmology have indicated that aberrant DNA methylation disrupts the expression of key genes, such as RHO, OPN1LW and TUBA1A (227,228), contributing to the pathogenesis of various ocular diseases, such as corneal and conjunctival disorders, glaucoma (229), cataracts (230), retinal diseases (231), and ocular tumours (232). As the leading global cause of blindness, cataracts, particularly ARCs, serve as a paradigm of ocular aging, wherein epigenetic dysregulation synergizes with cumulative environmental stressors, including UV radiation and chemical exposures (233,234). While congenital and traumatic cataracts result from genetic mutations or physical injury, ARCs stem from progressive lens opacification associated with multiple factors, including oxidative stress, protein aggregation, and critically, DNA methylation-mediated silencing of protective genes in LECs (Table SIV) (108,235,236). For example, age-dependent hypermethylation of the Klotho gene, a key anti-aging factor, progressively silences its expression, leading to marked reduction or even complete loss of Klotho expression at both mRNA and protein levels, ultimately participating in ARC pathogenesis (237). Furthermore, both mRNA and protein expression of DNA methylation-related genes (DNMT3B and MBD3) are elevated in LECs of ARCs, further substantiating the involvement of DNA methylation in ARC development (238).

To characterize the epigenetic landscape of ARCs, several studies have examined genome-wide methylation patterns in ARC samples. Chen et al (239) employed the MethylationEPIC BeadChip (850K) to profile DNA methylation in anterior lens capsule membranes from patients with ARCs and control subjects. By integrating GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses with their previously published transcriptome sequencing data, the authors identified differentially methylated genes. Their analysis revealed 52,705 differentially methylated sites in the ARC group (13,858 hypermethylated and 38,847 hypomethylated) compared with the control group, which consisted of non-ARC patients, with GO and KEGG analyses highlighting functional associations with cell membrane components, calcium signalling pathways and their potential molecular mechanisms (239). Similarly, a study conducted in 2018 used DNA methylation microarrays, a high-throughput sequencing approach, to identify methylated genes, and revealed that aberrant methylation of collagen type IV α1 chain (COL4A1), gap junction protein α3 and signal induced proliferation associated 1 like 3 was associated with ARCs (240). Further investigation is warranted to elucidate the role of these DNA methylation changes in ARC pathogenesis.

Oxidative stress is a well-established driver of LEC pathophysiology and a key contributor to ARC pathogenesis (7-9). Emerging evidence has further linked oxidative stress-induced DNA damage in LECs to cataract formation (241,242). Therefore, DNA methylation-mediated dysregulation of oxidative stress-related genes and DNA damage repair genes has emerged as a primary research focus, given its pivotal role in ARC development.

Oxidative stress-related genes

Methylation changes in oxidative stress-related genes, as epigenetic modifications of the antioxidant defence system, likely contribute to oxidative stress-induced lens damage in ARC (223,235,243).

One of the main antioxidant defence mechanisms is nuclear factor erythroid 2-related factor 2 (Nrf2) activation. Nrf2 is a central nuclear transcriptional factor, which controls the transcription of >200 protective genes, including 20 genes encoding antioxidant enzymes (244). The activity of the Nrf2 pathway is regulated through multiple mechanisms, both upstream and downstream, as well as by other signalling pathways (245). Kelch-like ECH-associated protein 1 (Keap1) is an oxidative stress sensor and a negative regulator of Nrf2 (246). Under basal conditions, Keap1 binds to Nrf2 in the cytoplasm, promoting its ubiquitin-mediated proteasomal degradation, and thus, maintaining Nrf2 at basal levels (247). During oxidative stress, elevated ROS disrupt the Keap1-Nrf2 interaction, enabling Nrf2 nuclear translocation. Within the nucleus, Nrf2 binds to antioxidant response elements in target gene promoters, inducing the expression of antioxidant enzymes that mediate ROS clearance (248). The Nrf2/Keap1 pathway serves as a master regulator of redox homeostasis, orchestrating the expression of key antioxidant enzymes, including heme oxygenase-1, GPX and superoxide dismutase (SOD), to mitigate oxidative damage (249,250).

