Proteomic Analysis Of Smoking Effects On RPE Cells With ARMS2/HTRA1 High-risk Alleles And Establishment Of Optimized Stem Cell Lines

Purpose 1.Protein spectrum based on iTRAQ technology was used to evaluated the effect of smoker's serum and non-smoker's serum on the RPE cells with ARMS2/ HTRA1 high-risk and low risk alleles.The differentially expressed proteins(DEPs)between groups were screened and the function and pathway enrichment analysis were carried out to compare the proteomic changes after the smoker' serum stimulations,to explore the mechanism of smoking and ARMS2/ HTRA1 high-risk alleles in the pathogenesis of AMD,further to find the direct evidence of their association and influence.2.CRISPR/Cas9 gene editing technique was applied to modify the target gene of stem cells to establish optimizing homozygous stem cell lines with ARMS2 high risk alleles and HTRA1 high risk alleles,so that we can separate the two closely linked genes.To provide a cell model for the specific analysis of the molecular biological characteristics of ARMS2 and HTRA1 and the identification of the real pathogenic genes of AMD.Methods 1.The serum of smokers and non-smokers were collected from the pre-screened healthy population to explore the optimal serum concentration.Primary human RPE cells derived from ARMS2/HTRA1 high-risk individuals and wild-type individuals were isolated and cultured from eyeballs of corneal donors.At the same time,i PSCs derived from ARMS2/HTRA1 high-risk individuals and wild-type individuals were induced to differentiate to RPE cells.After ARMS2/HTRA1 highrisk and wild-type primary RPE cells were exposed to pre-mixed smoker's serum and non-smoker's serum for 7 days,the total protein of each group was extracted and the protein profile was detected by protein spectrum based on iTRAQ technique.The DEPs were obtained by data acquisition and analysis with Thermofisher Proteome Discoverer 1.3 ~ 1.4 software for the peptide signal detected by QExactive mass spectrometer.P_value < 0.05 and ratio value 1.2 times(up-regulation 1.2,down-regulation 0.833)were screened as credible DEPs.Credible DEPs were analyzed for GO functional and KEGG pathway enrichment analysis.The same action conditions was repeated on i PSCs-RPE cells with ARMS2/HTRA1 high-risk and wild-type alleles to validate the DEPs through Realtime PCR,Western blot,Immunofluorescence and so on.Afer exposed to smoker's serum and non-smoker's serum,the phagocytic function of i PSCs-RPE cells was detected by co-culturing with POS labeled with FITC.2.To construct of HTRA1 overexpressing lentivirus and infect ARPE-19 cells.After puromycin screening,HTRA1 over-expressing stable cell lines were obtained and the Caveolin-1 protein expression was detected by Real-time PCR,Western blot,Immunofluorescence.The relationship between HTRA1 expression and Caveolin-1 expression was explored.3.According to the sequence of the target gene,sg RNA was designed to construct the target vector.The CRISPR/Cas9 gene editing technology was applied to modify the target gene of stem cells with ARMS2/HTRA1 high risk alleles to establish ARMS2 high risk alleles and HTRA1 high risk alleles homozygous optimization stem cell lines.Results 1.The analysis of protein mass spectrometry showed that after exposure to smoker's serum,there were 400 DEPs in the high risk group,which were different from the wild type group.7 credible proteins with significances differences were obtained after screened and eliminate individual difference proteins.After exposure to smoker's serum,we verified that the expression of Caveo1in-1 in RPE cells with high risk alleles was up-regulated at the m RNA level and protein level,and the phagocytic function of RPE cells was enhanced.2.The expression of Caveolin-1 was significantly up-regulated at m RNA level and protein level in stable cell lines with over-expression of HTRA1.The overexpression of AMD high-risk gene HTRA1 and smokers' serum had a synergistic effect on the up-regulation of Caveolin-1 protein expression.3.ARMS2 high risk alleles and HTRA1 high risk alleles homozygous optimized stem cell lines were successfully established to provide a cell model for further study on the pathogenesis of AMD susceptibility genes.Conclusions Proteomic analysis showed that the DEPs in ARMS2/HTRA1 high risk group was significantly more than that in wild type group after smoker's serum stimulations,and the expression of these DEPs was probably related to the pathogenesis of AMD.Our study demonstrated that the up-regutation of AMD high-risk gene HTRA1 and AMD high-risk environmental factor,smoking,could up-regulate the expression of Caveolin-1 and compensated increase the phagocytosis of RPE cells,which will increase the burden of oxidation and lead to the accumulation of waste.The elimination or subsequent inflammatory reaction eventually leads to the destruction of photosensitive cells,thus promoting the occurrence and development of AMD.