METTL16 Inhibits Lens Epithelial Cells Function in Diabetic Cataract via m⁶A-Modified DKK1-Mediated Wnt/β-Catenin Signaling

Abstract

Purpose

To explore the role of methyltransferase-like protein 16 (METTL16), an N⁶-methyladenosine (m⁶A) methyltransferase, in the development and progression of diabetic cataracts (DCs), as its underlying molecular mechanisms remain unknown.

Methods

We evaluated m⁶A methylation levels in total RNA isolated from DC anterior capsule tissue and high glucose (HG)-induced human lens epithelial cells (HLECs) using m⁶A quantification and dot blot analysis. The expression levels of METTL16 in the anterior capsule tissue were detected using western blot analysis. Methylated RNA immunoprecipitation (MeRIP), RNA stability assays, and other relevant experiments were performed to investigate the regulatory mechanisms of METTL16 on methylation levels in HLECs. Additionally, cellular functions including proliferation, migration, and cell-cycle progression were assessed.

Results

The expression level of METTL16 and the level of m⁶A methylation were highly elevated in the DC anterior capsule tissue and HG-induced HLECs. MeRIP analysis revealed that Dickkopf-1 (DKK1) might act as the target of METTL16 via the Wnt/β-catenin pathway. Notably, the expression of DKK1 increased with the increase in METTL16 expression in HG-induced HLECs. Furthermore, DKK1 expression was negatively correlated with the nuclear translocation of β-catenin, thereby regulating cell proliferation, migration, and cell-cycle progression in HLECs. In addition, HG-induced lens opacity contributes to cataract formation, and treatment with the DKK1 inhibitor WAY-262611 effectively prevented the development of this pathological process.

Conclusions

The METTL16–DKK1–Wnt/β-catenin axis inhibits the proliferation, migration, and cell-cycle progression of HG-induced HLECs. These findings provide an epigenetic insight into the pathogenesis of DC.

Diabetes is one of the leading causes of death and disability worldwide and affects people regardless of country, age group, or sex.¹ International Diabetes Federation projections show that one in eight adults—approximately 783 million people—will be living with diabetes by 2045, an increase of 46%.² The incidence of diabetic cataracts (DCs) has been on the rise in tandem with the continuous growth in the number of individuals suffering from type 2 diabetes. DC is characterized by the disruption of lens optical transparency due to activation of the polyol pathway,³ oxidative stress,⁴ and accumulation of advanced glycation end products in a continuously chronic hyperglycemic environment.^5,6 The current treatment for DC is phacoemulsification cataract aspiration combined with intraocular lens implantation. Patients with diabetes represent a higher risk population than healthy patients for cataract surgery and are associated with a variety of other ocular complications.⁷

N ⁶-methyladenosine (m⁶A) is the most prevalent post-transcriptional modification in eukaryotic RNAs and has emerged as a widespread regulatory mechanism for RNA splicing, translation, stability, translocation, and high-level structure.^8–10 The m⁶A modification is primarily added by methyltransferases (writers), removed by demethylases (erasers), and recognized and bound by m⁶A-binding proteins (readers), thereby influencing gene expression. The installation of m⁶A is primarily mediated by a methyltransferase complex that includes methyltransferase-like protein 3 (METTL3),¹¹ methyltransferase-like protein 14 (METTL14),¹² Wilms tumor 1-associated protein,¹³ KIAA1429 protein, methyltransferase-like protein 16 (METTL16),¹⁴ RNA-binding motif protein,¹⁵ and Zinc finger CCCH domain-containing protein,^13,15 and it is removed by demethylases such as fat mass and obesity-associated protein and AlkB homolog 5.^16,17 The fates and functions of m⁶A-methylated RNAs are mediated primarily through “readers,” including the YT521-B homology domain family proteins and also the insulin-like growth factor 2 mRNA binding proteins.¹⁸ Although the METTL3–METTL14 complex has been established as the core nuclear m⁶A writer and its involvement in high glucose (HG)-induced lens epithelial cell injury has been documented,^19,20 our work has shifted the focus to METTL16. Unlike the nuclear-localized METTL3–METTL14 complex, METTL16 is preferentially enriched in the cytosol, enabling it to function dually as an m⁶A writer and translational facilitator, with potentially greater efficacy in regulating cytoplasmic mRNA fate.

A previous study by our team demonstrated that METTL16 expression is increased in the anterior capsule of patients with DC compared with the anterior capsule of patients with age-related cataract (ARC), determined by four-dimensional data-independent acquisition (4D-DIA)-based quantitative proteomics.²¹ A sequence-based m⁶A modification site predictor (SRAMP, http://www.cuilab.cn/sramp),²² methylated RNA immunoprecipitation (MeRIP), and RNA sequencing (RNA-seq) indicate that METTL16-mediated m⁶A modifications are directed toward the 3′ untranslated region of Dickkopf-1 (DKK1) mRNA, thereby promoting its protein translation. This study aimed to clarify the regulatory mechanism of METTL16-mediated m⁶A modification in HG-induced human lens epithelial cells (HLECs), which may serve as a potential target for the non-surgical treatment of DC.