The Nrf2/Keap1 pathway is a critical regulator of antioxidant defence in the lens, and its dysregulation may contribute to ARC pathogenesis (246). Age-dependent demethylation of the Keap1 promoter (223) upregulates Keap1 expression, enhancing Nrf2 proteasomal degradation and impairing Nrf2-mediated antioxidant defences. This redox imbalance, driven by Keap1 promoter demethylation, gives rise to ROS accumulation in LECs, ultimately promoting ARC development (223). The methylation status of the Keap1 promoter can be influenced by multiple exogenous and endogenous compounds. Valproic acid (VPA), an antiepileptic drug, alters the expression profiles of passive DNA demethylation pathway enzymes such as Dnmt1, Dnmt3a and Dnmt3b, and the active DNA demethylation pathway enzyme ten-eleven translocation 1 (TET1), leading to DNA demethylation in the Keap1 promoter of LECs (251). Similarly, sodium selenite, a widely used experimental cataract-inducing agent, suppresses Nrf2-dependent antioxidant protection while facilitating lenticular protein oxidation and cataract formation (246). Furthermore, methylglyoxal treatment in LECs results in loss of Keap1 promoter methylation and impairment of the Nrf2-dependent antioxidant system (252). By contrast, ferulic acid, a compound with well-documented antioxidant activity (253-255), protects LECs against ultraviolet A-triggered oxidative stress via inhibition of Keap1 promoter demethylation (245). Furthermore, co-treatment with exogenous acetyl-L-carnitine effectively counteracts homocysteine (Hcy)-induced Keap1 gene demethylation in LECs, while regulating Nrf2/Keap1 mRNA expression, restoring antioxidant protein levels and mitigating Hcy-driven ROS overproduction (256).

Under physiological conditions, the lens maintains high endogenous glutathione (GSH) levels, which serve an essential role in antioxidant defence by scavenging ROS and preserving proteins in their reduced state (257). GSH-S-transferases (GSTs) are a diverse group of phase II detoxification enzymes that catalyse the conjugation of GSH to both endogenous oxidative stress products and exogenous electrophilic compounds (258,259). All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, and the cytosolic enzymes are encoded by at least five distantly related gene families (α, μ, π, σ and θ classes) (260). Furthermore, several GST isozymes modulate the MAPK signalling cascade, which mediates cellular responses to oxidative stress (261,262).

GSH S-transferase Mu 3 (GSTM3), a member of the μ-class subfamily, exhibits potent antioxidant activity (263). Li et al (243) investigated GSTM3 expression and epigenetic regulation in LECs and lens cortex tissues from patients with ARCs. Their findings revealed GSTM3 downregulation in ARC lenses compared with transparent lenses extracted from patients with vitreoretinal diseases, concomitant with hypermethylation of its promoter region (243). Given the critical role of GSTM3 in antioxidant defence, GSTM3 dysfunction induced by promoter methylation may impair cellular redox homeostasis, thereby exacerbating oxidative stress-mediated lens damage (243). In addition to GSTM3, π-class GSH S-transferase pi-1 (GSTP1) participates in the removal of oxidative adducts by transferring them to GSH (235). GSTP1 mRNA and protein levels are decreased in the lens epithelium and cortex of patients with ARNC compared with age-matched controls, consistent with hypermethylation of GSTP1 promoter CpG islands, suggesting a potential role for GSTP1 epigenetic alterations in ARC lenses (235).

DNA repair genes

Excessive ROS induce extensive oxidative DNA lesions in LECs (264), and oxidative stress-induced DNA damage is recognized as a pivotal factor in ARC pathogenesis (265). Higher levels of 8-hydroxy-2-deoxyguanosine, an established biomarker of oxidative DNA damage, have been observed in ARC lenses compared with normal controls (266). DNA repair pathways are essential for repairing DNA damage, which includes nucleotide excision repair (NER), base excision repair (BER), double-strand break (DSB) repair and direct repair, and their normal functions mainly depend on the expression and function of DNA repair genes (267,268).

