Research Based On ITRAQ Technology In CNV Rat And Protective Role Of TAB1 In CNV

Background and Aims:Age related macular disease(AMD)is one of the most severe eye diseases leading to blindness among the people over 50 in the western countries.Choroidal neovascularization(CNV)is the most severe pathological ending of AMD and is also a common cause of vision impair and blindness.CNV formats from the micro vessels in the choroid,breaks through the Bruch’s membrane and lays underneath the retinal neuroepithelium layer.The fluid and blood in the CNV could leak from the vessel because of the incomplete structure of CNV.The leaked fluid or blood will accumulate around the macular and impact vision.The anti-VEGF therapy has gained great success.However,it also has some limitations like repeated injections and infection after injections.What’s more,Some patients don’t even react to the anti-VEGF therapy very well,which hinted us there may remain some other CNV pathological mechanisms besides elevated VEGF.So,we have to investigate the mechanisms of CNV and look for some other therapy targets besides VEGF.As is known to all,protein is the executor of life function.The concept of proteomics is presented in 1995.Proteomics is defined as the whole gene expression of a kind of cell,a tissue or an organism,including all the subtypes and posttranslational proteins.Many kinds of protein are involved in a disease,which allows proteomics technologies arise rapidly in looking for disease markers.Isobaric tags for relative and absolutely quantification(iTRAQ)technology is an in vitro labeled technology launched by American applied biosystems in 2004.ITRAQ technology has several advantages including high sensitivity,high resolution,good repeatability.Thus,iTRAQ technology has been applied in seeking biomarkers and mechanisms of diseases.To the best of our knowledge,the RPE-choroidal-sclera complex protein has not been studied with iTRAQ technology yet.Thus,the aim of our project is to analyze the whole protein of rat RPE-choroidal-sclera complex,hoping to enrich the understanding of the mechanisms of CNV and to find new treating targets.Methods:(1)Laser-induced CNV rat model.The CNV rat model was induced by 532 nm laser coagulation.Nine normal rats and nine CNV rats were sacrificed for the iTRAQ detection.(2)iTRAQ labeling and protein identification.(3)Bioinformatic analysis.(4)Protein verification.Eight of the changed proteins were chosen for WB test for verification.(5)Choroidal flat amount was used to test the size of CNV lesion.ELISA was used to evaluate IL-18 and IL-6 in vivo and in vitro.(6)CCK-8 was used to test the proliferation of cells and WB was used to test the relevant Nf-k B signal molecules.Main results:(1)We have identified 4380 proteins in total and found 49 elevated proteins and 241 downregulated proteins in CNV rats compared to normal rats.(2)The different proteins were mainly involved in the process of mitotic nuclear division,immune response-regulating cell surface receptor signaling pathway,negative regulation of transcription,DNA-templated,negative regulation of cell death,cytokine production.chromatin binding,transmembrane receptor activity,helicase activity,kinase regulator activity,enzyme inhibitor activity.(3)KEGG analysis results showed that the pathway of human papillomavirus infection,PI3K-Akt signaling pathway,herpes simplex infection,tuberculosis,pathways in cancer were significantly changed.(4)MCM7,TRIM32,RPIA and TAB1 were positively tested among MCM7,YES1,SEPT9,p27kip1,p57kip2,RPIA,TRIM32 and TAB1.(5)CNV lesions were significantly smaller in CNV+AAV-TAB1 group than that in CNV group at day 14,day 30 and day 60(p<0.01,p<0.001,p<0.001)while there is no statistical difference between CNV and CNV+AAV group at any time point.(6)The IL-18 expression level in the RPE-choroid-sclera in CNV+AAV-TAB1 was significantly higer than that in the control group at day 14,day 30 and day 60(p<0.01,p<0.01,p<0.001).There was no significant difference between CNV and CNV+AAV group at each time point(p > 0.05).the IL-6 expression level in the RPE-choroid-sclera in CNV+AAV-TAB1 was significantly lower than that in the control group at day 14,day 30 and day 60(p<0.01,p<0.01,p < 0.01).There was no significant difference between CNV and CNV+AAV group at each time point.(p>0.05)(7)IL-18 expression significantly reduced at hour 24,hour 48 and hour 72 after TAB1 overexpression transfection(p<0.01,p<0.001,p<0.001),while it was not significantly changed at hour 12 after transfection(p>0.05).In the part of IL-6,IL-6 expression was significantly reduced at hour 24,hour 48 and hour 72 after TAB1 overexpression transfection(p<0.01,p<0.01,p<0.01),while it was not significantly changed at hour 12 after transfection.(8)Cell proliferation was upregulated in TAB1-Adv group than that in Adv group at hour 24,hour 48 and hour 72(p<0.05,p<0.01,p<0.01),while it was not significantly changed in the two groups at hour 12(p>0.05).Main Conclusions:(1)iTRAQ analysis is practicable in CNV study.(2)The changed proteins were mainly associated with immune activity and cell proliferation,which means immune activity is an important reaction in CNV.Our study provides new insights to CNV study and presents new ideas in treating targets.(3)TAB1 can reduce the CNV lesion,upregulate IL-18 expression,downregulate IL-16 expression,which means TAB1 has a potential protective role in CNV.Our study provided a new insight into CNV mechanisms and provided a new potential therapy target.