Methods

Study Participants and Approval

Our study was approved by the Ethics Committee of the Hangzhou Branch of the Eye Hospital, Wenzhou Medical University (approval no. H2024-020-K-15) and complied with all relevant ethical regulations, including the tenets of the Declaration of Helsinki. The anterior capsule specimens used for RNA and protein extraction were obtained from patients with ARC or DC who underwent cataract surgery at the same institution. Informed consent was obtained from all participants before the surgical procedure. Clinical information of all samples used in this study is provided in Supplementary Table S1.

Cell Culture and Treatment

HLECs were purchased from American Type Culture Collection (CRL-11421; ATCC, Manassas, VA, USA). HLECs were cultured in Gibco Dulbecco's Modified Eagle's Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5.5-mM glucose, 10% Gibco fetal bovine serum (FBS), and 1% Gibco penicillin–streptomycin at 37°C in a 5% CO₂ environment. The cell-culture medium was refreshed every 2 days and passaged at 80% to 90% confluency. In the normal glucose (NG) experimental group, the HLEC medium contained 5.5-mM glucose. In the HG groups, additional glucose (Sigma-Aldrich, St. Louis, MO, USA) was added to achieve final concentrations ranging from 25 to 150 mM across four groups. WAY-262611 (MedChemExpress, Monmouth Junction, NJ, USA), a DKK1 inhibitor, was prepared as a stock solution in dimethyl sulfoxide (DMSO) at a concentration of 1 mM. The final working concentration of WAY-262611 was set at 0.1 µM, and this solution was added 4 hours before HG stress (Supplementary Fig. S2A).

Experimental Animals and Lens Culture In Vitro

Two-month-old male Sprague Dawley rats were purchased from Charles River Laboratories (Zhejiang, China). All experimental protocols involving animals were approved by the Institutional Animal Care and Ethics Committee of Wenzhou Medical University (approval no. AP2024-07-0095) and followed the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The eyeballs of adult male rats were enucleated after euthanasia via inhalation of excess carbon dioxide. The NG group was cultured in 5.5-mM glucose DMEM at 37°C with 5% CO₂, and the HG group was exposed to 150-mM glucose DMEM. The infiltration control group was cultured in 5.5-mM glucose DMEM supplemented with 150-mM mannitol. Lens opacity was quantified through measurement and calculation using ImageJ (National Institutes of Health, Bethesda, MD, USA), enabling an objective assessment of the experimental outcomes related to lens conditions under different treatments.

Lentiviral Transfection

Twenty-four hours before transfection, the cells were seeded in six-well plates at a density of 40,000 cells per well to achieve 70% to 80% confluency. Overexpression or knockdown of short-hairpin RNA (shRNA) targeting METTL16, along with empty vector viral controls, were synthesized by GeneChem (Shanghai, China). HLECs were transfected with the lentivirus at a multiplicity of infection of 15 in the DMEM containing puromycin (10 µg/mL) for 16 hours, which would go through 72 hours of screening by puromycin (10 µg/mL). The shRNA sequences are listed in Supplementary Tables S2 and S3.

RNA Isolation, Complementary DNA Synthesis, and Quantitative PCR

HLECs and anterior capsular tissues were harvested for RNA isolation using Invitrogen TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The RNA concentration was determined by spectrophotometry using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). Subsequently, complementary DNA (cDNA) was reverse-transcribed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer's instructions. Quantitative reverse-transcription PCR (qRT-PCR) was performed using SYBR Green Master Mix (Vazyme) on a CFX96 Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). β-Actin was used as the internal control for normalizing the mRNA levels. The relative expression levels of RNA were calculated using the 2^–ΔΔCt method. Primer sequences are listed in Supplementary Table S4. All qRT-PCR experiments were performed with at least three biological replicates.

Measurement of m⁶A Level

Total RNA was extracted from HLECs using TRIzol reagent according to the manufacturer's instructions. The total m⁶A was quantified using a calorimetric assay and m⁶A dot blot assay. The quantity of m⁶A in the total RNA was assayed using an m⁶A RNA Methylation Quantification Kit (Colorimetric) (P-9005; EpigenTek, Farmingdale, NY, USA). Briefly, positive control, negative control, and 200 ng of the isolated mRNA were added to each well containing the capture antibody. Next, a detection antibody was added. After several incubations, m⁶A levels were quantified calorimetrically at a wavelength of 450 nm.

For m⁶A dot blot assay, the concentration of all RNA samples was adjusted to 400 ng/µL and diluted in a gradient to 200 ng/µL and 100 ng/µL, and then spotted onto a nylon membrane (FFN10; Beyotime Biotech, Shanghai, China) in 1-µL aliquots. After cross-linking the nylon membranes for 2 hours under ultraviolet conditions, the membranes were stained with a solution of 0.02% methylene blue in 0.3-M sodium acetate (pH 5.2) for 5 minutes. Subsequently, they were thoroughly washed with nuclease-free water for 30 minutes to remove excess methylene blue and blocked with 5% non-fat milk for 2 hours at room temperature. The membranes were then incubated overnight at 4°C with m⁶A antibody (1:1000, HA721152; HuaBio, Zhejiang, China). They were then incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (1:10,000, GB23303; Servicebio, Wuhan, China) for 1 hour at room temperature.