NER is a highly conserved DNA repair pathway responsible for processing helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts (269). During NER, Cockayne syndrome complementation group B (CSB), encoded by the ERCC6 gene, recruits NER repair factors to DNA damage sites and facilitates subsequent repair (67). CSB protein deficiency has been implicated in Cockayne syndrome (270). The expression of ERCC6 at both mRNA and protein levels is reduced in LECs of ARNCs, and is associated with methylation of a CpG site in the ERCC6 promoter, suggesting epigenetic regulation of ERCC6 in LECs of ARNCs (108). Furthermore, ultraviolet-B (UVB) exposure contributes to ARNC development by inducing DNA damage, which is typically repaired via the NER mechanism (108). UVB-treated human lens epithelium B3 (HLE-B3) and 239T cells exhibit hypermethylation at the-441 CpG site (relative to the transcription start site) within the Sp1 transcription factor binding region of the ERCC6 promoter, along with increased recruitment of DNMT3B to this site (108).

OGG1 is a key enzyme in the BER pathway that specifically recognizes and excises the oxidatively damaged base 8-oxo-7,8-dihydroguanine, a mutagenic lesion that induces G:C→T:A transversions (271). Loss of functional OGG1 is associated with the pathogenesis of multiple age-related diseases, including Alzheimer's disease, age-related macular degeneration and ARCs (272-274). Hypermethylation of the first-exon CpG island of the OGG1 gene has been observed in the lens cortex of ARCs, and was associated with low OGG1 expression and impaired DNA repair function, and eventually contributed to lens opacity (275).

In addition, two other DNA repair genes exhibit epigenetic silencing in ARC: The Werner syndrome gene (WRN) and O6-methylguanine-methyltransferase (MGMT). WRN, which maintains genomic stability by repairing DNA DSBs (276), is hypermethylated in the promoter CpG island of ARC anterior lens capsules and its expression is decreased compared with those in normal controls (43). Similarly, MGMT, an enzyme that removes mutagenic adducts from the O-6 position of guanine, thereby protecting the genome against guanine-to-adenine transitions (277), has been observed to be hypermethylated and downregulated in ARCs (278). These deficiencies lead to the accumulation of unrepaired DNA lesions, driving cell dysfunction and genomic instability, which further promotes ARC progression (108).

Other genes

Numerous studies have established the indispensable role of lens crystallins in maintaining lens clarity, optical transparency and refractive function (279,280). CRYAA and αB-crystallin, the subunits of oligomeric α-crystallin, constitute ~35% of total lens proteins (281). In addition to serving as a major structural protein component of the lens, CRYAA exhibits molecular chaperone-like activity that protects other crystallins from thermally induced inactivation or aggregation, thereby being essential for maintaining lens transparency (280-282). Furthermore, CRYAA mediates cytoprotective effects against both thermal and oxidative stress in LECs (283), and a study has demonstrated that CRYAA can delay cataract formation by trapping denatured proteins prone to aggregation (279). CRYAA expression levels have been reported to be reduced in ARC lenses, with hypermethylation of the CRYAA gene promoter CpG islands representing a key mechanism for CRYAA downregulation (230). Evidence indicates that methylation of CpG sites in the CRYAA promoter directly reduces the DNA-binding capacity of the transcription factor Sp1 (236). Treatment with the DNA demethylating agent zebularine increases CRYAA expression in LECs, suggesting a potential novel therapeutic strategy for ARCs (230,236).

COL4A1 encodes the α1 chain of type IV collagen, the major collagen component of basement membranes (284). Genome-wide methylation analysis has revealed hypermethylation of the COL4A1 promoter region in anterior lens capsule samples from patients with ARCs (240). In both HLE-B3 cells and anterior lens capsules of rats, UVB irradiation induces DNA hypermethylation of COL4A1 promoter CpG islands, and thus, suppresses COL4A1 expression, indicating that epigenetic alterations of the COL4A1 promoter might be involved in UVB-induced lens damage in ARCs (285).

