State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, No. 7 Jinsui Road, Tianhe District, Guangzhou, China.
Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China.
Invest Ophthalmol Vis Sci. 2023 Apr 3;64(4):10. doi: 10.1167/iovs.64.4.10.
Chemokine receptor 4 (CXCR4) plays an essential role in the early stage of corneal neovascularization (CNV), but the underlying key molecular mechanism has yet to be addressed. This study aimed to explore the new molecular mechanism of CXCR4 in CNV and the related pathological events.
CXCR4 was assayed by immunofluorescence or Western blotting. The function of the supernatant from hypoxia-treated human corneal epithelial cells (HCE-T) cells was examined by culturing with human umbilical vein endothelial cells. MicroRNA sequencing was used to detect the downstream microRNAs upon CXCR4 knockdown and analyzed by preliminary bioinformatics. The proangiogenic functions and downstream target genes of microRNA were investigated by gene interference and luciferase assay. An alkali-burned murine model was introduced to examine the function and mechanism of miR-1910-5p in vivo.
High CXCR4 expression was confirmed in corneal tissues of patients with CNV and hypoxic HCE-T cells. The supernatant from hypoxia-treated HCE-T cells is involved in the CXCR4-mediated angiogenesis of human umbilical vein endothelial cells. Notably, miR-1910-5p was demonstrated to be at a high level in wild-type HCE-T cells and its supernatant, and in CNV patient tears. The proangiogenic functions of miR-1910-5p were demonstrated with the assays of cell migration, tube formation, and aortic ring. Moreover, miR-1910-5p significantly inhibited multimerin-2 expression by targeting its 3' untranslated region and caused significant extracellular junctional defects in human umbilical vein endothelial cells. MiR-1910-5p antagomir could significantly increase multimerin-2 level and decrease vascular leakage, and ultimately inhibit CNV in a murine model.
Our results revealed a novel CXCR4-mediated mechanism and proved that targeting the miR-1910-5p/multimerin-2 pathway could be a promising therapeutic target for CNV.
趋化因子受体 4（CXCR4）在角膜新生血管化（CNV）的早期阶段发挥重要作用，但潜在的关键分子机制尚未得到解决。本研究旨在探讨 CXCR4 在 CNV 中的新分子机制及相关病理事件。
通过免疫荧光或 Western blot 检测 CXCR4。通过培养人脐静脉内皮细胞检测缺氧处理的人角膜上皮细胞（HCE-T）细胞上清液的功能。采用 microRNA 测序检测 CXCR4 敲低后的下游 microRNA，并进行初步的生物信息学分析。通过基因干扰和荧光素酶测定研究 microRNA 的促血管生成功能和下游靶基因。引入碱烧伤小鼠模型，体内研究 miR-1910-5p 的功能和机制。
在 CNV 患者和缺氧 HCE-T 细胞的角膜组织中证实了高 CXCR4 表达。缺氧处理的 HCE-T 细胞上清液参与了 CXCR4 介导的人脐静脉内皮细胞血管生成。值得注意的是，miR-1910-5p 在野生型 HCE-T 细胞及其上清液和 CNV 患者泪液中均处于高水平。通过细胞迁移、管形成和主动脉环实验证实了 miR-1910-5p 的促血管生成功能。此外，miR-1910-5p 通过靶向其 3'非翻译区显著抑制多聚蛋白-2 的表达，并导致人脐静脉内皮细胞的细胞外连接明显缺陷。miR-1910-5p 拮抗剂可显著增加多聚蛋白-2 水平，减少血管渗漏，最终抑制小鼠模型中的 CNV。
本研究揭示了一种新的 CXCR4 介导的机制，并证明靶向 miR-1910-5p/多聚蛋白-2 通路可能是 CNV 的一种有前途的治疗靶点。
