背景:糖尿病视网膜病变(DR)是工作成年人致盲眼病的主要原因,主要归因于微血管的过度增殖,导致玻璃体出血和视网膜牵引,从而严重损害患者的视力。NSUN2介导的RNAm5C甲基化与各种疾病有关,在这次调查中,我们专注于阐明NSUN2对下游基因MUC1表达调控的影响,特别是通过RNAm5C甲基化,关于DR的进展。
方法:利用微阵列分析,我们检查了患者的玻璃体液,以确定DR的潜在治疗靶点.通过qRT-PCR验证NSUN2的差异表达,蛋白质印迹,和人体组织中的免疫荧光,动物组织,和DR的细胞模型。通过基因敲低和过表达,在体外和体内探索了NSUN2与DR之间的关系。各种技术,如MeRIP-qPCR和斑点印迹,用于揭示NSUN2的下游靶标和作用机理。
结果:在DR模型中,NSUN2和RNAm5C甲基化水平均显著升高。NSUN2的敲除在体外和体内都减轻了DR病变的形成。机械上,NSUN2通过与RNAm5C读取器ALYREF结合促进MUC1表达。敲除ALYREF导致与NSUN2敲除观察到的类似的DR病变改变。此外,MUC1过表达成功逆转了由NSUN2沉默诱导的一系列DR改变。
结论:NSUN2通过ALYREF介导的RNAm5C甲基化调节MUC1的表达,从而调节DR的进展,为今后DR的治疗提供新的选择。
BACKGROUND: Diabetic retinopathy (DR) is the leading cause of blinding eye disease among working adults and is primarily attributed to the excessive proliferation of microvessels, which leads to vitreous hemorrhage and retinal traction, thereby significantly impairing patient vision. NSUN2-mediated RNA m5C methylation is implicated in various diseases, and in this investigation, we focused on elucidating the impact of NSUN2 on the regulation of the expression of the downstream gene
MUC1, specifically through RNA m5C methylation, on the progression of DR.
METHODS: Utilizing Microarray analysis, we examined patient vitreous fluid to pinpoint potential therapeutic targets for DR. Differential expression of NSUN2 was validated through qRT-PCR, Western blot, and immunofluorescence in human tissue, animal tissue, and cell model of DR. The relationship between NSUN2 and DR was explored in vitro and in vivo through gene knockdown and overexpression. Various techniques, such as MeRIP-qPCR and dot blot, were applied to reveal the downstream targets and mechanism of action of NSUN2.
RESULTS: The levels of both NSUN2 and RNA m5C methylation were significantly elevated in the DR model. Knockdown of NSUN2 mitigated DR lesion formation both in vitro and in vivo. Mechanistically, NSUN2 promoted
MUC1 expression by binding to the RNA m5C reader ALYREF. Knockdown of ALYREF resulted in DR lesion alterations similar to those observed with NSUN2 knockdown. Moreover,
MUC1 overexpression successfully reversed a series of DR alterations induced by NSUN2 silencing.
CONCLUSIONS: NSUN2 regulates the expression of
MUC1 through ALYREF-mediated RNA m5C methylation, thereby regulating the progression of DR and providing a new option for the treatment of DR in the future.