The Study On The Role Of HIF-1α/miR-381-3p Pathway In Retinal Vascular Diseases

Objective:Retinal neovascularization（RNV）is a major cause of vision loss in patients with several ocular diseases,including diabetic retinopathy（DR）,retinal vein occlusion（RVO）,retinopathy of prematurity（ROP）,and sickle cell retinopathy,which is caused by the stable expression of the hypoxia-inducible factor-1α（HIF-1α）in retinal tissues under pathological conditions such as ischemia and hypoxia.Hypoxia-inducible factor-1α（HIF-1α）is a key transcription factor in retinal tissues in response to hypoxic signals,and HIF-1αhas been found to regulate the expression of Micro RNAs（miRNAs）and they play an important role in the development of vascular neovascular disease.It has been studied that the expression of Micro RNA-381-3p（miR-381-3p）is increased in retinal tissues of retinal angiogenesis model mice,but the mechanism of miR-381-3p in retinal angiogenesis is unclear.The aim of this study was to evaluate the functional role of miR-381-3p on the disease phenotype of retinal neovascularization mice and to investigate the potential regulatory mechanisms of miR-381-3p in the pathological progression of retinal vascular diseases,thus providing new targets for the treatment of retinal vascular diseases.Methods:1.The differentially expressed miRNAs in the retinal neovascularization mouse model were analyzed by bioinformatics methods,and the oxygen-induced retinopathy（OIR）mouse model was constructed to investigate the functional role of miR-381-3p in retinal vascular diseases.2.The effect of miR-381-3p expression on retinal tissue vascularization was evaluated by q RT-PCR,retinal isolation vascular staining,and hematoxylin-eosin staining;the expression of inflammatory factors IL-1β,IL-6,and TNF-αwas measured by immunohistochemistry staining and Western blot assay to assess the inflammatory response of retinal tissue;TUNEL staining and Western blot was performed to assess the apoptosis of retinal tissue.3.An in vitro model of cellular hypoxia was constructed based on 200μmol/L cobalt chloride（Co Cl₂）treatment of human retinal pigment epithelial cells（ARPE-19）to investigate the regulatory effect of HIF-1αon miR-381-3p expression.The expression of miR-381-3p in ARPE-19 cells under hypoxic conditions was examined by q RT-PCR.Then,the binding of HIF-1αto the promoter region of miR-381-3p was examined by the dual-luciferase reporter gene assay.4.The gene expression in the retinal tissues of OIR mice after reduced miR-381-3p expression was analyzed by transcriptome sequencing.The downstream target genes of miR-381-3p were screened and validated in an in vitro cellular hypoxia model and the OIR mouse model.Results:1.The expression of miR-381-3p was elevated in a mouse model of retinal neovascularization.2.In the OIR mouse model,downregulation of miR-381-3p expression in mouse retinal tissue inhibited retinal tissue vascularization in the OIR group of mice,decreased the expression of inflammation-related factors IL-1β,IL-6 and TNF-αexpression,and reduced apoptosis in mouse retinal tissue.3.In an in vitro cellular hypoxia model,the expression of miR-381-3p was upregulated in ARPE-19 cells after 200μmol/L Co Cl₂ treatment for 2 h and 8 h.In addition,dual luciferase reporter gene assays showed that HIF-1αwas able to bind directly to the miR-381-3p promoter to promote its expression.4.Analysis of transcriptome sequencing results showed that inhibition of miR-381-3p expression in retinal tissues of OIR mice caused upregulation of steap4 gene expression,suggesting that steap4 may be a downstream miR-381-3p target gene.In an in vitro cellular hypoxia model,inhibition of miR-381-3p expression in ARPE-19 cells upregulated steap4expression;inhibition of miR-381-3p expression in retinal tissues of OIR mice upregulated steap4 expression.Conclusion:Hypoxia induces elevated miR-381-3p expression in ARPE-19 cells and mouse retinal tissues,and the results of dual luciferase reporter gene assays suggest that the elevated miR-381-3p expression under hypoxic conditions is due to the binding of the transcription factor HIF-1αto the HRE element of the miR-381-3p promoter region.In addition,the HIF-1α/miR-381-3p pathway promotes the pathological progression of retinal vascular disease,and inhibition of miR-381-3p expression in the retinal tissue of OIR mice upregulates STEAP4 expression and attenuates the pathological changes in OIR mice.