氧化应激诱导的大鼠Müller细胞微小RNA表达谱的初步研究

目的 研究在氧化应激条件下视网膜Müller细胞的miRNA表达谱,为寻找氧化应激条件下Müller细胞的靶向分子提供线索。方法 用2mmol乙二醛处理大鼠Müller细胞48h,然后收集细胞,抽提总RNA,本研究采用小RNA两端加接头序列,反转录,克隆,随机测序的方法构建大鼠Müller细胞系的小RNA文库,序列经Genebank Blastn和miRBase比对分析,Mfold软件在线预测二级结构验证miRNA,经qRT-PCR鉴定miRNA,通过miRPath分析müller细胞表达的miRNA相关信号通路。结果 本文库获得163个有效序列,31个克隆为已知miRNA序列,代表13种miRNA,它们与PI3K-AKT,MAPK,细胞外基质的合成及相互作用等多条信号通路密切相关。发现1个新miRNA序列。结论 氧化应激条件下表达的miRNA为更好地了解在生理和疾病条件下的Müller细胞功能提供有益的线索。
Objective To investigate the miRNA expression profile of rat Müller cells under oxidative stress. Methods Rat Müller cells were treated with 2mM of glyoxal for 48 h and total RNA was extracted. The small RNA library was generated by adding linkers to both ends of small RNA, reverse transcription, cloning and random sequencing. The sequences were subjected to Genebank Blastn and miRBase for sequence alignment analysis and Mfold online software for secondary structure prediction in order to screen out potential miRNAs. The results were finally confirmed by qRT-PCR. The signaling pathways related to discovered miRNAs were evaluated by miRPath. ResultsFrom this library, 163 clones were sequenced. 31 clones of them were known miRNAs, representing 13 families of miRNAs. They were closely related to PI3K-AKT pathway, MAPK pathway, extracellular matrix synthesis and interaction related pathway. A new miRNA sequence was found. Conclusion Highly expressed miRNAs of Müller cell under oxidative stress provide useful clues for better understanding its function in physiological and pathological context