WTAP-mediated N6-methyladenosine mRNA methylation regulates laser-induced macular neovascularization

Abstract

Purpose: Neovascular age-related macular degeneration (nAMD) is now a major cause of central vision loss in older adults worldwide. The primary characteristic of nAMD is the formation of macular neovascularization (MNV), which is a pathologic form of angiogenesis. Epigenetics plays a role in multiple pathological physiologic processes. N6-methyladenosine (m6A) modification is the most common, abundant, and reversible modification in eukaryotic mRNAs, and it plays a role in various pathological angiogenesis processes. This study intends to reveal the expression and functions of m6A during the macular neovascularization (MNV) process.

Methods: A laser-induced MNV mouse model was used in this study. m6A quantitative analysis was performed to detect the expression of m6A. Subsequently, the expression of various m6A writers and erasers was detected using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Immunohistochemistry was used to detect Wilms' tumor 1-associating protein (WTAP) expression in the MNV lesions. Intravitreal injection of WTAP siRNA in MNV mice to silence the WTAP gene. Hematoxylin and eosin (H&E) were used to determine the thickness and length of the MNV. Fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA) were examined to measure the leakage area of the MNV. Proliferating cell nuclear antigen (PCNA) expression was detected with a western blot. The mRNA and protein levels of β-catenin were tested with qRT-PCR and western blot.

Results: We found increased m6A modification levels after laser induction compared with the normal control group. Subsequently, the expression of various m6A writers and erasers was detected. The results showed that WTAP increased in the MNV model in mice. After the injection of WTAP siRNA into the vitreous body, the expression of WTAP significantly decreased, subsequently decreasing the m6A modification levels. The width, breadth, and leakage area of MNV damage markedly decreased, and endothelial cell proliferation was inhibited. After laser-induced MNV, the expression of β-catenin increased, and that of β-catenin significantly decreased after WTAP knockout.

Conclusions: In conclusion, this study suggests that WTAP-mediated m6A methylation can regulate pathological angiogenesis during MNV and that WTAP may participate in the formation of MNV through the wingless-related integration site (Wnt) pathway. WTAP may be a potential target for MNV treatment.

Figure 1

m6A modification levels and WTAP expression increased after laser photocoagulation. A: Quantitative analysis of m6A showed a trend in the m6A level compared with the normal control group. B: Using qRT-PCR analysis to detect the expression of ALKBH5, WTAP, FTO, METTL3, and METTL14. Western blot was used to detect the expression of ALKBH5 (C), FTO (D), METTL3 (E), WTAP (F), and METTL14 (G). The statistical analyses were compared with the normal group. H: Immunohistochemistry was used to analyze the expression of WTAP in the normal control group and the 7-day group. Scale bar = 100 microns [ganglion cell layer (GC), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), inner segment/outer segment (IS/OS); retinal pigment epithelium (RPE)].

Figure 2

WTAP siRNA downregulates WTAP expression and m6A modification levels. A: After injecting three WTAP siRNAs into the vitreous body, qRT-PCR analysis was used to detect the expression of WTAP. QRT-PCR analysis (B) and protein immunoblotting (C and D) were used to measure WTAP expression after intraocular injection of WTAP siRNA. E: Quantitative analysis suggests that compared to the 7-day group, the m6A level was reduced after WTAP siRNA was injected into the vitreous body.

Figure 3

WTAP siRNA reduces the area of MNV lesions and alleviates MNV leakage. A: H&E staining showed the size of the MNV lesions in each group. B, C: Compared with the 7-day group after laser induction, the fluorescein leakage around the laser spot after the intravitreal injection of WTAP siRNA was weaker. Scale bar = 100 microns. Statistical analysis of the MNV length (D), thickness (E), and leakage area (F) in the 7-day group, the vehicle group, the scramble siRNA group, and the WTAP siRNA group compared with the 7-day group.

Figure 4

WTAP siRNA inhibits the proliferation of CECs in MNV. A: Using western blot to detect PCNA levels before and after laser induction. B: Statistical analysis revealed a trend in PCNA expression compared with the normal group. C: Using western blot to detect the protein level of PCNA in the control group and the WTAP siRNA group injected into the vitreous body. D: Statistical analysis showing the trend of PCNA expression.

Figure 5

WTAP may affect the MNV pathological process through the Wnt pathway. A: QRT-PCR analysis showed a significant decrease in β-catenin expression after intravitreal injection of WTAP siRNA. B: Using western blot to measure the expression of β-catenin. C: Statistical analysis showing the trend of β-catenin expression.