Project description:Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers with high mortality rate. Smoking is one of the established risk factors of ESCC. However, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. Understanding the effects of cigarette smoke on esophageal squamous epithelial cells at a molecular level would lead to a better understanding of the pathobiology of ESCC which has implications for identification of early biomarkers and therapeutic targets. To investigate the effect of cigarette smoke exposure, we developed a cell line model where Het1A cells (non-neoplastic human esophageal epithelial cells) were chronically treated with cigarette smoke condensate (CSC) for 2 months, 4 months, 6 months and 8 months. We carried out comparative proteomic, phosphoproteomic and whole exome sequencing analyses on CSC treated and untreated cells. Increased cell proliferation and invasion of Het1A cells was observed after chronic exposure to cigarette smoke. Using quantitative proteomic and phosphoproteomic analyses, we identified 56 proteins and 296 phosphoproteins that showed differential expression. Bioinformatics analysis of differentially expressed proteins and phosphoproteins showed enrichment of molecules involved in DNA damage response pathway. Whole exome sequencing (WES) of CSC treated and untreated cells also revealed mutations and copy number alterations in genes associated with DNA damage response. By correlating WES, proteomic and phosphoproteomic results, we observed potential loss of function in HMGN2 and MED1 that were reported as potential tumor suppressors and are known to play important role in DNA damage response. We also observed decreased expression of HMGN2 in tissue section of ESCC. Overexpression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of CSC treated cells. These findings suggest that cigarette smoke affects genes and proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.

Project description:Background Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Recent studies have demonstrated strong genetic associations between AMD and single nucleotide polymorphisms (SNPs) within genes such as CFH and HTRA1. However, we have identified monozygotic twins discordant for AMD phenotypes (one with disease, the other without disease), suggesting that an epigenetic mechanism may also contribute to the pathogenesis of AMD. Methods We identified two twin pairs with phenotypic discordance of AMD. We obtained genomic DNA from their peripheral blood mononuclear cells (PBMCs) and subjected them to DNA methylation-chip analysis (MeDIP-chip) that profiled genome-wide DNA methylation patterns on promoters of all genes and microRNAs. We next utilized the Methyl-Profiler DNA methylation assays to detect the methylation status of selected promoters. Flow cytometry and quantitative real-time PCR were used to detect the expression of the selected gene in peripheral blood cells, as well as in retinal and choroidal tissues of AMD patients and non-AMD controls. Results Our MeDIP-chip analysis identified 256 genes with hypo-methylated promoters only in the twins with AMD and 744 genes with hyper-methylated promoters only in the twins with AMD. Importantly, the promoter region of IL17RC was associated with hypo-methylated CpG sites only in the twins with AMD but not in the twins without AMD. We also found the association of the hypo-methylated IL17RC promoter with AMD in 7 pairs of siblings with discordant AMD pathology (P=0.003), as well as an independent patient cohort (95 neovascular wet and 107 geographic atrophy dry AMD patients as well as 96 non-AMD controls (95% CI, 0.01-0.10, P=9.8x10-8 for wet AMD vs. control; 95% CI, 0.05-0.32, P=2.2x10-5 for dry AMD vs. control)). Interestingly, we did not find an association between the levels of methylation on the IL17RC promoter with the previously identified genetic risk alleles in CFH, HTRA1, and ARMS2. We demonstrated an elevated expression of the IL-17RC protein in CD14+ monocytes in the peripheral blood of AMD patients as compared to non-AMD controls. These IL-17RC+ monocytes have elevated expression of CXCR1, CXCR2, and CXCR4. In addition, IL17RC was expressed only in the retinal and choroidal tissues from AMD patients but not from age-matched controls. Conclusions We show an association of the hypo-methylated IL17RC promoter with AMD, which resulted in the elevated expression of IL-17RC in peripheral blood cells as well as the retinal and choroidal tissues of AMD patients. Our study suggests that the hypo-methylated IL17RC promoter and elevated expression of IL-17RC can potentially serve as biomarkers for the diagnosis of AMD, while IL-17RC can be a new therapeutic target for AMD. In addition, our results strongly suggest an epigenetic control mechanism of AMD pathogenesis. The Affymetrix MOE430 2.0 GeneChip was used to detect gene expression patterns within the whole eye of C57B/6 normal mice.

Project description:Background Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Recent studies have demonstrated strong genetic associations between AMD and single nucleotide polymorphisms (SNPs) within genes such as CFH and HTRA1. However, we have identified monozygotic twins discordant for AMD phenotypes (one with disease, the other without disease), suggesting that an epigenetic mechanism may also contribute to the pathogenesis of AMD. Methods We identified two twin pairs with phenotypic discordance of AMD. We obtained genomic DNA from their peripheral blood mononuclear cells (PBMCs) and subjected them to DNA methylation-chip analysis (MeDIP-chip) that profiled genome-wide DNA methylation patterns on promoters of all genes and microRNAs. We next utilized the Methyl-Profiler DNA methylation assays to detect the methylation status of selected promoters. Flow cytometry and quantitative real-time PCR were used to detect the expression of the selected gene in peripheral blood cells, as well as in retinal and choroidal tissues of AMD patients and non-AMD controls. Results Our MeDIP-chip analysis identified 256 genes with hypo-methylated promoters only in the twins with AMD and 744 genes with hyper-methylated promoters only in the twins with AMD. Importantly, the promoter region of IL17RC was associated with hypo-methylated CpG sites only in the twins with AMD but not in the twins without AMD. We also found the association of the hypo-methylated IL17RC promoter with AMD in 7 pairs of siblings with discordant AMD pathology (P=0.003), as well as an independent patient cohort (95 neovascular wet and 107 geographic atrophy dry AMD patients as well as 96 non-AMD controls (95% CI, 0.01-0.10, P=9.8x10-8 for wet AMD vs. control; 95% CI, 0.05-0.32, P=2.2x10-5 for dry AMD vs. control)). Interestingly, we did not find an association between the levels of methylation on the IL17RC promoter with the previously identified genetic risk alleles in CFH, HTRA1, and ARMS2. We demonstrated an elevated expression of the IL-17RC protein in CD14+ monocytes in the peripheral blood of AMD patients as compared to non-AMD controls. These IL-17RC+ monocytes have elevated expression of CXCR1, CXCR2, and CXCR4. In addition, IL17RC was expressed only in the retinal and choroidal tissues from AMD patients but not from age-matched controls. Conclusions We show an association of the hypo-methylated IL17RC promoter with AMD, which resulted in the elevated expression of IL-17RC in peripheral blood cells as well as the retinal and choroidal tissues of AMD patients. Our study suggests that the hypo-methylated IL17RC promoter and elevated expression of IL-17RC can potentially serve as biomarkers for the diagnosis of AMD, while IL-17RC can be a new therapeutic target for AMD. In addition, our results strongly suggest an epigenetic control mechanism of AMD pathogenesis.

Dataset Information

Proteomics based identification and validation of biomarkers for AMD

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