RNA Sequencing

Total RNA was extracted from the NC HLECs and METTL16 overexpression (METTL16-OE) HLECs. RNA-seq analysis was performed by LC-Bio Technology (Zhejiang, China).

RNA Stability

Twenty-four hours before adding actinomycin D (MedChemExpress) at a concentration of 10 µg/mL, cells were seeded in 24-well plates at 50,000 cells per well to achieve 70% to 80% confluency. Subsequently, at designated time points of 0, 2, 4, 6, and 8 hours post-treatment, the cells were collected, and total RNA was extracted using the TRIzol method. Following RNA extraction, qRT-PCR was performed to measure DKK1 mRNA levels.

MeRIP Assay

The MeRIP assay was conducted using a Magna MeRIP m⁶A Kit (17-10499; Sigma-Aldrich) according to the manufacturer's instructions. Briefly, the isolated mRNA was chemically fragmented into 100 nucleotides or fewer and immunoprecipitated with an anti-m⁶A antibody or anti-human IgG linked to Magna IP Protein A/G Magnetic Beads. Ten percent of the fragmented RNA was saved as input. Immunoprecipitated DKK1 RNA was analyzed using qRT-PCR.

Flow Cytometry

The cell cycle of HLECs was analyzed using flow cytometry. After they were washed with phosphate-buffered saline (PBS), HLECs were fixed with 70% ethanol and incubated at 4°C overnight. The 4′,6-diamidino-2-phenylindole (DAPI) staining solution (0.5 mL) was added to each cell sample tube and incubated for 30 minutes at room temperature in the dark. Cells were analyzed using a FACS Canto II Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The cell-sorting strategy is shown in Supplementary Figure S1.

HLEC Proliferative Assay

The proliferative ability of the cells was assessed using a Cell Counting Kit-8 (CCK8; Beyotime) assay. Cells were seeded in 96-well plates at 5000 cells per well and subsequently incubated in HG medium for 24, 48, 72, and 96 hours. After the exposure to HG stress, 10 µL CCK-8 was thoroughly mixed with 90 µL DMEM and then added to each well. After a 2-hour incubation at 37°C with 5% CO₂, the absorbance of the cells at a wavelength of 450 nm was measured using a multimode microplate reader (Tecan, Zürich, Switzerland).

Wound Healing Assay

HLECs were seeded in 12-well plates at 1.5 × 10⁵ cells per well and cultured in complete medium until reaching 80% to 90% confluence. Sterile 200-µL pipette tips were used to create uniform linear scratches perpendicular to the reference lines, ensuring minimal damage to the culture substrate. Cellular debris was removed by gentle washing with PBS three times, and the medium was replaced with DMEM containing 1% FBS to eliminate the influence of cell proliferation on migration. Cell migration was observed and photographed after 0 and 12 hours using an EVOS M5000 microscope (Thermo Fisher Scientific). The scratch area was calculated using ImageJ software.

Protein Extraction and Western Blotting

Cells and DC anterior capsular tissues were harvested for protein extraction using radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% protease inhibitors (Beyotime). The total protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). The protein samples were separated by 8% to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to a 0.22-µm polyvinylidene fluoride (PVDF) membrane (BioRad Laboratories). The primary antibodies were as follows: anti-METTL16 (1:1000, HPA020352; Sigma-Aldrich), anti-DKK1 (1:1000, #21112-1-AP; Proteintech, Wuhan, China), anti–β-catenin (1:1000, ET1601-5; HuaBio), anti–β-tubulin (1:1000, MA0002; Nature Bioscience, Zhejiang, China), and anti-lamin B1 (1:1000, GB115715; Servicebio). They were then incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (1:10,000, GB23303; Servicebio) for 1 hour at room temperature. Specific protein bands were visualized using enhanced chemiluminescence reagents (Nature Bioscience) and scanned using a gel imaging scanner (Cytiva, Uppsala, Sweden). Quantitative analysis of the protein bands was performed using ImageJ software.

Immunofluorescence of Cultured Cells

For immunofluorescence, cells were seeded in 96-well confocal orifice plates (Cellvis, Mountain View, CA, USA) at 3000 cells per well and incubated in NG medium (5.5 mM) or HG medium (150 mM) for 24 hours. The HLECs were fixed with 4% paraformaldehyde at room temperature for 30 minutes, followed by permeabilization with 0.3% Triton X-100 for 10 minutes. The cells were incubated with blocking buffer containing 5% bovine serum albumin (BSA) at room temperature for 1 hour and then incubated overnight at 4°C with the primary antibody diluted in blocking buffer. After three washes with PBS, the cells were incubated with the corresponding secondary antibody, Alexa Fluor 594–conjugated donkey anti-rabbit IgG (1:400, ab150080; Abcam, Cambridge, UK), at room temperature for 2 hours in the dark. The cells were mounted with an anti-fluorescent light quencher containing DAPI (Beyotime). All images were captured using a ZEISS LSM 900 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).

Statistical Analysis

All experiments were independently replicated at least three times. The data are expressed as mean ± SD. Statistical analysis was performed using Prism 9.5 (GraphPad Software, Boston, MA, USA). For all quantitative data, Student’s t-test (for comparisons between two groups), one-way ANOVA (for comparisons among multiple groups with one variable), or two-way ANOVA (for comparisons among multiple groups with two or more variables) was employed, and P < 0.05 was considered statistically significant.

