PDF(Pubmed)
Abstract：
OBJECTIVE: Retinal vascular endothelial cell (RVECs) injury is a major cause of morbidity and mortality among the patients with diabetes. RVECs dysfunction is the predominant pathological manifestation of vascular complication in diabetic retinopathy. N6-methyladenosine (m6A) serves as the most prevalent modification in eukaryotic mRNAs. However, the role of m6A RNA modification in RVECs dysfunction is still unclear.
METHODS: RT-qPCR analysis and western blot were conducted to detect the change of m6A RNA modification in diabetic retinopathy. CCK-8 assay, transwell experiment, wound healing assay, tube formation experiment, m6A-IP-qPCR were performed to determine the role of YTHDC1 in RVECs. Retinal trypsin digestion test and H&E staining were used to evaluate histopathological changes.
RESULTS: The levels of m6A RNA methylation were significantly up-regulated in HG-induced RVECs, which were caused by increased expression of YTHDC1. YTHDC1 regulated the viability, proliferation, migration and tube formation ability in vitro. YTHDC1 overexpression impaired RVECs function by repressing CDK6 expression, which was mediated by YTHDC1-dependent mRNA decay. Moreover, it showed sh-YTHDC1 inhibited CDK6 nuclear export. Sh-YTHDC1 promotes the mRNA degradation of CDK6 in the nucleus but does not affect the cytoplasmic CDK6 mRNA. In vivo experiments showed that overexpression of CDK6 reversed the protective effect of sh-YTHDC1 on STZ-induced retinal tissue damage.
CONCLUSIONS: YTHDC1-mediated m6A methylation regulates diabetes-induced RVECs dysfunction. YTHDC1-CDK6 signaling axis could be therapeutically targeted for treating DR.
METHODS: RT-qPCR analysis and western blot were conducted to detect the change of m6A RNA modification in diabetic retinopathy. CCK-8 assay, transwell experiment, wound healing assay, tube formation experiment, m6A-IP-qPCR were performed to determine the role of YTHDC1 in RVECs. Retinal trypsin digestion test and H&E staining were used to evaluate histopathological changes.
RESULTS: The levels of m6A RNA methylation were significantly up-regulated in HG-induced RVECs, which were caused by increased expression of YTHDC1. YTHDC1 regulated the viability, proliferation, migration and tube formation ability in vitro. YTHDC1 overexpression impaired RVECs function by repressing CDK6 expression, which was mediated by YTHDC1-dependent mRNA decay. Moreover, it showed sh-YTHDC1 inhibited CDK6 nuclear export. Sh-YTHDC1 promotes the mRNA degradation of CDK6 in the nucleus but does not affect the cytoplasmic CDK6 mRNA. In vivo experiments showed that overexpression of CDK6 reversed the protective effect of sh-YTHDC1 on STZ-induced retinal tissue damage.
CONCLUSIONS: YTHDC1-mediated m6A methylation regulates diabetes-induced RVECs dysfunction. YTHDC1-CDK6 signaling axis could be therapeutically targeted for treating DR.
摘要：
目的：视网膜血管内皮细胞（RVECs）损伤是糖尿病患者发病和死亡的主要原因。RVECs功能障碍是糖尿病视网膜病变血管并发症的主要病理表现。N6-甲基腺苷(m6A)是真核mRNA中最普遍的修饰。然而,m6ARNA修饰在RVECs功能障碍中的作用尚不清楚.
方法：采用RT-qPCR和Westernblot方法检测糖尿病视网膜病变m6ARNA修饰的变化。CCK-8测定，Transwell实验,伤口愈合试验，管形成实验,进行m6A-IP-qPCR以确定YTHDC1在RVEC中的作用。采用视网膜胰蛋白酶消化试验和H&E染色评价组织病理学变化。
结果：在HG诱导的RVECs中m6ARNA甲基化水平显著上调，这是由YTHDC1的表达增加引起的。YTHDC1调节生存能力，扩散,体外迁移和管形成能力。YTHDC1过表达通过抑制CDK6表达而损害RVECs功能，这是由YTHDC1依赖性mRNA衰变介导的。此外，它显示SH-YTHDC1抑制CDK6核出口。Sh-YTHDC1促进细胞核中CDK6的mRNA降解，但不影响细胞质CDK6mRNA。体内实验表明，CDK6的过表达逆转了sh-YTHDC1对STZ诱导的视网膜组织损伤的保护作用。
结论：YTHDC1介导的m6A甲基化调节糖尿病诱导的RVECs功能障碍。YTHDC1-CDK6信号传导轴可以是治疗DR的治疗靶向。
方法：采用RT-qPCR和Westernblot方法检测糖尿病视网膜病变m6ARNA修饰的变化。CCK-8测定，Transwell实验,伤口愈合试验，管形成实验,进行m6A-IP-qPCR以确定YTHDC1在RVEC中的作用。采用视网膜胰蛋白酶消化试验和H&E染色评价组织病理学变化。
结果：在HG诱导的RVECs中m6ARNA甲基化水平显著上调，这是由YTHDC1的表达增加引起的。YTHDC1调节生存能力，扩散,体外迁移和管形成能力。YTHDC1过表达通过抑制CDK6表达而损害RVECs功能，这是由YTHDC1依赖性mRNA衰变介导的。此外，它显示SH-YTHDC1抑制CDK6核出口。Sh-YTHDC1促进细胞核中CDK6的mRNA降解，但不影响细胞质CDK6mRNA。体内实验表明，CDK6的过表达逆转了sh-YTHDC1对STZ诱导的视网膜组织损伤的保护作用。
结论：YTHDC1介导的m6A甲基化调节糖尿病诱导的RVECs功能障碍。YTHDC1-CDK6信号传导轴可以是治疗DR的治疗靶向。
