CAJ | 학술논문

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 H2 O2处理细胞24h后,Trizol试剂抽提细胞总RNA,使用Exiqon miRCURY LNATM 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1425个已知miRNA中,共有367个在不同浓度H2 O2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H2 O2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：H2 O2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。
목적：응용miRNA표체보심편사선시망막색소상피세포여양화응격상관적miRNA,위경가전면심입지연구년령상관성황반병변( age-related macular degeneration,AMD)발생적분자궤제제공신적사로。 　　방법：배양 D407세포,분별사용100、200、400μmol/L H2 O2처리세포24h후,Trizol시제추제세포총RNA,사용Exiqon miRCURY LNATM microRNA표체보심편( miRBase 16.0수거고)검측불동농도처리후D407세포miRNA적표체차이,병장불동농도처리후적변화진행취류분석。사용Stem loop realtime PCR대심편결과진행험증,병운용생물신식학방법예측차이miRNA조공적파기인。 　　결과：심편소포함적1425개이지miRNA중,공유367개재불동농도H2 O2처리후표체발생변화。통과Treeview연건진행취류분석발현miR-31등17개miRNA수H2 O2농도적승고정현축점강저적추세,miR-206등7개칙축점승고。 PCR험증현시심편결과준학성교호。 　　결론：H2 O2작용전후RPE적miRNA표체존재명현차이, miRNA작위전록후수평적조절분자,삼여료세포양화응격반응,가능재AMD적발생발전중기유중요작용。
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 H2 O2 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 LNATM 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 H2 O2 treatment. The Treeview Clustering showed that 17 miRNAs, including miR-31, were downregulated along with the increase of H2 O2 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 H2 O2 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.