目的 筛选马兜铃酸I(AAI)致大鼠肾损伤后血清中差异表达的微小RNA(micro RNA,miRNAs),为阐明miRNAs调控马兜铃酸肾病的机制研究提供靶标并奠定研究基础。方法 SD雄性大鼠随机分为对照组及AAI(2.25 mg/kg)组,连续ip给药14 d造成肾损伤模型后,对大鼠肾脏进行HE染色观察肾损伤程度。利用miRNAs芯片技术寻找AAI组及对照组大鼠血清中差异表达的miRNAs,预测靶基因,采用实时定量PCR(qRT-PCR)验证芯片结果的可靠性,并对部分结果进行信号通路分析。结果 HE染色显示AAI组大鼠肾脏出现不同程度的病变;miRNAs芯片结果显示有5个miRNAs(miR-10a-3p、miR-207、miR-3594-3p、miR-874-3p、miR-9a-3p)表达明显上调,无明显下调的miRNAs,并预测Rhebl1、Usp10等16种基因为差异表达的miRNAs靶基因;qRT-PCR验证结果与芯片结果基本一致;信号通路分析结果显示MAPK信号通路等相关性较大。结论 芯片分析得到的差异表达的miRNAs可能与AAI致肾毒性机制相关,为进一步深入研究特定miRNAs的调控机制提供新的靶点,并为后期生物信息学层次的研究提供有效依据。

Objective To screen the miRNA differential expression profile in serum of rats after renal injury induced by aristolochic acid I, (AAI) to identify the target of miRNA in the mechanism of aristolochic acid nephropathy (AAN), and to make further study on the mechanism of AAN. Methods Eighteen male SD rats were randomly divided into two groups, control group and AAI group (2.25 mg/kg). Rats were ip injected with AAI once daily for 14 d. On day 14, blood collected was used for the determination of miRNA in serum by miRNA microarray; Renal pathological changes were examined by HE staining. The miRNA that expressed significantly different would be verified by real time quantitative PCR (qPT-PCR). Target genes were predicted using bioinformatics as well as Pathway analysis did. Results HE staining revealed that kidneys were damaged with different degrees. By significance analysis of microarray based on microarray screening, significantly different expression of five miRNA (miR-10a-3p, miR-207, miR-3594-3p, miR-874-3p, and miR-9a-3p) which were up-regulated were obtained, while there were no miRNAs which were down-regulated. qPT-PCR approved the results as well. It was suggested that 16 genes, such as Rhebl1 and Usp10, might be the target genes. Pathway analysis showed a greater correlation between MAPK signaling pathways. Conclusion The differential expressed miRNAs obtained by gene analysis might be related with AAI and further studies on the mechanism of some miRNA would be a new target of gene therapy and provide an effective basis for the further studies of bioinformatics.

国家自然科学青年基金资助项目(30701106);江苏省科技厅自然科学基金面上项目(BK2012852);江苏省高校优势学科建设工程资助项目(2011XYZ4-003)