Results

METTL16 Was Upregulated in DC Tissue and HG-Induced HLECs

In the DC anterior capsules tissues, RT-PCR indicated that METTL16 mRNA expression was upregulated 5.2-fold (P = 0.001) relative to that in ARC anterior capsule tissues (Fig. 1A). Western blot analysis further confirmed that METTL16 protein expression increased 2.2-fold (P = 0.0116) in DC anterior capsule tissues (Figs. 1B, 1C). We further established a glucose concentration gradient for culturing HLECs (5.5, 25, 50, 100, and 150 mM). Under increasing glucose concentrations, METTL16 mRNA expression exhibited progressive upregulation compared to the 5.5-mM group. Notably, a significant increase in METTL16 levels was observed in the groups treated with 100-mM and 150-mM glucose. RT-PCR analysis revealed a significant (P < 0.0001) increase in METTL16 mRNA levels (Fig. 1D). Consistent with this trend, western blot analysis confirmed that METTL16 protein levels were significantly elevated by 1.7-fold (P = 0.0291) in the 100-mM group and 1.9-fold (P = 0.0055) in the 150-mM group compared to the 5.5-mM control group (Figs. 1E, 1F). Based on these results, 150-mM glucose was selected as the optimal HG concentration for subsequent experiments. Overall, these findings demonstrate that METTL16 is upregulated under HG conditions in both the cellular and clinical contexts.

Figure 1.

METTL16 was upregulated in DC tissue and HG-induced HLECs. (A) RT-PCR revealed the METTL16 mRNA expression in anterior capsule tissues from ARC or DC patients. (B, C) Western blot analysis revealed the METTL16 protein expression in anterior capsule tissues from ARC or DC patients. (D) The qRT-PCR analysis of METTL16 mRNA expression in HLECs cultured in various concentrations of glucose (5.5, 25, 50, 100, and 150 mM). (E, F) Western blot analysis of METTL16 protein expression in HLECs cultured in various concentrations of glucose. (G, H) Dot blot analysis showed the m⁶A quantity in anterior capsule tissues from ARC or DC patients. (I) m⁶A quantitative analysis illustrated the m⁶A level in anterior capsule tissues from ARC or DC patients. (J, K) Dot blot analysis revealed the m⁶A quantity in the NG or HG-induced HLECs. (L) Colorimetric quantification of m⁶A levels in the NG or HG-induced HLECs. Data are representative of three independent experiments. The data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as compared with the 5.5-mM glucose group or ARC group.

METTL16 Elevates m⁶A RNA Modification Levels in HLECs

m⁶A is the most prevalent post-transcriptional modification in eukaryotic RNAs.⁸ Dot blot assays and m⁶A quantification demonstrated higher m⁶A levels in DC tissues (4.5-fold by dot blot, P = 0.0035; 25.5% by quantification, P = 0.0321) (Figs. 1G–I). Correspondingly, dot blot assays and m⁶A quantification revealed that m⁶A levels were elevated in HG-induced HLECs (1.5-fold increase by dot blot, P = 0.0011; 8.84% increase by m⁶A quantification, P = 0.0001) (Figs. 1J–L).

To validate the correlation between METTL16 and m⁶A levels, we established stable HLEC lines with METTL16-OE or METTL16 knockdown (METTL16-KD) via lentiviral transfection using empty vector-transfected cells as controls. Transfection efficiency was confirmed by qRT-PCR and western blotting. METTL16-OE significantly increased METTL16 mRNA expression by more than 19-fold (P < 0.0001) and protein levels by 6.7-fold (P = 0.0005) (Figs. 2A–C). Both the HG-cultured negative control (HG-induced NC) and normal glucose-cultured METTL16-OE (NG-induced METTL16-OE) groups showed elevated m⁶A modifications relative to the normal glucose-cultured negative control (NG-induced NC) group. Specifically, m⁶A quantification demonstrated an increase of 13.75% (P = 0.0032) in the HG-induced NC group and 8.18% (P = 0.0336) in the NG-induced METTL16-OE group, relative to the NG-induced NC group (Fig. 2D).

Figure 2.

METTL16 was correlated with the m⁶A content. (A–C) RT-PCR and western blot revealed the METTL16 mRNA and protein expression in HLECs with METTL16-OE or control transfection. (D) m⁶A quantitative analysis illustrated the m⁶A level in HLECs with METTL16-OE or control transfection. (E–G) RT-PCR and western blot revealed the METTL16 mRNA and protein expression in HLECs with METTL16-KD or control transfection. (H) m⁶A quantitative analysis illustrated the m⁶A level in HLECs with METTL16-KD or control transfection. (I, J) Dot blot analysis revealed the m⁶A quantity in HLECs with METTL16-OE or control transfection. (K, L) Dot blot analysis revealed the m⁶A quantity in HLECs with METTL16-KD or control transfection. Data are representative of three independent experiments. The data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as compared with the control group.

METTL16-KD decreased mRNA expression by 59.8% (P = 0.0003) and protein levels by 33.9% (P = 0.0019) compared to the controls (Figs. 2E–G). METTL16 knockdown reduced m⁶A levels under HG conditions. The HG-cultured METTL16-KD group showed no significant increase in m⁶A levels compared to the NG-induced NC group but exhibited a marked reduction (46.35%, P = 0.0158) relative to the HG-induced NC group (Fig. 2H).

