| Objective: Retinopathy of prematurity(ROP)is one of the blinding eye diseases in children and is characterized by the formation of neovascularization within the retina.If left undetected and untreated in time,it can lead to retinal detachment and severe long-term visual impairment.The pathogenesis of ROP is complex and is not very clear at present,but is closely related to the lack of intact retinal vascular development in premature infants.Currently,laser,condensation,and anti-vascular endothelial growth factor treatments are still the main tools,but the treatment process may damage the retinal tissue and lead to visual impairment.Metastasis-associated Lung Adenocarcinoma Transcript 1(MALAT1)is widely expressed in mammals and is involved in the development of many diseases.Studies have shown that MALAT1 is involved in cardiovascular disease,abnormal endothelial cell function,tumor angiogenesis and metastasis.In addition,MALAT1 is sensitive to changes in the oxygen environment,and ischemic hypoxia can induce its high expression and thus cause lesions.Since ischemia and hypoxia are the pathological basis of Retinal neovascularization(RNV)formation in ROP.Therefore,we further explored the role played by MALAT1 in RNV formation.These findings help to find a more effective clinical approach to treat ROP/RNV and provide insight into its pathogenesis.Methods: Human retinal endothelial cells(HRECs)were first cultured and treated with200 μ mol/L cobalt chloride for 24 h to simulate hypoxia,transfected with plasmid MALAT1-RNAi to knock down the expression level of MALAT1,and the cells were divided into the following four groups: normal control group,hypoxia group,knockdown control group,and knockdown MALAT1 group;transfected with miR-106a-5p mimics overexpressing miR-106a-5p,were also divided into four groups.Real-time quantitative PCR was taken to detect the m RNA expression of MALAT1,miR-106a-5p and matrix metalloproteinase 2(MMP-2)in the cells,and Western blot technique was used to detect the expression levels of MMP-2,VEGF,IL-1β,TNF-α,Caspase-3,Blc-2 protein in the cells,and Ed U method was used to detect the The proliferation ability of the cells,the apoptosis level of the cells by flow cytometry,the migration ability of the cells by scratch assay and Transwell assay,and the angiogenesis ability of the cells by tubule formation assay.Dual luciferase reporter gene assay was performed to verify the binding relationship between MALAT1 and miR-106a-5p and miR-106a-5p and MMP-2.To construct an oxygen-induced retinopathy(OIR)model,mice in the OIR group were injected with miR-106a-5p mimics in the vitreous cavity and divided into normal control group,OIR group,drug-injected control group and drug-injected group,and MALAT1,miR-106a-5p and MMP-2 were detected in the retinal tissue by PCR technique.miR-106a-5p and MMP-2 m RNA expression in retinal tissues by PCR,and MMP-2,VEGF and HIF-1α protein expression by Western Blot and immunohistochemical assays.The morphology of the retina was observed using retinal patch staining and HE staining to assess the extent of the lesions.Results: The expression of MALAT1 in the retinal tissues of mice in the OIR group was significantly higher than that in the control group(P < 0.05),while the expression of miR-106a-5p was lower than that in the control group(P < 0.05).Hypoxia promoted MALAT1 m RNA expression in HRECs,and this trend could be reversed by MALAT1-RNAi.In addition,downregulation of MALAT1 attenuated the proliferation,migration and angiogenic ability of HRECs(P < 0.05).Western Blot results showed that the expression levels of VEGF,IL-1β and TNF-α were significantly lower in the knockdown MALTA1 group compared with the knockdown control group(P < 0.05).miR-106a-5p and MMP-2 were also differentially expressed in HRECs.were also differentially expressed in HRECs.qRT-PCR results showed that MMP-2 m RNA expression was significantly lower in the knockdown MALAT1 group compared with its control group(P < 0.05),and Western blot results showed that MMP-2 expression was reduced in the transfected group compared with the transfected control group(P < 0.05).Similarly,overexpression of miR-106a-5p in hypoxic HRECs significantly decreased the expression of MMP-2,VEGF,IL-1β,TNF-α and Caspase-3,and elevated Bcl-2expression(P < 0.05),and the apoptosis rate,tube-forming ability and cell migration ability were significantly decreased(P < 0.05)relative to their controls.The results of dual luciferase reporter gene assay also demonstrated the existence of direct binding sites between miR-106a-5p and MALAT1 and MMP-2(P < 0.05).HE staining showed that the number of nuclei breaking through the inner retinal boundary membrane was significantly higher in the OIR group than in the normal control group(P < 0.05),and the number of endothelial nuclei breaking through the inner retinal boundary membrane was significantly lower in the drug-injected group.The number of endothelial nuclei breaking through the inner retinal boundary membrane was significantly lower than that of the drug-injected control group(P < 0.05).The results of retinal vascular staining pavement indicated that the area of retinal neovascularization and the area of non-perfused area were significantly higher in the OIR group than in the normal control group(P < 0.05),and the area of retinal neovascularization and the area of non-perfused area were significantly lower in the overexpression miR-106a-5p group than in its control group(P< 0.05).Meanwhile,relative to the normal group,Western Blot and immunohistochemical results showed that the expression levels of MMP-2,VEGF,HIF-α,IL-1β,TNF-α and Caspase-3 were significantly higher(P < 0.05)and the expression of Bcl-2 was lower(P < 0.05)in the OIR group.In the injection group,the trend was opposite(P < 0.05),which indicated that overexpression of miR-106a-5p could effectively reduce the expression levels of MMP-2,VEGF and inflammatory factors.Conclusion: Knockdown of MALAT1 and overexpression of miR-106a-5p can reduce the proliferation,migration,apoptosis,angiogenesis and inflammation of HRECs induced by hypoxia.Overexpression of miR-106a-5p can reduce the degree of retinopathy in OIR mice.MALAT1 may promote the occurrence and development of ROP by sponging miR-106a-5p to enhance the expression of MMP-2,thus providing a new perspective for the targeted therapy of ROP. |
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