Proteomic Analysis In Traumatic Proliferative Vitreoretinopathy And The Molecular Mechanism In It

Background:As one of the serious complication of ocular trauma,proliferative vitreoretinopathy(PVR)is the leading cause of retinal detachment failure,which is characterized with excessive proliferation,migration and contraction of retinal pigment epithelium.The mechanism of PVR development is complex and still not fully understood.Vitreous proteins are abnormally expressed during the whole disease process of PVR.As an identificative and quantitative technique,proteomics can be used in early diagnosis and clinical treatment of a variety of diseases,including PVR,based on which,we could further study the differentially expressed proteins.Aim:For the first,we aim to identify the differentially expressed proteins in retinal tissue between the traumatic proliferative vitreoretinopathy patients and the normal through proteomics analysis,then to provide the novel theoretical basis for the pathogenesis of traumatic PVR and find new breakthrough for its prophylaxis and treatment.Then,it was aimed to knock down the expression of ABCA4 in human retinal pigment epithelial(RPE),ARPE-19 cells,and study its effect on the viability,proliferation,apoptosis and migration of RPE cells,and finally,to provide some new ideas to prevent and treat traumatic PVR.Methods:During the proteomic analysis in this study,the following experiments were performed:(1)Extracting the retinal samples from the traumatic PVR patients and the normal persons,then assaying the proteins concertation using BCA method.(2)SDS-PAGE and coomassie blue staining were performed to evaluate the extracting effect.(3)High-performance liquid chromatography and tandem mass spectrometry test was conducted to analysis the extracted proteins.(4)Label-free analysis was used for the qualitative and quantitative analysis of proteins.(5)The bioinformatics analysis of the identified differential expression proteins was performed,including GO enrichment analysis,KEGG pathway analysis,and hierarchical cluster analysis.(6)RT-qPCR and western blot was used to verify the expression level of ABCA4 in the retina tissues of the traumatic PVR patients.When the function of ABCA4 was studied,(1)the small interference RNA,specific targeting ABCA4,siABCA4 or non-specific siRNA,siNC,was transferred to the RPE cell line ARPE-19.(2)The expression level of ABCA4 was measured by qRT-PCR and Western blotting.(3)The cell viability was detected by MTT colorimetric assay in the ABCA4 knockdown cells or control cells.The changes of cell cycle and apoptosis in two kinds of cells were compared by flow cytometry combined with Annexin V-FITC or PI staining.(4)The migration ability of ABCA4 knockdown and control cells was measured by scratch test.(5)In addition,the expression level of cell proliferation,cycle and apoptosis related proteins in ABCA4 knockdown and control cells were detected by Western blotting assay.Results:After the proteomics,we found(1)The retina protein concertation in traumatic PVR patients was 19.7ug/ul,which was a little more than that of the normal persons corresponding to 14.1 ug/ul.(2)The protein extracting effect in this experiment was very good,besides,comparing with the retina in control group,the traumatic PVR patients are of a higher retina protein concertation,which expressed in the greater proportion of high abundance proteins.(3)A total of 25305 peptide fragments and 3541 proteomics was separated and identified in the retina samples collected from traumatic PVR patients and normal persons using LC-MS/MS technology.(4)A total of 3518 proteins was identified using Maxquant software,of which,there were 241 differentially expressed proteins.(5)The result of GO analysis suggested that the identified the differentially expressed proteins was associated with 18 biological processes,15 molecular functions,and 9 cellular components.Specially,the first five biological processes that contain most differential proteins are as follows:multicellular organismal process,single-multicellular organism process,cell periphery,plasma membrane,response to external stimulus.(6)The result of KEGG suggested that the top five pathways are related with the identified proteins are as follows:huntington's disease,oxidative phosphorylation,focal adhesion,Alzheime's disease,non-alcoholic fatty lover disease.(7)The result of hierarchical cluster analysis indicated that there is a total of 88 up-regulated expression proteins and 153 down-regulated expression proteins in 241 differentially expressed proteins.(8)The result of RT-qPCR and western blot showed that ABCA4 was significantly upregulated at both mRNA level and protein level in retina tissues of traumatic PVR patients.The findings in the function study showed that(1)Transfection of ABCA4 specific siRNA,siABCA4 successfully inhibited ABCA4 expression in RPE cell line ARPE-19,and constructed ARPE-19 cells with ABCA4 low expression.Compared with the cells transfected with control nonspecific siRNA,the increase of cell viability alone with culture time of the cells transfected with siABCA4 was significantly inhibited.Western blotting assay of proliferating cell associated antigen,proliferating cell nuclear antigen(PCNA)and Ki67 showed that the expression of PCNA and Ki67 decreased significantly in cells transfected siABCA4,indicating that the number of normal proliferating cells decreased.(2)PI staining combined with cell cycle analysis by flow cytometry showed that,compared with cells transfected with control siRNA,the percentage of cells in G0/G1 phase of cells transfected with siABCA4 was significantly increased,and cells in S and G2/M phases decreased significantly,indicating transfection of siABCA4 induced ABCA4 downregulation in ARPE-19 cells resulted in cell cycle arrest in G0/G1 stage.Study on the molecular mechanism showed that p21,also known as cyclin dependent kinase inhibitors(CDKN1)or CDK-interacting protein 1(CDK1)is significantly upregulated in ARPE-19 cells with low expression of ABCA4,whereas two kinds of cell cycle promoting proteins,cyclin-dependent kinase 4(CDK4)and cyclin D1 was significantly upregulated.(3)Annexin V FITC staining was used to study apoptosis with flow cytometry in two kinds of cells and found that compared with cells transfected with nonspecific siRNA cells,cells transfected with siABCA4,the percentage of apoptotic cells in early and late stage were both significantly increased.The molecular mechanism study found that compared to the cells with normal ABCA4 expression,cell apoptosis promoting proteins,Bax and bad protein expression were increased significantly in ARPE 19 cells with lower ABCA4 expression.(4)The results of scratch test showed that compared with cells transfected with non-specific control siRNA,the migration ability of RPE cells transfected with specific siABCA4 decreased significantly.Conclusion:(1)There are a total of 241 differentially expressed proteins in retina tissues of traumatic PVR patients comparing that in normal persons,which include 153 upregulated expression proteins and 88 downregulated expression proteins.(2)The expression level of ABCA4 was significantly upregulated in retina of traumatic PVR patients,which implied that ABCA4 may be played an important role in the development and process of traumatic PVR.(3)Transfection of ABCA4 specific siRNA,siABCA4 reduces the expression of ABCA4 and construction of ARPE-19 cells with low expression of ABCA4.(4)Transfection of siABCA4 in RPE cells inhibits cell viability increase with the increase of culture time,cell proliferation and induces cell apoptosis.(5)The effect of siABCA4 on the viability,proliferation and apoptosis of RPE cells is related to the abnormal expression of cell cycle regulatory proteins,p21,CDK4 and Cyclin D1,as well as the increased expression of Bax and Bad.(6)Transfection of siABCA4 reduces the migration ability of RPE cells.(7)ABCA4 has the potential to be a target for the prevention and treatment of traumatic PVR.