ALKBH5-Mediated m6A Modification of A20 Regulates Microglia Polarization in Diabetic Retinopathy

Abstract

BackgroundTo investigate the role of microglia polarization in the pathogenesis of diabetic retinopathy, and study the mechanism of ALKBH5-mediated m6A modification of A20 of retinal microglia polarization.MethodsDiabetics rats were constructed and the M1/M2 polarization of retinal microglia was determined using immunofluorescence, flow cytometry, and quantitative real-time PCR (qRT-PCR). Glucose at different concentrations was added to treat the microglia, and the polarization rate was detected. RNA sequencing was performed to identify the differentially expressed gene in glucose treated microglia, and A20 expression was confirmed by qRT-PCR and western blotting. Lentiviruses encoding shRNA for A20 or overexpressing A20 were constructed to clarify the role of A20 in microglia polarization in vitro and vivo. N6-methyladenosine (m6A) modification level and degradation rate of A20 were determined and m6A related proteins were detected.ResultsDiabetics rats showed a higher M1 polarization rate but lower M2 polarization rate of retinal microglia. With the increase of glucose concentration, microglia tend to polarize into M1 inflammatory type rather than M2 anti-inflammatory type. Shown by RNA sequencing, glucose treated microglia showed a differentially expressed gene profile, which was enriched in kinds of inflammatory categories and pathways. A20 expression was lower in microglia with glucose treatment, which was demonstrated to negatively regulate the M1 polarization. Moreover, intraocular injection of A20-overexpression lentiviruses (OE-A20) rectified the enhanced M1 retinal microglia polarization of diabetes rats. The higher m6A modification level and faster degradation rate of A20 was observed in glucose treated microglia, which was mediated by m6A demethylase ALKBH5.ConclusionLower expression A20 resulted in the enhanced M1 polarization of retinal microglia in diabetic retinopathy, which was caused by ALKBH5 mediated m6A modification. This study may provide new perspectives on not only the pathogenesis but also the diagnosis and treatment for diabetic retinopathy.

Transcriptome Profiling Revealed the Relationship Between Immune-Related Genes and m6A Modifiers in Polycystic Ovary Syndrome

“…It has been shown that the dysfunctional demethylation of ALKBH could reduce the production of in ammatory factors in ARDS (71). In diabetic retinopathy, the change of ALKBH5 expression could cause m6A modi cation to be dysfunctional (72). In cancers, it was shown that ALKBH5 and its related genes were enriched in the in ammatory, metabolic, and immune signaling pathways in gliomas, suggesting that ALKBH5 might be a potential treatment for this disease (73).…”

Emerging Role of Epitranscriptomics in Diabetes Mellitus and Its Complications

“…Indeed, a higher rate of M1 inflammatory type but lower rate of M2 anti-inflammatory type were observed in both diabetic rats and BV2 cells (a type of mouse microglia cell line) treated with glucose (213). High glucose repressed expression of Alkbh5 in microglia, which in turn contributed to lower A20 (also known as tumor necrosis factor-a induced protein 3 (TNFAIP3)) expression and increased M1 polarization; these changes were the results of higher m 6 A modification level and faster degradation rate of A20 mRNA, which ultimately contributed to the initiation and development of DR (213). Overall, epigenetic regulation of these targets may help control and delay the development and progression of DR, which provides new insights into the pathogenesis and therapy of this disease.…”

“…Dysregulation of microglia (tissue-specific macrophages in the retina) polarization was considered to be one of the primary pathogenesis of DR (211,212). Indeed, a higher rate of M1 inflammatory type but lower rate of M2 anti-inflammatory type were observed in both diabetic rats and BV2 cells (a type of mouse microglia cell line) treated with glucose (213). High glucose repressed expression of Alkbh5 in microglia, which in turn contributed to lower A20 (also known as tumor necrosis factor-a induced protein 3 (TNFAIP3)) expression and increased M1 polarization; these changes were the results of higher m 6 A modification level and faster degradation rate of A20 mRNA, which ultimately contributed to the initiation and development of DR (213).…”

N6-methyladenosine modification: A potential regulatory mechanism in spinal cord injury

“…Therefore, reducing the polarization of M1 glial cells or driving their conversion to the anti-inflammatory M2 phenotype can aid nerve recovery and lessen secondary damage (Liu W. et al, 2020). According to recent studies, m6A modification plays a critical role in glial phagocytosis and polarization (Figure 2; Li et al, 2021;Zhou et al, 2021;Chen et al, 2022). A study on uveitis found that deletion of the m6A reader YTHDC1 enhances the M1 polarization of microglia and accelerates inflammation (Zhou et al, 2021).…”