Consistently, dot blot analysis showed 1.9-fold (P = 0.0103) higher m⁶A levels in the HG+NC group and a 2.0-fold (P = 0.0081) increase in the NG-induced METTL16-OE group compared to the NG-induced NC group (Figs. 2I, 2J), whereas dot blot analysis showed a 0.4-fold (P = 0.0127) decrease of m⁶A content in the HG-induced METTL16-KD group compared to the HG-induced NC group (Figs. 2K, 2L). Taken together, METTL16 overexpression elevated m⁶A levels in the total RNA from HLECs, whereas knockdown had the opposite effect.

METTL16 Inhibits HLECs Proliferation, Migration, and Cell-Cycle Progression

To investigate the biological function of METTL16, we assessed cell proliferation capacity and cell-cycle progression in human lens epithelial cells under high-glucose conditions, as well as in the METTL16-OE and METTL16-KD groups. Compared to the NG group, HG-cultured HLECs showed significantly (P < 0.0001) suppressed cell proliferation on days 2, 3, and 4 (Fig. 3A). The percentage of HLECs in the G1 phase was significantly higher (P = 0.0295) than that of the NG-induced HLECs, whereas the percentage of HLECs in the S phase was significantly lower (P = 0.0008) (Figs. 3B, 3C). Alterations in the G1, S, and G2 phases represent core regulatory nodes by which cells respond to internal and external environmental stress.^23,24 Notably, sustained G1 phase arrest can induce cells to enter a senescent state, manifested by irreversible cell-cycle arrest.²⁴ The wound healing assay revealed that, under HG conditions, the migrated area was reduced by at least 21% after 12 hours relative to that in the NG group (P < 0.0001) (Figs. 3D, 3E). Similarly, the NG-induced METTL16-OE group showed decreased cell proliferation (day 2, P = 0.0238; day 3, P = 0.0288; day 4, P = 0.0351) compared to the NG-induced NC group (Fig. 3F). Correspondingly, in the NG-induced METTL16-OE and HG-induced NC groups, the number of cells in the G1 phase was significantly increased (P = 0.0433) (Figs. 3G, 3H). The wound healing assay results showed that, compared with the NG-induced NC group, cell migration in the HG-induced NC group and the NG-induced METTL16-OE group was reduced by 22% (P = 0.0001) and 23% (P = 0.0001), respectively, after 12 hours (Figs. 3I, 3J).

Figure 3.

Overexpression of METTL16 inhibits HLEC proliferation, migration, and cell-cycle progression. (A) Proliferation of HLECs under NG (5.5 mM) and HG (150 mM) conditions after 1, 2, 3, and 4 days. (B, C) Flow cytometry revealed the cell cycle of HLECs under NG and HG conditions. (D, E) Wound healing rate of HLECs under NG and HG conditions after 12 hours. Scale bar: 200 µm. (F) Proliferation of HLECs with NG or HG administration and METTL16-OE or NC transfection after 1, 2, 3, and 4 days. (G, H) Flow cytometry revealed the cell cycle of HLECs with NG or HG administration and METTL16-OE or NC transfection. (I, J) Wound healing under NG and HG conditions after 12 hours for the NC and METTL16-OE groups. Scale bar: 200 µm. Data are representative of three independent experiments. The data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as compared with the control group.

In contrast, the proliferation of the HG-induced METTL16-KD group showed no significant difference from that in the NG-induced NC group. However, compared to the HG-induced NC group, the HG-induced METTL16-KD group significantly rescued cell proliferation on days 2, 3, and 4 (day 2, P = 0.0004; days 3 and 4, P < 0.0001) (Fig. 4A). The percentage of cell-cycle phases in the NG-induced METTL16-KD group was significantly lower (P < 0.047) than that in the NG-induced NC group in the G1 phase, and the percentage of cells in the G2/M phase was significantly higher (P = 0.0054) (Figs. 4B, 4C). Cell migration in the HG-induced METTL16-KD group exhibited no significant difference compared with the NG-induced NC group. However, compared to the HG-induced NC group, the HG-induced METTL16-KD group showed significantly (P = 0.0419) rescued cell migration by 15% after 12 hours (Figs. 4D, 4E). Collectively, these data indicate that METTL16 is involved in regulating the proliferation, migration, and cell cycle of HG-induced HLECs.

Figure 4.

Knockdown of METTL16 enhances HLECs proliferation, migration, and cell-cycle progression. (A) Proliferation of HLECs with NG or HG administration and METTL16-KD or NC transfection after 1, 2, 3, and 4 days. (B, C) Flow cytometry revealed the cell cycle of HLECs with NG or HG administration and METTL16-KD or NC transfection. (D, E) Wound healing under NG and HG conditions after 12 hours for the NC and METTL16-KD groups. Scale bar: 200 µm. Data are representative of three independent experiments. The data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as compared with the control group.

