| ObjectiveAge-related macular degeneration(AMD)is a progressive chronic disease of macula of the retina characterized by impairment or loss of central vision.And choroidal neovascularization(CNV)in the macula is the main pathological feature of AMD.Based on the research,lipocalin 2(LCN2),as a marker of endothelial injury,plays a key role in CNV of AMD.To discuss the role and mechanism of LCN2 in CNV-induced AMD,we investigated the effect of LCN2 on CNV process from CNV formation and inflammatory response in hypoxic cell model and laser-induced CNV mouse model.And it is expected to clarify the molecular mechanism of the pathogenesis of AMD,seek new therapeutic targets,and provide a theoretical basis for the diagnosis and treatment of the disease.MethodsPartⅠThe expression and related role of LCN2 in CNV formationHuman retinal pigment epithelial cells(ARPE-19)and human umbilical vein endothelial cells(HUVECs)were selected as the research objects to establish hypoxic cell model and CNV mouse model induced by laser.(1)The effect of LCN2 on biological activity of hypoxic cells: the expression of LCN2 in hypoxic cells was detected by Western blot;The effects of silencing or overexpression of LCN2 on VEGF production,inflammatory response,cell proliferation,apoptosis,migration and lumen formation were detected by ELISA,CCK-8,flow cytometry,Transwell and lumen formation experiment.(2)The effect of LCN2 expression on choroid / RPE tissue of CNV mice induced by laser: the expression of LCN2 in choroid / RPE tissue of control group and CNV mice was detected by Western blot;After intravitreal injection of LCN2 inhibitor RGDS,the CNV area of mouse choroid / RPE was detected by immunofluorescence staining.PartⅡ SOX9 activates LCN2 gene transcriptionARPE-19 was selected as the research object to establish the hypoxic cell model and carry out relevant project experiments.(1)Transcription factor prediction of LCN2: Based on AMD sample sequencing data sets GSE29801 and GSE103060;The transcription factors of LCN2 were predicted by knocktf and cistromedb databases.(2)SOX9 regulates LCN2 expression: chromatin immunoprecipitation and double luciferase gene detection verify the regulation of SOX9 on LCN2;The expression of SOX9 and its effect on the expression of LCN2 in hypoxic cells were detected by Western blot.PartⅢ The mechanism of SIRT1 / SOX9 / LCN2 signal axis regulating AMDARPE-19 and HUVECs were selected as the research objects to establish hypoxic cell model and CNV mouse model induced by laser.(1)The effect of SIRT1 / SOX9 / LCN2 signal axis on biological activity of hypoxic cells: the expression of SIRT1 and its effect on SOX9 acetylation were detected by Western blot;SOX9 nuclear translocation was detected by immunofluorescence;The SIRT1 / SOX9 / LCN2 signal axis and its effects on VEGF production,inflammatory response,cell proliferation activity,apoptosis,migration and lumen formation were verified by immunoblotting,ELISA,CCK-8,flow cytometry,Transwell and cell lumen formation experiments.(2)The effect of SIRT1 / SOX9 / LCN2 signal axis on choroid / RPE tissue of CNV mice induced by laser: the expression of SIRT1,acetylated SOX9 and LCN2 in choroid / RPE tissue of CNV mice and their upstream and downstream relationship were detected by Western blot;The area of CNV in retinal choroid / RPE tissue of CNV mice was detected by immunofluorescence staining.ResultsPartⅠ(1)LCN2 promotes the development of hypoxic cytopathy: in the hypoxic cell model,the expression of LCN2 increases;Knockdown of LCN2 can inhibit the expression of VEGF,TNF-α and IL-6 and the ability of apoptosis,migration and lumen formation promote cell proliferation;overexpression of LCN2 showed the opposite effect.(2)LCN2 promotes the development of laser-induced lesions in CNV mice: the expression of LCN2 in choroid / RPE tissue of CNV mice increased;LCN2 inhibitor can reduce the CNV area of mouse choroid / RPE tissue.Part Ⅱ(1)SOX9 is the transcription factor of LCN2: SOX9 is predicted to be the potential transcription factor of LCN2 by Shengxin analysis.The experiment verifies that there is a direct targeting relationship between SOX9 and LCN2 gene promoter.(2)SOX9 regulates the expression of LCN2: the expression of SOX9 is up-regulated in hypoxic cell model;Silencing SOX9 can inhibit the expression of LCN2,while overexpression of OX9 has the opposite effect..Part Ⅲ(1)SIRT1 / SOX9 / LCN2 signal axis promotes the development of hypoxic cytopathy: the expression of SIRT1 is increased in hypoxic cell model;Overexpression of SIRT1 could reduce the level of acetylated SOX9 and increase nuclear Sox9,while silencing SIRT1 had the opposite effect,but both had no effect on the expression of total SOX9;Silencing SIRT1 decreased the expression of LCN2 and inhibited intracellular VEGF and TNF-α the expression of IL-6 can inhibit cell apoptosis,migration and lumen formation,and promote cell proliferation;Silencing SIRT1 and overexpression of LCN2 reversed the above trend.(2)SIRT1 / SOX9 / LCN2 signal axis regulation the development of laser-induced lesions in CNV mice: the expression of SIRT1 and LCN2 increased and acetylated SOX9 decreased in choroid / RPE of CNV mice;Silencing SIRT1 could increase the level of acetylated SOX9 and reduce the expression of LCN2;At the same time,overexpression of LCN2 can reverse the above trend;Silencing SIRT1 can reduce CNV area,overexpression of LCN2 can increase CNV area,while silencing SIRT1 and overexpression of LCN2 can reverse the above trend.Conclusions(1)LCN2 can inhibit the proliferation of hypoxic retinal epithelial cells,promote VEGF production,inflammatory response,apoptosis,migration and lumen formation,and promote the development of AMD;Inhibition of LCN2 expression can reduce the CNV area of choroid / RPE tissue in CNV mouse model and alleviate the condition of AMD.(2)SOX9 is a transcription factor of LCN2,which can activate the transcription of LCN2 gene.(3)SIRT1 deacetylates SOX9 into the nucleus,up regulates the transcriptional expression of LCN2,and then promotes the formation of CNV and the development of AMD. |
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