ALKBH5 Regulates Corneal Neovascularization by Mediating FOXM1 M6A Demethylation

Abstract

Purpose: This study aims to explore the regulatory role and potential mechanisms of ALKBH5-mediated N6-methyladenosine (m6A) demethylation modification in corneal neovascularization (CNV).

Methods: A mouse CNV model was established through corneal alkali burns. Total m6A levels were measured using an m6A RNA methylation quantification kit. The mRNA expression of candidate m6A-related enzymes was quantified by quantitative RT-PCR. Small interfering RNA targeting ALKBH5 was injected subconjunctivally into alkali-burned mice. The CNV area, corneal epithelial thickness, and pathological changes were evaluated. Protein expression was detected by western blot and immunofluorescence. Human umbilical vein endothelial cells (HUVECs) were treated with IL-6. Plasmid transfection knocked down ALKBH5 or overexpressed FOXM1 in IL-6-induced HUVECs. The assays of CCK8, wound healing, and tube formation evaluated the cell proliferation, migration, and tube formation abilities, respectively. The dual-luciferase assay examined the binding between ALKBH5 and FOXM1. Methylated RNA immunoprecipitation-qPCR detected the m6A levels of FOXM1.

Results: Significant CNV was observed on the seventh day. Total m6A levels were reduced, and ALKBH5 expression was increased in CNV corneas and IL-6-induced HUVECs. ALKBH5 knockdown alleviated corneal neovascularization and inflammation and countered IL-6-induced promotion of cell proliferation, migration, and tube formation in HUVECs. ALKBH5 depletion increased m6A levels and decreased VEGFA and CD31 expression both in vivo and in vitro. This knockdown in HUVECs elevated m6A levels on FOXM1 mRNA while reducing its mRNA and protein expression. Notably, FOXM1 overexpression can reverse ALKBH5 depletion effects.

Conclusions: ALKBH5 modulates FOXM1 m6A demethylation, influencing CNV progression and highlighting its potential as a therapeutic target.

Figure 1.

The 14-day monitoring of the mouse corneal alkali burns model. (A) Representative images of the cornea obtained through slit-lamp examination (top two rows, scale bar: 0.5 mm) and HE staining (bottom row, scale bar: 100 µm), n = 12. (B–D) Quantification results of CNV area, length, and corneal epithelial thickness. n is the total number of test animals used. **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group.

Figure 2.

Downregulated m6A levels and upregulated ALKBH5 expression in CNV corneas. (A) Quantitative analysis of total m6A levels in normal and CNV corneas (n = 6). (B) The qRT–PCR analysis of candidate m6A-related enzymes in normal and CNV corneas, with the fold ratio of the control group normalized to 1 (n = 6). (C) Western blot analysis (left graph) and quantitative analysis (right graph) of ALKBH5 protein levels in corneas on days 3, 7, and 14 after alkali burns, with the fold ratio of the control group normalized to 1 (n = 12). (D) IHF analysis (left graph) and quantitative analysis (right graph) of ALKBH5 in normal and CNV corneas (n = 6). ALKBH5, green; nucleus, blue. Scale bar: 100 µm. n is the total number of test animals used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference, compared with the control group.

Figure 3.

Subconjunctival injection of siALKBH5 alleviates CNV in vivo. (A) Western blot analysis (left graph) and quantitative analysis (right graph) of ALKBH5 protein levels in the control group, CNV group, CNV + saline group, and CNV + siALKBH5 group, with the fold ratio of the control group normalized to 1 (n = 12). (B) Quantitative analysis of total m6A levels in four groups (n = 12). (C) Representative images of the cornea obtained through slit-lamp examination (top two rows, scale bar: 0.5 mm) and HE staining (bottom row, scale bar: 100 µm) in four groups, n = 12. (D–F) Quantitative analysis of CNV area, length, and corneal epithelial thickness in four groups, with the fold ratio of the control group set to 1. (G) Western blot analysis (leftmost image) and quantitative analysis (right two images) of VEGF-A and CD31 protein levels in four groups, with the fold ratio of the control group normalized to 1. (H, I) IHF analysis (left graph) and quantitative analysis (right graph) of VEGF-A and CD31 in four groups. The relative fluorescence intensity was determined by comparing the average fluorescence intensity values of the samples, expressed as folds relative to the control (n = 12). VEGF-A and CD31, green; nucleus, blue. Scale bar: 20 µm. n is the total number of test animals used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference.