DKK1 Acted as a Target of m⁶A Modification by METTL16 in the HLECs

To explore the molecular mechanism by which METTL16 regulates the phenotype of HLECs, we performed RNA-seq on the NC and METTL16-OE groups. Differentially expressed genes (DEGs) were defined as those with log fold change (logFC) > 1 and P < 0.05. In total, 203 DEGs were identified, including 78 upregulated and 125 downregulated genes (Fig. 5A). Gene Ontology (GO)²⁵ enrichment analysis significantly enriched the terms “negative regulation of cell population proliferation” and “wound healing,” supporting the conclusion that METTL16 upregulation contributes to cell-cycle arrest, specifically during the G1-to-S phase transition (Fig. 5B). Enrichment maps were derived from the Kyoto Encyclopedia of Genes and Genomes (KEGG).²⁶ The database demonstrated significant enrichment of the advanced glycation end-product (AGE)–AGE receptor (RAGE) signaling pathway in diabetic complications (Fig. 5C),^27,28 indicating that elevated METTL16 expression is closely associated with high glucose-induced alterations in the cellular environment.

Figure 5.

DKK1 acted as a target of m⁶A modification by METTL16 in the HLECs. (A) The heat map shows the top 100 DEGs between the control group and the METTL16-OE group. (B, C) GO and KEGG pathway analysis between the control group and the METTL16-OE group. (D) Confident DKK1 sequences of m⁶A modification by SRAMP. (E) MeRIP qPCR showed the DKK1 mRNA enrichment in HLECs with METTL16-OE or control transfection precipitated by m⁶A antibody. (F, I) RNA stability assay showed the DKK1 mRNA half-life (t_1/2) with METTL16-OE or METTL16-KD. (G, H) Western blot revealed the DKK1 protein expression in HLECs with NG or HG administration and METTL16-OE or NC transfection. (J, K) Western blot revealed the DKK1 protein expression in HLECs with NG or HG administration and METTL16-KD or NC transfection. Data are representative of three independent experiments. The data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as compared with the control group.

Further comparison of these DEGs with genes associated with cell proliferation and cell-cycle processes revealed that DKK1 expression was positively correlated with METTL16 expression. DKK1 is a secreted protein and one of the key physiological inhibitors of the canonical Wnt/β-catenin signaling pathway.^29,30 Activation of this pathway requires Wnt ligands to bind to membrane-bound Frizzled receptors and low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) co-receptors, forming a trimeric complex that initiates downstream signaling.³¹ A hallmark of Wnt pathway activation is the nuclear translocation of β-catenin.²⁹ DKK1 competes with Wnt ligands for binding to LRP5/6, thereby preventing effective assembly of the Wnt signaling complex.^32,33 This maintains the activity of the β-catenin degradation complex, reducing cytoplasmic β-catenin levels and impairing its nuclear translocation. Consequently, downstream transcriptional activity is diminished, affecting critical biological processes such as cell proliferation, differentiation, and apoptosis.

m⁶A is the most prevalent internal RNA modification, and METTL16 modulates m⁶A RNA modification levels in HLECs. The online platform SRAMP was used to predict the abundance of m⁶A methylation sites in DKK1. In humans, DKK1 harbors numerous high-confidence m⁶A modification sites within its coding sequence (CDS) region, suggesting that DKK1 expression may be regulated by m⁶A methylation (Fig. 5D).

To confirm and extend these results, we assessed the enrichment of DKK1 pairs in the METTL16-OE group relative to the NC group and the effect of METTL16 on the half-life of DKK1. MeRIP qPCR analysis demonstrated significantly higher m⁶A enrichment in DKK1 mRNA in the METTL16-OE group than in the control group (P = 0.0436) (Fig. 5E). RNA stability assays revealed that METTL16-OE prolonged the half-life (t_1/2) of DKK1 mRNA relative to empty lentiviral vector transfection (Fig. 5F). Western blot analysis showed that METTL16-OE transfection increased DKK1 protein levels compared to those in the control group (P = 0.0024) (Figs. 5G, 5H). Conversely, METTL16-KD shortened the DKK1 mRNA half-life (t_1/2) (Fig. 5I) and reduced DKK1 protein expression (P = 0.0191) (Figs. 5J, 5K). Collectively, these results confirmed that DKK1 is a target of METTL16-mediated m⁶A modification in HLECs.

DKK1 Inhibits HLECs Proliferation, Migration, and Cell-Cycle Progression via Wnt/β‐Catenin Signaling

Nuclear translocation of β-catenin represents a critical step in canonical Wnt signaling. Immunofluorescence staining revealed reduced colocalization between DAPI and β-catenin in the METTL16-OE group (Fig. 6A). Consistent with these results, western blot analysis of nuclear and cytoplasmic β-catenin (CTNNB1) showed that the METTL16-OE group exhibited lower nuclear β-catenin levels compared to control cells, with no significant difference in cytoplasmic β-catenin levels between the two groups (P = 0.0026) (Figs. 6B–D). Conversely, the opposite trend in β-catenin subcellular localization was observed in the METTL16-KD group (Fig. 6E). Specifically, the METTL16-KD group displayed higher nuclear β-catenin levels, but cytoplasmic β-catenin levels remained comparable between the two groups (P = 0.0456) (Figs. 6F–H).

Figure 6.