In summary, emerging evidence has highlighted the role of DNA methylation in the pathogenesis of ARCs, linking epigenetic dysregulation to oxidative stress, DNA damage and cellular dysfunction of LECs. Aberrant DNA methylation patterns in key genes may serve as potential biomarkers or therapeutic targets. Further studies based on large-scale clinical research and model systems are needed to identify and validate additional methylation signatures, elucidate underlying mechanisms and explore targeted epigenetic interventions.

Histone modification

As essential structural components, histone proteins form the fundamental building blocks of chromatin. Histones undergo posttranslational modifications (PTMs), which modulate their affinity for DNA, and thus, influence chromatin compaction states (286). These modifications are highly dynamic and continuously changing in response to histone-modifying enzyme activity, ultimately generating specific PTM patterns linked to epigenetic diseases (287). Posttranslational histone modifications include acetylation, methylation, phosphorylation, citrullination, ubiquitination and adenosine diphosphate ribosylation (288,289). Of these, histone acetylation and methylation have been most extensively studied, with substantial evidence supporting their associations with ARCs (Table SV) (43,108,235,243,290).

Histone acetylation is a reversible modification of multiple lysine residues on histone tails, and dynamically regulated by histone acetyltransferases (HATs) and HDACs (291). HATs are a class of enzymes that catalyse the transfer of acetyl groups to lysine residues on histone tails, promoting transcriptional activation by disrupting the interaction between histone tails and nucleosomal DNA, thus facilitating chromatin opening (292). By contrast, HDACs catalyse the removal of acetyl groups from histone tails and suppress transcriptional activity by producing a more condensed chromatin state (293). To date, HDACs have been classified into four major groups (classes I-IV) (294). Among these, SIRT1, a class III NAD+-dependent deacetylase and a member of the sirtuin family, may be critical in protecting against oxidative stress-induced ocular damage, including ARCs (295). Notably, SIRT1 expression is upregulated in human ARCs, suggesting a compensatory mechanism in response to stressors such as UV radiation and oxidative damage. This upregulation may confer protection by modulating oxidative stress responses and inhibiting apoptosis of LECs (296,297).

Histone methylation refers to the transfer of methyl groups from SAM to specific lysine or arginine residues on histone proteins (298). This epigenetic modification is catalysed by histone methyltransferases, while its removal is mediated by histone demethylases (299). Lysine residues can undergo mono-, di- or tri-methylation, whereas arginine methylation is limited to mono- or di-methylated states (299). The functional impact of histone methylation on transcriptional regulation depends on both the modified residue and the degree of methylation (298). For instance, methylation at H3K4, H3K36 and H3K79 is associated with transcriptional activation, whereas methylation at H3K9, H3K27 and H4K20 promotes transcriptional repression (300). As histone methylation can either activate or repress gene expression, it serves as a critical regulator of transcriptional activity in LECs (235).

Histone modifications, along with DNA methylation, influence the expression of certain genes in LECs. Dysregulation of these genes exacerbates oxidative stress damage and DNA damage while impairing normal cellular functions, thereby promoting the development and progression of ARC (43,108,235-237,243,275,278,285,290) (Fig. 2). Notably, numerous genes implicated in ARCs are dually regulated by both DNA methylation and histone modifications (43,108,235,243). These epigenetic mechanisms do not operate in isolation but instead engage in dynamic crosstalk, wherein each modification reciprocally enhances or inhibits the other to modulate gene expression (301). For instance, one study has demonstrated that endogenous DNMT3A and DNMT3B are associated with HDAC activity, where DNMT3A facilitates transcriptional repression via specific molecular interactions between its ATRX-like PHD domain and both HDAC1 and the co-repressor RP58 (302). Consequently, future research examining the effects of DNA methylation and histone modifications on target genes should systematically consider the crosstalk between these two epigenetic mechanisms.