Figure 3.

Continued.

Figure 4.

Downregulated m6A levels and upregulated ALKBH5 expression in IL-6-induced HUVECs. (A) Western blot analysis (left graph) and quantitative analysis (right graph) of IL-6 and IL-1β protein levels in normal and CNV corneas, with the fold ratio of the control group normalized to 1 (n = 6). (B) The CCK8 assay was used to analyze the cell viability of HUVECs treated with varying concentrations of IL-6 for 24 hours. (C) The qRT–PCR analysis of VEGF-A in HUVECs treated with different IL-6 concentrations for 24 hours, with the fold ratio of the control group normalized to 1. (D) The qRT–PCR analysis of ALKBH5 in HUVECs treated with different IL-6 concentrations for 24 hours, with the fold ratio of the control group normalized to 1. (E) Quantitative analysis of total m6A levels in HUVECs after 24-hour treatment with varying IL-6 concentrations. n is the total number of test animals used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference, compared with the control group.

Figure 5.

The anti-angiogenic effect of siALKBH5 in IL-6-induced HUVECs. (A) Quantitative analysis of total m6A levels in the control group, IL-6 group, IL-6 + siNC group, and IL-6 + siALKBH5 group. (B) The CCK8 assay was used to analyze the cell viability of HUVECs in four groups. (C, D) The images taken at 0 and 24 hours after scratching in the wound healing assay and the assessment of wound healing rates. Scale bar: 200 µm. (E) The formation of tubes was visualized after cell seeding on Matrigel and incubated for four hours in four groups. Scale bar: 100 µm. Quantification results of capillary lengths (F) and branch points (G). (H) Western blot analysis (leftmost image) and quantitative analysis (right two images) of VEGF-A and CD31 protein levels in four groups, with the fold ratio of the control group normalized to 1. (I, J) ICF analysis (left graph) and quantitative analysis (right graph) of VEGF-A and CD31 in four groups. The relative fluorescence intensity was determined by comparing the average fluorescence intensity values of the samples, expressed as folds relative to the control. VEGF-A and CD31, red; nucleus, blue. Scale bar: 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference.

Figure 5.

Continued.

Figure 6.

ALKBH5 mediates m6A modification of FOXM1. (A) The qRT–PCR analysis of FOXM1 in the control group, IL-6 group, IL-6 + siNC group, and IL-6 + siALKBH5 group, with the fold ratio of the control group normalized to 1. (B) Western blot analysis (left graph) and quantitative analysis (right graph) of FOXM1 protein levels in four groups, with the fold ratio of the control group normalized to 1. (C) The SRAMP online tool identified the m6A modification site on FOXM1 mRNA. The prediction results were sorted by position on the horizontal axis, and the vertical axis showed the combined prediction scores by three random forest classifiers. (D) The schematic of the most likely binding site generated by SRAMP. (E) MeRIP–qPCR analysis of FOXM1 m6A enrichment in normal and CNV corneas, with the fold ratio of the negative control normalized to 1. (F) MeRIP–qPCR analysis of FOXM1 m6A enrichment in IL-6-induced HUVECs with or without ALKBH5 knockdown, with the fold ratio of the negative control normalized to 1. (G) The interaction between ALKBH5 and FOXM1 using the dual-luciferase reporter assay in IL-6-induced HUVECs with or without ALKBH5 knockdown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference.

Figure 7.

FOXM1 overexpression reverses ALKBH5 depletion effects. (A) Western blot analysis (leftmost image) and quantitative analysis (right two images) of ALKBH5 and FOXM1 protein levels, with the fold ratio of the control group normalized to 1. (B) The CCK8 assay was used to analyze the cell viability of HUVECs. (C, D) The images taken at 0 and 24 hours after scratching in the wound healing assay and the assessment of wound healing rates. Scale bar: 200 µm. (E) The formation of tubes was visualized after cell seeding on Matrigel and incubated for four hours. Scale bar: 100 µm. Quantification results of capillary lengths (F) and branch points (G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference.