Long Non-coding RNA TUG1 Regulates Retinal Neovascularization In Retinopathy Of Prematurity

Objective:Retinopathy of prematurity（ROP）is a common retinovascular proliferative disease in low weight and premature infant.Newborn pediatricians are more and more comprehensive in cognition of ROP,and the oxygen usage of newborns is also becoming more standardized.However,under the condition of perinatal medicine,some children with small gestational age and very low weight are born with retinopathy of prematurity.It is difficult to avoid retinopathy of prematurity in recent years.The incidence rate of ROP in premature infants is not decreasing.Although the anti VEGF drugs widely used in clinic have some curative effect,they have the risk of visual function damage and high recurrence rate.Taurine up regulated genes-1（TUG1）was first discovered in the retina,and its function was involved in the development of retina.The results showed that TUG1 promoted tumor angiogenesis,but the role of TUG1 in retinal neovascularization（RNV）formation was not clear.The main purpose of this study is to clarify the mechanism of TUG1 regulating VEGF and RNV formation,and to provide new research ideas and targets for the treatment of ROP/RNV disease.Methods:Firstly,the oxygen induced retinopathy（OIR）model in mice was established and divided into normal control group,OIR group,TUG1 control group and sh-TUG1group.The sh-TUG1 group was injected lentivirus to knock down TUG1,and the TUG1control group was used as lentivirus control group.The interference effect of TUG1 was detected by PCR.The degree of retinopathy was evaluated by retinal vascular staining and he staining.The apoptosis of retinal cells was evaluated by TUNEL assay.The expressions of VEGF,BAX,Bcl-2,Caspase-3,IL-1β,IL-6 and TNF-αwere detected by Western blot,immunohistochemistry and immunofluorescence.Human retinal endothelial cells were cultured and treated with 200μmol/L Co Cl₂ to simulate hypoxia.After overexpression of has-mir-299-3p,the expression of VEGF was measured by RT-PCR.Cell apoptosis was measured by flow cytometry.The ability of tubule formation was measured by tubule formation assay.Cell migration was observed by scratch assay.The expression of TUG1 and VEGF was detected by dual luciferase reporter gene To explore the relationship between tug1 and miR-299/VEGF and the generation of RNV.Results:The expression of TUG1 in the retina of OIR group was significantly higher than that of control group（P<0.05）,and the expression of TUG1 in sh-TUG1 group was significantly lower than that of OIR group and TUG1 control group（P<0.05）.HE staining results showed that the number of nuclei in OIR group was significantly higher than that in control group（P<0.05）,and the number of endothelial cells in sh-TUG1group was significantly lower than that in TUG1 control group（P<0.05）.The retinal neovascularization area and non-perfusion area in OIR group were significantly higher than those in control group（P<0.05）.The retinal neovascularization area and non-perfusion area in sh-TUG1 group were significantly lower than those in TUG1control group（P<0.05）.The results of HE staining and retinal patch showed that knockdown of TUG1 expression was helpful to alleviate the pathological changes of retina.TUNEL results showed that the apoptosis rate of retinal tissue cells in sh-TUG1group was significantly lower than that in TUG1 control group（P<0.05）.At the same time,compared with the normal group,Western blot results showed that the expression levels of BAX and Caspase-3 in OIR group were significantly increased（P<0.05）,and the expression of Bcl-2 was significantly decreased（P<0.05）.The apoptosis rate of sh-TUG1 group was significantly lower than that of TUG1 control group（P<0.05）.Knockdown of TUG1 can effectively reduce the apoptosis rate.Compared with the normal control group,the expressions of VEGF,IL-1β,IL-6 and TNF-αin OIR group were significantly increased（P<0.05）.The expressions of VEGF,IL-1β,IL-6 and TNF-αin sh-TUG1 group were significantly lower than those in TUG1 control group（P<0.05）.Knockdown of TUG1 could effectively reduce the expressions of VEGF and inflammatory factors.When miR-299 was overexpressed in hypoxic HREC cells,compared with Co Cl₂ control group,the expression of VEGF was significantly decreased（P<0.05）,and the apoptosis rate,tubulation ability and migration ability of cells were significantly decreased（P<0.05）.The results of dual luciferase assay indicated that wild-type TUG1 and VEGF had targeted binding sites with has-miR-299-3p.Conclusion:This study shows that hypoxia can increase the expression of inflammatory factors in endothelial cells,promote cell apoptosis,and cause retinal neovascular diseases.miR-299-3p can bind to 3’-UTR of VEGF and down regulate the expression of VEGF m RNA.Under hypoxia,the expression of TUG1 in retinal endothelial cells is high,and the competitive adsorption of miR-299-3p leads to the high expression of VEGF.Knockdown of TUG1 can effectively reduce the expression of VEGF,which may provide a new treatment idea for retinopathy of prematurity and other retinal neovascular diseases.