Figure 2 DNA methylation and histone modifications in ARC pathogenesis. ARC, age-related cataract; DNMT, DNA methyltransferase; Me, methyl group; HAT, histone acetyltransferase; Ac, acetyl group; HMT, histone methyltransferase; HDAC, histone deacetylase; HDM, histone demethylase; ERCC6, excision repair cross-complementing rodent repair deficiency, complementation group 6; OGG1, 8-oxoguanine DNA glycosylase 1; WRN, Werner syndrome gene; MGMT, O6-methylguanine-DNA methyl-transferase; GSTP1, glutathione S-transferase P1; GSTM3, glutathione S-transferase Mu 3; SOD1, superoxide dismutase 1; CRYAA, crystallin αA; COL4A1, collagen type IV α1 chain.

m6A modification

Similar to DNA modifications, RNA modifications are dynamically regulated by specific enzymes that catalyse the addition or removal of chemical groups on RNA nucleotides. These modifications serve critical roles in molecular processes, including pre-mRNA splicing, nuclear export, transcript stability maintenance and translation initiation (303). Prevalent RNA modifications include m6A, N1-methyladenosine, m5C and 7-methylguanosine (m7G) (304). Among these modifications, m6A is the best-characterized modification, and is the primary focus in this section.

m6A modification refers to the addition of a methyl group to the sixth nitrogen atom of adenosine (305), which regulates diverse cellular processes during development, differentiation and disease (306). m6A modification is predominantly found in RRACH motifs (R=A/G; H=A/C/U) within the transcriptome, and shows enrichment in coding sequences, 3' UTRs and particularly the region around the stop codon (307,308). m6A modification is a reversible and dynamic RNA modification added by 'writers', such as METTL3, METTL14 and Wilms tumor associated protein, and removed by 'erasers', including fat-mass and obesity-associated protein and α-ketoglutarate-dependent dioxygenase alkB homologue 5 (ALKBH5) (303). The 'readers', which recognize m6A methylation, mainly consist of IGF2 mRNA binding proteins (IGF2BP1/2/3), the YTH domain protein family (YTHDC1/2 and YTHDF1/2/3) and the heterogeneous nuclear ribonucleoprotein family (HNRNPC and HNRNPG) (309). m6A modification is involved in cell proliferation, apoptosis and oxidative stress, which leads to the development of various ocular diseases, including ARCs (310,311). Research has revealed the role of m6A modification dysregulation in ARC pathogenesis, where it modifies the epigenetic profile of the lens genome, thus increasing the susceptibility to ARCs (41).

In eukaryotic mRNA, m6A is the most abundant posttranscriptional RNA modification, regulating RNA translation, splicing, stability and translocation through interactions with its methyltransferase, demethylase and methyl-binding protein (312). For example, METTL14 has been identified as a factor promoting m6A modification of NEIL1, which leads to YTHDF2 recruitment, facilitating NEIL1 mRNA degradation, and ultimately compromising the protective functions of NEIL1 against oxidative damage, apoptosis and mitochondrial dysfunction (313). Furthermore, METTL3 elevates CPB1 expression by regulating the methylation levels of BACH transcriptional regulator 2, thereby accelerating apoptosis and oxidative stress in H2O2-treated LECs (311).

Emerging studies have demonstrated that m6A modifications are widely present in circRNAs and lncRNAs (41,175,314,315). A study conducted in 2020 reported decreased m6A abundance in total circRNAs from LECs of patients with ARCs, while m6A regulators, including ALKBH5 and METTL14, were upregulated in ARC samples compared with normal controls (41). In 2023, Li et al (175) investigated the function and mechanism of m6A modification of circRNAs in ARCs for the first time, and found that METTL3-mediated m6A modification of hsa_circ_0007905 promoted apoptosis and inhibited the proliferation of LECs through the miR-6749-3p/EIF4EBP1 axis, contributing to ARC progression. Similar to circRNAs, m6A methylation levels of lncRNAs could serve important roles in ARC pathogenesis, as confirmed by methylated RNA immunoprecipitation sequencing and RNA sequencing performed on ARC tissues (314). Furthermore, m6A-modified lncRNA ENST00000586817 might regulate the expression of GPX4 through a cis mechanism and could thus be involved in ferroptosis in ARCs (315).

