Annals of clinical and laboratory science · 2023
The Transcription Factor RUNX1 Aggravates Hypoxia-Induced Human Retinal Microvascular Endothelial Cell Dysfunction by Negatively Regulating TFF1 Promoter.
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Zhang Yanli, Qiu Shuang, Liu Lingyan, Qiu Lifen
Аннотация
OBJECTIVE: As a retinal vaso-proliferative disorder, retinopathy of prematurity (ROP) is characterized by neovascularization and angiogenesis, causing irreversible retinal damage and even visual loss among premature infants. Trefoil factor 1 (TFF1) has been identified as a key regulator in mediating retinal angiogenesis in diabetic retinopathy. However, whether TFF1 can mediate the angiogenic process in ROP remains unknown. Here, we aimed to investigate the regulatory function of TFF1 and its underlying mechanisms in hypoxia-exposed human retinal vascular endothelial cells (HRVECs) in vitro.
METHODS: HRVECs were exposed to hypoxia condition to establish the in vitro ROP models. HRVEC viability was validated using CCK-8 assay. The migratory and angiogenic capacities of HRVECs were assessed by wound healing and tube formation assays, respectively. RT-qPCR was performed to detect gene levels. Western blotting was used to measure the protein levels of TFF1 and Runt-related transcription factor 1 (RUNX1). The binding relationship between RUNX1 to TFF1 promoter was confirmed by chromatin immunoprecipitation and luciferase reporter assays.
RESULTS: Hypoxia downregulated TFF1 expression and elevated RUNX1 expression in HRVECs. Moreover, hypoxic condition increased HRVEC viability and accelerated HRVEC migration and angiogenesis, which were antagonized by TFF1 elevation or RUNX1 knockdown. RUNX1 as a transcription factor bound to TFF1 promoter and transcriptionally repressed TFF1 expression in HRVECs. In rescue assays, overexpression of TFF1 counteracted the promotive effect of RUNX1 overexpression on the viability, migratory and angiogenic abilities of HRVECs under hypoxia.
CONCLUSIONS: RUNX1 transcriptionally suppresses TFF1 expression to aggravate hypoxia-induced HRVEC dysfunction.
METHODS: HRVECs were exposed to hypoxia condition to establish the in vitro ROP models. HRVEC viability was validated using CCK-8 assay. The migratory and angiogenic capacities of HRVECs were assessed by wound healing and tube formation assays, respectively. RT-qPCR was performed to detect gene levels. Western blotting was used to measure the protein levels of TFF1 and Runt-related transcription factor 1 (RUNX1). The binding relationship between RUNX1 to TFF1 promoter was confirmed by chromatin immunoprecipitation and luciferase reporter assays.
RESULTS: Hypoxia downregulated TFF1 expression and elevated RUNX1 expression in HRVECs. Moreover, hypoxic condition increased HRVEC viability and accelerated HRVEC migration and angiogenesis, which were antagonized by TFF1 elevation or RUNX1 knockdown. RUNX1 as a transcription factor bound to TFF1 promoter and transcriptionally repressed TFF1 expression in HRVECs. In rescue assays, overexpression of TFF1 counteracted the promotive effect of RUNX1 overexpression on the viability, migratory and angiogenic abilities of HRVECs under hypoxia.
CONCLUSIONS: RUNX1 transcriptionally suppresses TFF1 expression to aggravate hypoxia-induced HRVEC dysfunction.