DKK1 regulates HG-induced dysfunction in HLECs via Wnt/β-catenin signaling. (A) Representative immunofluorescence images of CTNNB1 in HLECs with NG or HG administration and METTL16-OE or NC transfection. Scale bar: 50 µm. (B–D) Western blot revealed the Cyt-CTNNB1 and Nuc-CTNNB1 protein expression in HLECs with NG or HG administration and METTL16-OE or NC transfection. (E) Representative immunofluorescence images of CTNNB1 in HLECs with NG or HG administration and METTL16-KD or NC transfection. Scale bar: 50 µm. (F–H) WB revealed the Cyt-CTNNB1 and Nuc-CTNNB1 protein expression in HLECs with NG or HG administration and METTL16-KD or NC transfection. Data are representative of three independent experiments. The data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as compared with the control group.

WAY-262611 is a potent, selective DKK1 antagonist that acts by alleviating DKK1-mediated inhibition of the Wnt/β-catenin pathway.³⁴ Western blot analysis demonstrated a 0.83-fold (P = 0.0058) decrease of DKK1 protein in the HG-induced HLECs with WAY-262611 treatment for 4 hours relative to the HG-induced HLECs, but the WAY-262611 treatment did not affect METTL16 expression in HG-induced HLECs. (Supplementary Figs. S2B, S2C). However, the wound healing assay revealed that, by treating HG-induced HLECs with WAY-262611, the migrated area was rescued by at least 15% after 12 hours relative to the HG group (P = 0.0267) (Figs. 7A, 7B). Consistently, treatment of HG-induced HLECs with WAY-262611 resulted in a significant reduction (P = 0.0138) in the G1 phase and a marked increase (P < 0.0001) in the G2 phase compared with the HG group (Figs. 7C, 7D). The treatment of HLECs with WAY-262611 for 4 hours significantly rescued (P = 0.0262) the impaired cell proliferation observed under HG conditions alone (P = 0.0262) (Fig. 7E).

Figure 7.

DKK1 regulates HG-induced dysfunction in HLECs via Wnt/β-catenin signaling. (A, B) Wound healing of HLECs under NG and HG conditions with DMSO or WAY-262611 supplement after 12 hours. Scale bar: 200 µm. (C, D) Flow cytometry revealed the cell cycle of HLECs under NG or HG conditions with DMSO or WAY-262611 supplement. (E) Proliferation of HLECs under NG or HG conditions with DMSO or WAY-262611 supplement. (F, G) Rat lenses were extracted and cultivated in 150-mM glucose or 150-mM mannitol media for 7 days. The addition of WAY-262611 to the HG-stressed lenses prevented the formation of cataracts (n = 3 per group). Scale bar: 1000 µm. Data are representative of three independent experiments. The data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as compared with the control group.

We extracted lenses from 2-month-old rats and cultured them for 7 days in media containing 5.5-mM glucose (NG), 150-mM glucose (HG), 150-mM mannitol (osmotic control), or 150-mM glucose supplemented with WAY-262611 (HG+WAY-262611). The HG+WAY-262611 group was used to assess whether WAY-262611 partially reversed cataract formation. During the first three days of culture, all four groups maintained their relative transparency. On day 7, the lenses in the NG group remained near-transparent; by day 7 (Figs. 7F, 7G). From day 3 onward, the HG group exhibited lens clouding; the opacity increased by day 5 and progressed to the lens nucleus by day 7. In contrast, the HG+WAY-262611 group showed markedly slowed cataract progression, with only mild cortical clouding observed between days 5 and 7. These results suggest that pharmacological inhibition of DKK1 may represent a potential strategy to counteract hyperglycemia-induced suppression of lens epithelial cell proliferation and cell-cycle progression, thereby delaying the progression of diabetic cataracts.

Discussion

Our study reveals a critical role for the RNA methyltransferase METTL16 in the pathogenesis of DC, and its mechanism of action involves mediating m⁶A modification of DKK1 to regulate the Wnt/β-catenin signaling pathway—thereby controlling lens epithelial cell (LEC) function via a previously unrecognized epitranscriptomic mechanism. METTL16 was recently identified as an m⁶A methyltransferase; however, this was only reported to add m⁶A to several noncoding RNAs-U6 small nuclear RNA (snRNA),³⁵ MALAT1,³⁶ and MAT2A.³⁷ Beyond its previously recognized role as a sole m⁶A methyltransferase, METTL16 is emerging as a sensor and homeostatic regulator of cellular metabolism, primarily by controlling S-adenosylmethionine (SAM) levels—the universal methyl donor^35,36—and by modifying noncoding RNAs such as U6 snRNA^35,38 and MALAT1 to influence their stability and function.^39,40

In the anterior capsule of DC, we observed a significant upregulation of METTL16 expression at both the mRNA and protein levels in patient samples and HG-cultured HLECs, which correlated with a global increase in m⁶A modification. Functionally, HG exposure suppressed HLEC proliferation and migration and induced G1/S phase arrest. Crucially, the METTL16-OE group mimicked these deleterious effects, whereas METTL16-KD attenuated them, concomitantly with a reduction in global m⁶A levels. Mechanistically, METTL16 senses and maintains intracellular SAM homeostasis by catalyzing the m⁶A modification of U6 snRNA and regulating MAT2A pre-mRNA intron retention.^35,41 Disruption of this METTL16-dependent regulatory pathway significantly impairs cellular growth, suggesting that METTL16 is a key mediator driving DC pathogenesis under metabolic stress.