Research on the role and molecular mechanisms of m6A modification in ARC progression remains in its early stages, and further investigation is required to identify more potential molecular targets to develop intervention strategies against ARCs. m6A modification is regulated by methyltransferases, demethylases and m6A recognition proteins, and consequently, the phenotypic changes of LECs in ARC pathogenesis may result from the combined effects of various m6A modifications, necessitating further studies to elucidate the underlying regulatory networks. In addition, other types of RNA modifications, including m5C and m7G, may be implicated in certain ocular disorders such as uveal melanoma and ocular angiogenesis (304,316,317), and their potential connections with ARCs remain to be explored.

Epigenetic modifications and oxidative stress in ARCs

Oxidative stress is a well-documented etiological factor in ARCs (7-9), and thus, it is critical to further investigate the interplay between oxidative stress and epigenetic modifications in ARC pathogenesis.

Epigenetic regulation is implicated in oxidative stress-related processes crucial in the initiation and development of ARCs (223). ncRNA networks regulate the expression of oxidative stress-related genes such as NOX4, SIRT1 and GPX4, and aberrant levels of ncRNAs alter the levels of pro-oxidant markers and antioxidant enzymes in LECs (68,79,140). Clinically, associations have been observed between abnormal ncRNA expression and changes in oxidative stress markers in patients with ARCs (76). Wu et al (318) explored the relationship between miRNAs and oxidative stress-related genes, and found that cataract-regulated miRNAs could promote cataract formation not only by targeting the 3' UTR, which is a classic regulatory pathway, but also by binding to the TATA-box region of oxidative stress-related genes, leading to the subsequent elevation of pro-oxidative genes and inhibition of anti-oxidative genes. Furthermore, numerous critical genes that serve protective roles in oxidative stress processes, such as GSTP1, GSTM3 and SOD1, are regulated by DNA methylation and histone modifications (235,243,290). Additional oxidative stress-related genes implicated in ARC pathogenesis remain to be fully characterized, particularly regarding their functional contributions to disease progression and regulation through epigenetic mechanisms.

Oxidative stress induced by exogenous stimuli can also induce epigenetic alterations, since ROS can regulate the activity of epigenetic enzymes involved in DNA, RNA and histone modifications (319-321). For example, UVB exposure induces the recruitment of DNMT3b and HDAC1 at the ERCC6 promoter, thereby resulting in epigenetic alterations, including specific hypermethylation of the CpG site and H3K9 deacetylation of ERCC6 in LECs (108). At both cellular and organismal levels, UVB exposure enhances the expression of DNMTs, including DNMT1/2/3, and reduces the expression of TETs, including TET1/2/3, leading to hypermethylation of COL4A1 promoter CpG islands and subsequent inhibition of COL4A1 expression (285). Furthermore, ROS can directly induce the oxidation of m5C to 5-hydroxymethylcytosine (5hmC) (322). 5hmC may activate DNA demethylation processes and serve as an intermediate of DNA demethylation, thus causing DNA hypomethylation (322). Numerous studies have indicated that dysregulation of 5hmC could be involved in multiple diseases, including cancer and neurodegenerative diseases (323,324). However, the association between 5hmC and ARCs remains to be fully elucidated.

