Evaluation of Global Differential Gene and Protein Expression in Primary Pterygium: S100A8 and S100A9 as Possible Drivers of a Signaling Network

PurposePterygium is a wing shaped fibrovascular growth on the ocular surface, characterized by fibrosis, angiogenesis, extracellular matrix remodeling, and inflammatory infiltrates. Epidemiologic studies have linked pterygium formation to various chronic inflammatory conditions, such as ultraviolet radiation, sawdust exposure, and dry eye disease. The purpose of this study is to identify proteins that are differentially expressed in primary pterygium by using a combination of gene microarray and proteomic platforms.MethodsPaired pterygium and uninvolved conjunctiva tissues of four patients were evaluated for differences in global gene transcript levels using a genechip microarray. Proteins extracted from another four pairs of tissues were quantified by iTRAQ approach. Western blot and immunofluorescent staining on additional patients were used to validate dysregulated protein expression obtained from microarray and proteomics data. In addition, primary conjunctival fibroblasts were treated with recombinant S100A8, S100A9 or both. Transcript level changes of a panel of potential target genes were evaluated by real time-PCR.ResultsThe following were up-regulated at both protein and transcript levels S100 A8 and A9, aldehyde dehydrogenase 3 family, member1 (ALDH3A1) and vimentin (VIM). Conversely, serpin peptidase inhibitor clade A member 1 (SERPINA1) and transferrin (TF) were down-regulated. Upon adding S100A8, S100A9 or both, the inflammatory chemokine CXCL1, matrix proteins vimentin, biglycan, and gelsolin, as well as annexin-A2, thymosin-β4, chymase (CMA1), member of Ras oncogene family RAB10 and SERPINA1 were found to be up-regulated.ConclusionsWe identified 3 up-regulated and 2 down-regulated proteins by using a stringent approach comparing microarray and proteomic data. On stimulating cells with S100A8/9, a repertoire of key genes found to be up-regulated in pterygium tissue, were induced in these cells. S100A8/9 may be an upstream trigger for inflammation and other disease pathways in pterygium.翼状胬肉（pterygium）是发生于眼表的翼状纤维血管增生性病变，以纤维化、血管生成、细胞外基质重塑及炎性浸润为典型特征。流行病学研究表明，翼状胬肉的形成与多种慢性炎症相关危险因素存在关联，包括紫外线辐射、木屑暴露以及干眼症。本研究旨在联合应用基因芯片与蛋白质组学平台，筛选原发性翼状胬肉组织中差异表达的蛋白质。 本研究选取4例患者的配对翼状胬肉组织与未受累结膜组织，通过基因芯片微阵列检测其全基因转录水平的差异。另外提取4对配对组织中的蛋白质，采用同量异位素标记相对和绝对定量（iTRAQ）技术进行定量分析。此外，针对额外入组的患者样本，采用蛋白质免疫印迹（Western blot）与免疫荧光染色，验证基因芯片及蛋白质组学数据中筛选得到的异常表达蛋白质。同时，将原代结膜成纤维细胞分别用重组S100A8、重组S100A9或二者联合处理，通过实时荧光定量PCR（real-time PCR）检测一组潜在靶基因的转录水平变化。 本研究发现，S100A8、S100A9、乙醛脱氢酶3家族成员1（ALDH3A1）以及波形蛋白（VIM）在蛋白质与转录水平均呈现上调表达。与之相反，丝氨酸蛋白酶抑制剂家族A亚家族成员1（SERPINA1）与转铁蛋白（TF）的表达水平则出现下调。在使用重组S100A8、S100A9或二者联合处理原代结膜成纤维细胞后，炎性趋化因子CXCL1、基质蛋白波形蛋白、双糖链蛋白聚糖、凝溶胶蛋白，以及膜联蛋白-A2、胸腺素β4、糜酶（CMA1）、Ras癌基因家族成员RAB10与SERPINA1的表达水平均被上调。 本研究通过联合分析基因芯片与蛋白质组学数据的严格分析流程，共筛选得到3种上调蛋白质与2种下调蛋白质。在使用S100A8/S100A9复合物刺激细胞后，翼状胬肉组织中筛选得到的一系列关键基因的表达均被诱导上调。S100A8/S100A9复合物可能是翼状胬肉炎症及其他疾病通路的上游触发因子。