目的：应用miRNA表达谱芯片筛选视网膜色素上皮细胞与氧化应激相关的miRNA，为更加全面深入地研究年龄相关性黄斑病变(age-related macular degeneration，AMD)发生的分子机制提供新的思路。

方法：培养D407细胞，分别使用100、200、400μmol/L H₂O₂处理细胞24h后，Trizol试剂抽提细胞总RNA，使用Exiqon miRCURY LNA^TM microRNA表达谱芯片(miRBase 16.0数据库)检测不同浓度处理后D407细胞miRNA的表达差异，并将不同浓度处理后的变化进行聚类分析。使用Stem loop realtime PCR对芯片结果进行验证，并运用生物信息学方法预测差异miRNA调控的靶基因。

结果：芯片所包含的1 425个已知miRNA中，共有367个在不同浓度H₂O₂处理后表达发生变化。通过Treeview软件进行聚类分析发现miR-31等17个miRNA随H₂O₂浓度的升高呈现逐渐降低的趋势，miR-206等7个则逐渐升高。PCR验证显示芯片结果准确性较好。

结论：H₂O₂作用前后RPE的miRNA表达存在明显差异，miRNA作为转录后水平的调节分子，参与了细胞氧化应激反应，可能在AMD的发生发展中起有重要作用。

AIM: To identify the oxidative stress related miRNA in retinal pigment epithelium(RPE)by miRNA expression profile chip and provide a new idea for comprehensive and deep research on the molecular mechanisms of age-related macular degeneration(AMD).

METHODS: Human RPE cell line D407 was treated by 100, 200, 400μmol/L H₂O₂ for 24h and harvested to isolate total RNA by Trizol reagent. The expression difference of D407 cell miRNA after processing of different concentrations was generated by Exiqon miRCURY LNA^TM microRNA expression profile chip and the changes after processing of different concentrations were conducted by Hierarchical Clustering analysis. The results of chips were verified through Stem loop realtime PCR, and the target genes of identified miRNAs were predicted by bioinformatics software.

RESULTS: Among the 1 425 known miRNAs listed on microarray, 367 miRNAs showed differential expression after H₂O₂ treatment. The Treeview Clustering showed that 17 miRNAs, including miR-31, were downregulated along with the increase of H₂O₂ concentration. Meanwhile, 7 miRNAs, including miR206, were upregulated. The results of qRT-PCR further validated the better results of microarray.

CONCLUSION:The miRNA expression of human RPE is dramatically changed after H₂O₂ treatment. miRNA adjusts the molecules level of micrornas transcription and it is involved in cell oxidative stress reaction, and miRNA may play a pivotal role in the pathogenesis and development of AMD.

国家自然科学基金青年基金项目(No.30901644)