Bioinformatic analyses of METTL16-OE cells revealed that DKK1 was a prominently upregulated target. GO terms indicated enrichment in processes such as negative regulation of cell proliferation, and KEGG pathway analysis highlighted the AGE–RAGE signaling axis.²⁸ We mechanistically demonstrated that METTL16 directly binds to and induces m⁶A modifications on DKK1 mRNA, enhancing its stability and thereby increasing its protein abundance. This aligns with recent reports of m⁶A-mediated DKK1 regulation in DC.⁴² As a secreted antagonist of canonical Wnt signaling, DKK1 binds to LRP5/6 co-receptors, preventing β-catenin nuclear translocation.^43,44 Consistent with this, both HG conditions and METTL16-OE led to cytoplasmic retention of β-catenin and suppression of Wnt pathway activity. METTL16-KD reverses this effect by restoring β-catenin nuclear translocation. In the canonical Wnt pathway, nuclear translocation of β-catenin following pathway activation promotes the expression of genes critical for cell proliferation, survival, differentiation, and migration.³³ The functional significance of this axis was confirmed through rescue experiments. Inhibition of DKK1 signaling with WAY-262611 not only ameliorated HG-induced deficits in HLEC proliferation, migration, and cell cycle progression in vitro but also delayed cataract formation in in vivo rat lens cultures. This finding provides compelling evidence that the METTL16–DKK1–Wnt/β-catenin axis is a central driver of pathological phenotypes.

The mature lens features a monolayer of epithelial cells on its anterior surface, extending to the equator and bathed by the aqueous humor, with the remainder consisting of densely packed fiber cells.^44,45 Cataractogenesis is characterized by aberrant proliferation and migration of lens epithelial cells.^46,47 The slowed proliferation and migration of LECs limit the production of new lens fibers, impairing the replacement of aged or damaged ones. This leads to protein aggregation, disrupting light refraction and causing localized lens opacities that progressively expand. When the cell cycle is arrested at the G0/G1 phase, LECs become functionally inactive, disrupting lens homeostasis.⁴⁸ This results in nutrient deficiency and the buildup of toxic metabolites, such as reactive oxygen species. During DC pathogenesis, hyperglycemia promotes abnormal LEC proliferation, migration, and cell-cycle dysregulation, resulting in lens metabolism disorders. Over time, the arrested state leads to depletion of LECs and impairs the repair capacity of the lens. These changes accelerate the cataract formation.

Our findings elegantly connect the hyperglycemic environment, epitranscriptomic regulation, and pivotal signaling pathways. As an initial insult, HG likely upregulates METTL16 expression, potentially by altering intracellular SAM levels or by activating specific stress-signaling pathways. METTL16, acting as a responsive effector, specifically targets the mRNA of key genes, such as DKK1, through its m⁶A writer activity, finely tuning its stability. The position of METTL16 is a crucial bridge connecting metabolic disturbances to cellular dysfunction. The subsequent increase in DKK1 protein effectively inhibits the Wnt/β-catenin pathway, which is vital for cell proliferation, migration, and homeostasis, ultimately contributing to HLEC dysfunction and cataractogenesis. Elucidating this mechanism provides a new integrative perspective on the complex pathogenesis of DC.

Furthermore, METTL16 may play analogous roles in other diabetic microvascular complications (e.g., as retinopathy and nephropathy), which also involve HG-induced dysfunction in specific cell types. Further investigations are required to determine whether METTL16 serves as a common epitranscriptomic hub.

In summary, we delineated a novel signaling pathway in which METTL16, which responds to diabetic stress, orchestrates an m⁶A-dependent post-transcriptional increase in DKK1 expression. This, in turn, suppresses Wnt/β-catenin signaling, leading to cell-cycle arrest and impaired regenerative capacity in HLECs. Our findings not only expand the understanding of epitranscriptomic regulation in diabetic complications but also indicate that the METTL16–DKK1–Wnt axis is a promising therapeutic target for intervention in diabetic cataract progression (Fig. 8). Future in vivo studies employing DKK1-neutralizing antibodies or small-molecule inhibitors are required to validate the translational potential of this pathway.

Figure 8.

Mechanism diagram of this study. In LECs of DC, METTL16 upregulation leads to elevated global m⁶A modification levels. This elevated m⁶A modification specifically targets DKK1 mRNA to regulate the DKK1-driven Wnt/β-catenin signal pathway, suppressing β-catenin nuclear translocation, which in turn impairs LEC proliferation, migration, and cell-cycle progression.

Limitations

This study delineated a METTL16–DKK1–Wnt/β-catenin axis that regulates the proliferation and cell-cycle progression of HG-induced HLECs. However, this study has some limitations. First, the anterior capsule of the human lens contains a limited number of HLECs, which precludes the acquisition of sufficient cellular material to assess cell proliferation, migration, and cell-cycle progression. Second, MeRIP qPCR relies on predesigned primers and can only detect fragments of the DKK1 transcript that contain predicted m⁶A sites. As a result, it may not cover all potential m⁶A sites in the entire DKK1 transcript.