microRNA在IFN-γ上调角膜上皮细胞MICA分子表达中的作用机制研究

Though keratoplasty is the most successful transplantation, corneal graft rejection is still the most frequent complication after corneal grafting and often leads to irreversible transplant failure. It is widely accepted that MHC class I and II antigen play crucial role in this rejection. In our recent study, we revealed for the first time that the proinflammatory cytokine, IFN-γ promoted surface MHC class I-related chain A (MICA) expression on corneal epithelium in a dose-dependent manner. IFN-γ also enhanced NK cell-mediated cytotoxicity against the corneal epithelium. On the contrary, Yadav et al. found that one of microRNAs, miR-520b was induced by IFN-γ, leading to a reduction in MICA surface protein levels. miR-520b acted on both the MICA 3'-untranslated region and the promoter region and caused a decrease in the levels of MICA transcript. The exact mechanism involved in the increased expression of MICA on corneal epithelium induced by IFN-γ is still unknown. In the current study, we will explore the changes of microRNAs in the human corneal epithelial cells upon IFN-γ treatment by Agilent whole human genome microarray analysi, flow cytometry, and Invitrogen TaqMan miRNA test. To identify miRNAs that regulate the expression of MICA, we will use the Sanger MicroCosm search engine (microrna.sanger. ac.uk), Gene Ontology (GO) analysis, and microRNA-gene-network analysis. We will then assess the MICA transcript and MICA-mediated cytotoxicity against the corneal epithelium by up-regulation or knock-down of the potential microRNA. Regulation of MICA on human corneal epithelium by IFN-γ-induced microRNAs may lead to a new vision into the pathogenesis of corneal graft rejection.

角膜排斥反应是角膜移植术后失败的主要原因。传统观点认为经典MHC抗原分子是参与角膜移植排斥反应的主要免疫分子。我们最近发现：人类角膜上皮细胞使用干扰素IFN-γ处理后表达非经典MHC分子MICA分子显著上调，并且NK细胞对角膜上皮细胞的细胞毒性作用显著增强。最近有学者报道了IFN-γ可以通过上调miRNA-520b下调肿瘤细胞表达MICA分子。这与IFN-γ上调角膜细胞MICA表达正好相反，但后者机制尚不明确。在本研究中，我们拟通过microRNA芯片、流式细胞仪、RT-PCR检测等方法进一步明确IFN-γ处理角膜细胞后microRNA的差异性表达；结合已有文献及数据库筛选出候选microRNA；检测增强及减弱候选microRNA情况下MICA的表达规律，并探讨MICA介导的细胞毒性作用的变化情况，从而明确IFN-γ上调角膜细胞表达MICA的机制，为揭示角膜排斥反应的发病机制提供新的思路。

角膜移植术后排斥反是角膜移植失败的主要原因，研究表明，经典MHC抗原（主要组织相容性复合物MHC-I分子、MHC-II分子及次要组织相容性复合物）是参与角膜移植排斥反应的主要免疫分子。但值得注意的是，除了经典MHC抗原外，非经典MHC分子—MHC-I类样分子A(MHC class I chain-related molecule A, MICA)正日益受到人们的关注。MICA分子的表达可能与热休克蛋白转录元件及局部细胞因子如干扰素-gamma（IFN-γ）、IL-2等的刺激有关，而IFN-γ可非特异性地促进下游炎症因子的释放促进角膜排斥反应的发生。本研究中，我们探索IFN-γ处理人角膜上皮细胞（HCEC）的最适浓度与最佳时间，以浓度梯度为100ng/ml、500ng/ml、1000ng/ml，时间为1min、5min、1h、12h、24h处理HCEC，发现在浓度为100ng/ml、500ng/ml、处理时间为1分钟时，MICA基因被瞬时激活，mRNA表达上调，其蛋白经过转录、翻译等合成过程，在IFN-γ处理后24小时表达上调。我们进一步探讨IFN-γ作用下人角膜上皮细胞中miRNA的表达规律，发现IFN-γ处理后，5种miRNA表达发生变化，其中2种miRNA表达上调，分别为：hsa-miR-4448、hsa-miR-29c-5p，3种miRNA表达下调，分别为hsa-let-7a-2-3p、hsa-miR-4481、hsa-miR-3136-5p。通过KEGG数据库分析、GO功能富集预测发现，表达下调的miRNA均可作用于MAPK信号通路，而MAPK信号通路的下游关键因子——AP-1，ATF-2，c-fos，c-jun是MICA基因的转录因子。综上，我们发现IFN-γ可通过下调人角膜上皮细胞中hsa-let-7a-2-3p、hsa-miR-4481、hsa-miR-3136-5p的表达，激活MAPK信号通路，从而活化MICA基因的转录因子，上调MICA的表达，影响角膜上皮细胞周期及细胞毒性，为IFN-γ及MICA基因在人角膜移植排斥反应中发挥的作用提供了可能的作用机制。