Xiao Qing, Zhao Yinu, Sun Hongjing, Xu Jia, Li Wenjie, Gao Limo
Department of Ophthalmology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 31009, Zhejiang Province, People's Republic of China.
Department of Ophthalmology, The Third Xiangya Hospital, Central South University, Changsha, 410013, Hunan Province, People's Republic of China.
Diabetol Metab Syndr. 2021 Dec 28;13(1):152. doi: 10.1186/s13098-021-00769-7.
Diabetic retinopathy (DR) is a diabetic complication and the primary cause of blindness in the world. However, the treatments of DR are challenging given its complicated pathogenesis. Here, we investigated the molecular mechanisms of DR by focusing on the function of E2F1/miR-423-5p/HIPK2/HIF1α/VEGF axis.
Cultured retinal endothelial cells (hRMECs, hRECs) were treated with 25 mM glucose to mimic the high glucose-induced DR in vitro. Streptozotocin (STZ) was injected into mice to induce DR in mice. qRT-PCR, western blotting, immunohistochemistry, and ELISA were employed to measure levels of E2F1, miR-423-5p, HIPK2, HIF1α, and VEGF. H&E staining was utilized to examine retinal neovascularization. CCK-8 assay, transwell assay, and vascular tube formation assay were used to assess the cell viability, migration, and angiogenesis. Dual luciferase assay was performed to validate interactions between E2F1 and miR-423-5p, miR-423-5p and HIPK2.
HG treatment increased the cell viability, migration, and angiogenesis accompanied by upregulation of E2F1, miR-423-5p, HIF1α, and VEGF levels, but reduction in HIPK2 expression. Knockdown of E2F1 or miR-423-5p suppressed the HG-induced increases in cell viability, migration, and angiogenesis. E2F1 transcriptionally activated miR-423-5p expression and miR-423-5p mimics blocked the effects of E2F1 knockdown on angiogenesis. Moreover, miR-423-5p directly targeted HIPK2 to disinhibit HIF1α/VEGF signaling. Knockdown of HIPK2 reversed the effects of miR-423-5p inhibitor on cell viability, migration, and angiogenesis. Knockdown of E2F1 suppressed neovascularization during DR in vivo.
E2F1 activates miR-423-5p transcription during DR to promote angiogenesis via suppressing HIPK2 expression to disinhibit HIF1α/VEGF signaling. Strategies targeting E2F1/miR-423-5p/HIPK2 axis could be potentially used for DR treatment.
糖尿病视网膜病变（DR）是一种糖尿病并发症，也是全球失明的主要原因。然而，鉴于其复杂的发病机制，DR的治疗具有挑战性。在此，我们通过聚焦E2F1/miR-423-5p/HIPK2/HIF1α/VEGF轴的功能来研究DR的分子机制。
用25 mM葡萄糖处理培养的视网膜内皮细胞（hRMECs、hRECs）以在体外模拟高糖诱导的DR。将链脲佐菌素（STZ）注射到小鼠体内以诱导小鼠发生DR。采用qRT-PCR、蛋白质印迹法、免疫组织化学和酶联免疫吸附测定法来检测E2F1、miR-423-5p、HIPK2、HIF1α和VEGF的水平。利用苏木精-伊红染色检查视网膜新生血管形成。采用CCK-8测定法、Transwell测定法和血管管形成测定法评估细胞活力、迁移和血管生成。进行双荧光素酶测定以验证E2F1与miR-423-5p、miR-423-5p与HIPK2之间的相互作用。
高糖处理增加了细胞活力、迁移和血管生成，同时E2F1、miR-423-5p、HIF1α和VEGF水平上调，但HIPK2表达降低。敲低E2F1或miR-423-5p可抑制高糖诱导的细胞活力、迁移和血管生成增加。E2F1转录激活miR-423-5p的表达，且miR-423-5p模拟物可阻断E2F1敲低对血管生成的影响。此外，miR-423-5p直接靶向HIPK2以解除对HIF1α/VEGF信号通路的抑制。敲低HIPK2可逆转miR-423-5p抑制剂对细胞活力、迁移和血管生成的影响。敲低E2F1可抑制体内DR期间的新生血管形成。
在DR期间，E2F1通过抑制HIPK2表达以解除对HIF1α/VEGF信号通路的抑制来激活miR-423-5p转录，从而促进血管生成。靶向E2F1/miR-423-5p/HIPK2轴的策略可能潜在地用于DR治疗。