Epigenetic regulation for the treatment of ARCs

Epigenetic modulators, including DNA methyltransferase inhibitors (DNMTis) and histone deacetylase inhibitors (HDACis), represent a novel class of therapeutic agents capable of correcting aberrant epigenetic modifications, providing innovative intervention strategies against molecular defects underlying cancer and other diseases. These compounds offer unique therapeutic advantages due to their reversible and targetable nature, enabling precise gene regulation without modifying DNA sequences (325-327). The DNMTis azacitidine and decitabine are approved by the US Food and Drug Administration (FDA) for the treatment of myelodysplastic syndromes and acute myeloid leukaemia, and exert their effects by inhibiting DNMTs and reversing tumour-suppressor gene silencing through DNA demethylation (328,329). Several other epigenetic drugs, such as vorinostat, belinostat and romidepsin, are FDA-approved for the treatment of cutaneous T-cell lymphoma and peripheral T-cell lymphomas (330-332). Although DNMTis and HDACis have been extensively tested in clinical and preclinical trials for human diseases (333-335), their effects in ARCs remain poorly understood. The DNMTi 5-aza-2'-deoxycytidine (decitabine) restores the transcriptional activities of GSTP1 in LECs (235), and trichostatin A (TSA; HDACi) corrects the anacardic acid (HAT inhibitor)-induced imbalance between HATs and HDACs, causing increased SOD1 expression by reversing histone acetylation (290). These findings provide evidence that DNMTis and HDACis could rescue LECs from dysregulated expression of key genes induced by aberrant epigenetic alterations during ARC development. Notably, HDACis also exhibit antioxidant properties, as evidenced by multiple studies (336,337). Qiu et al (338) compared the protective effects of four HDACis, β-hydroxybutyrate, TSA, suberoylanilide hydroxamic acid (SAHA) and VPA, on HLECs following UVB exposure, and found that low concentrations of HDACis (1 μmol/l SAHA) mildly attenuated oxidative stress. Although these promising in vitro findings support these drugs as possible candidates for ARC intervention, further validation through well-controlled in vivo studies is required for their clinical application.

Alterations in the expression patterns of various ncRNAs have been reported in ARCs (41,72,131,198), suggesting that targeted modulation of aberrant ncRNA activity may hold therapeutic potential. Over the past decade, miRNA mimics and anti-miRNAs have demonstrated therapeutic promise in clinical trials for various diseases, such as advanced solid tumours, ulcerative colitis and hepatitis C virus infection (339-341), whereas no lncRNA drug has reached the clinical development stage (342). miRNA mimics replicate endogenous miRNA functions to compensate for miRNA deficiency in pathological conditions, while anti-miRNAs (antagomirs) are engineered to suppress endogenous miRNAs that contribute to disease progression (342). As aforementioned, administration of anti-miRNAs targeting miR-326, miR-29a-3p and miR-187 independently enhanced lens transparency and ameliorated lens damage in ARC animal models, indicating their potential as viable non-surgical alternatives for ARC intervention (103-105). Furthermore, with increasing recognition of the regulatory functions of circRNAs in disease pathogenesis, circRNAs are increasingly being considered as targets for therapeutic intervention (56,176,182). For instance, circRNA 06209 overexpression through plasmid injection in a rat model of ARCs reduced lens opacity (182). However, before ncRNA-based therapies advance to the preclinical stage, several challenges must be addressed, including RNA instability, immune activation and off-target effects (342).

Epigenetic modifiers hold potential for the prevention and treatment of ARCs, but there is still a long way ahead before these agents can be routinely implemented in clinical practice. Future studies should focus on: i) Identifying novel therapeutic targets and developing corresponding treatments; ii) investigating and optimizing intraocular drug delivery systems with high efficiency and biocompatibility; and iii) evaluating long-term safety and efficacy in both animal models and clinical trials.

Future directions and perspectives

Emerging technologies in epigenetic studies of ARCs

Technological breakthroughs and cutting-edge research have sparked growing interest in the pivotal role of epigenetics in ocular disease. Advances in omics and high-throughput sequencing technologies have facilitated the elucidation of the role of epigenetics in disease pathogenesis and the identification of potential targets. This section highlights two emerging technologies, single-cell multi-omics and spatial transcriptomics, and discusses their transformative potential in deciphering the epigenetic landscape of ARCs.

