HIPK2对HIF-1α转录活性及泛素化的调控在低氧诱导视网膜新生血管中的作用

视网膜新生血管（RNV）的发生与低氧状态下HIF-1α稳定性增加密切相关。HIPK2是HIF-1α关键上游抑制分子，被认为是极具潜力的抗肿瘤治疗靶点，但在RNV中的作用尚不清楚。我们发现：HIPK2在OIR小鼠视网膜中低表达，其过表达显著抑制HIF-1α转录并促进其泛素化，抑制VEGF和RMECs细胞迁移及成管。研究报道HIF-1α也可以负调控HIPK2。故提出假说：HIPK2通过调控HIF-1α转录活性及泛素化影响RNV发生，且二者存在相互抑制的反馈回路。本课题拟在体外低氧模型中探讨HIPK2对HIF-1α转录活性和泛素化的调控作用，明确HIPK2结合HIF-1α的启动子位点以及HIPK2-HIF-1α相互抑制作用；并通过体内外靶向干预HIPK2表达，明确对HIF-1α/VEGF通路、RMECs生物学特性及RNV发生的影响。本研究将为RNV发病机制及靶向药物的开发提供新的理论和实验依据。

Retinal neovascularization (RNV) is a common and refractory eye disease that causes vision loss. HIF-1α accumulation caused by hypoxia is believed to play an important role in the pathological process of RNV. HIPK2 is an important upstream negative regulator of HIF-1α expression, which has been considered as a promising biomarker and a target for tumor therapy. However, the underlying role of HIPK2 in RNV development remains unclear. Our preliminary results suggested that hypoxia down-regulated the mRNA and protein expression of HIPK2 in retina from oxygen-induced retinopathy model and in vitro hypoxia-stimulated retinal microvascular endothelial cells (RMECs). Overexpression of HIPK2 significantly inhibited the transcriptional activity and promoted the ubiquitination of HIF-1α, decreased VEGF secretion and inhibited RMECs migration and lumen formation. On the contrary, it was reported that HIF-1α could negative regulate HIPK2 expression. Based on this, we put forward the hypothesis that HIPK2 affects the occurrence and development of RNV by regulating the transcriptional activity and ubiquitination of HIF-1α. Besides, there exist a negative feedback loop between HIF-1α and HIPK2. In this study, dual-luciferase reporter assay, co-immunoprecipitation and ubiquitination detection are used to investigate the regulation role of HIPK2 on the transcriptional activity and ubiquitination of HIF-1α in hypoxia-stimulated RMECs, then identify the HIF-1α promoter region repressed by HIPK2 and analyze the mutual negative regulation role between HIF-1α and HIPK2. Furthermore, HIPK2 knockout or overexpression RMECs induced by lentivirus and HIPK2 conditional knockout transgenic mice are used to determine the effects of HIPK2 on the HIF-1α/VEGF signaling pathway, RMECs proliferation, apoptosis, migration, lumen formation, inflammatory cytokines production and RNV development. This study is expected to provide new pathogenesis and therapeutic target for RNV, which has an important theoretical and experimental significance.