FTO-modulated m⁶A demethylation and upregulation of KIF11 mRNA promotes retinal microvascular dysfunction in diabetic...

The N6-methyladenosine (m⁶A) demethylation of mRNAs is critical for the progression of diabetic retinopathy (DR). Fat mass and obesity-associated protein (FTO) has been reported to be overexpressed in DR and is considered to be an important epitranscriptomic regulator (m⁶A eraser) in retinal angiogenesis. The principle of Kinesin family member 11 (KIF11) gene in DR is unclear.

Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was used to screen abnormal m⁶A modification in vitreous body samples of proliferative DR (PDR) patients. CCK-8 and EdU assays were employed to detect the proliferation and DNA synthesis in human retinal microvascular endothelial cells (hRECs), while Transwell, wound healing, and tube formation assays were used to evaluate migration and angiogenesis. In addition, the levels of RNA and m⁶A modified mRNA were tested using quantitative RT-PCR (qRT-PCR) and methylated RNA immunoprecipitation-qRT-PCR (MeRIP-qRT-PCR); the protein levels were tested via western blot. RNA binding protein immunoprecipitation-qRT-PCR (RIP-qRT-PCR) was used to test the interaction between FTO and m⁶A modified mRNA. A 16-week diabetic retinopathy (DR) rat model was induced by streptozotocin (STZ), and 5 µL of AAV9 virus (1 × 10¹² vg/mL) was intravitreally injected every 8 weeks. The retinas were extracted and subjected to Evans blue leakage and retinal trypsin digestion assays, while the retinal paraffin sections were subjected to hematoxylin and eosin (H&E) staining, immunohistochemical or immunofluorescence assays.

MeRIP-Seq and RIP-RT-qPCR indicated that KIF11 should be a downstream target of FTO. Regarding qRT-PCR and MeRIP-qRT-PCR, in vitreous body samples from patients with PDR and hRECs under hyperglycemic conditions, expressed enhanced levels of FTO and KIF11, whereas the m⁶A methylation of KIF11 was decreased. The overexpression of KIF11 enhanced the proliferation, DNA synthesis, migration, wound healing and tube formation of hRECs by affecting the downstream PI3K/AKT/mTOR and β-catenin/c-myc pathways, while the knockdown of KIF11 had the opposite effects. In STZ-induced DR rats, overexpression of FTO and KIF11 significantly promoted retinal leakage, acellular capillary formation, pericyte loss, fibrosis, and gliosis in DR progression, whereas the knockdown of FTO and KIF11 mitigated these phenomena.

Our findings demonstrated that the m⁶A demethylation of KIF11 mRNA, as modulated by FTO, provides a potential target for the clinical therapy of DR retinal microvascular dysfunction.