Single-cell multi-omics sequencing enables simultaneous single-cell profiling of chromatin accessibility, histone modifications, transcription factor binding, DNA methylation and transcriptome analysis (343). This approach has revealed cell type-specific epigenetic regulatory mechanisms in brain development (344), cancer evolution (345) and HIV-associated immune exhaustion (346). Tangeman et al (347) described the first single-cell multiomic atlas of lens development, depicting a comprehensive portrait of lens fibre cell differentiation and exploring congenital cataract-linked regulatory networks. However, to the best of our knowledge, single-cell multi-omics has not yet been applied in research on ARCs. Current research considers different omics levels individually, drawing conclusions from each level separately, without data integration for a cohesive understanding of the overall mechanisms. Single-cell multi-omics could revolutionize ARC research by offering a comprehensive understanding of the complex biological processes involved in ARC pathogenesis.

Spatial transcriptomics allows high-resolution profiling of gene expression in intact cell and tissue samples (348), and has made significant advances in recent years, exerting notable influence on diverse fields, such as tissue architecture, developmental biology and disease research, particularly in cancer and neurodegenerative diseases (349). Given the differential miRNA expression profiles among ARC subtypes defined by the location of lens opacity (72), there may be differences in gene expression profiles across different regions of the lens. To the best of our knowledge, spatial transcriptomics remains unexplored in epigenetic studies of ARCs. Spatial transcriptomics allows the quantification and illustration of gene expression profiles within the native spatial context (350), which could facilitate the identification of epigenetic biomarkers and microenvironmental interactions critical for understanding cataract pathogenesis.

Epigenetic clocks and ARCs

The epigenetic clock, also known as DNA methylation age (DNAmAge), is a biomarker for biological age prediction based on DNA methylation patterns at specific CpG sites (351). By modelling linear correlations between methylation levels and chronological aging, it serves as a molecular 'timer' that can estimate cellular aging rates more accurately than chronological age, with implications for disease risk and lifespan prediction (352). Epigenetic age acceleration (EAA) exhibits strong associations with age-related diseases by reflecting discrepancies between DNAmAge and chronological age, acting as a predictive biomarker for conditions such as cardiovascular disorders, neurodegenerative diseases and type 2 diabetes (353).

To investigate the causal relationship between epigenetic clocks and common age-related eye diseases or glaucoma endophenotypes, Chen et al (354) conducted bidirectional two-sample Mendelian randomization and found that ARCs were linked to decreased HannumAge, a measure of extrinsic aging based on 71 age-related CpGs from whole blood samples of adults. Further research is warranted to determine whether quantifying EAA can effectively identify ARC susceptibility at early stages and whether it may serve as a basis for developing targeted interventions to delay lens opacification.

Conclusion

Breakthroughs in omics, bioinformatics and high-throughput sequencing technologies have accelerated discoveries regarding the role of epigenetics in ARCs, enabling a deeper understanding of the initiation and progression mechanisms of ARCs while potentially revolutionizing disease management approaches. The present review comprehensively summarizes advances in epigenetic research on ARCs, examining how ncRNAs, DNA methylation, histone modifications and m6A modification contribute to ARC pathogenesis, and exploring their clinical potential for innovative therapeutic strategies. Epigenetics in ARCs is an emerging field with immense potential for diagnosis, treatment and prevention of lens opacities, as innovations over the past decade have identified numerous epigenetic alterations as potential biomarkers and intervention targets. Further studies are required to identify therapeutic targets and biomarkers, elucidate the precise mechanisms through which epigenetics influences disease pathogenesis, evaluate the efficacy and safety of novel epigenetic therapies, and translate these therapies into clinical practice.

Supplementary Data

Availability of data and materials

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Authors' contributions

WY collected the literature and drafted the manuscript. YZ, SC, JG and ZP reviewed the manuscript and made revisions to the manuscript. YY directed the work and revised the manuscript. Data authentication is not applicable. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 82371036), and the Key Research and Development Program of Zhejiang Province (grant no. 2025C02156).

References