microRNA microarray profiling in the livers of control db/+ and diabetic db/db mice. Mus musculus

Analysis revealed a set of 13 microRNAs to be significantly altered between livers of control db/+ and diabetic db/db mice. Of these, miR-34a, miR-107, miR-378, miR-378*, miR-31, miR-31*, miR-151-5p, miR-676, miR-22, miR-93, let-7b were up-regulated and let-7e and miR-227 were down-regulated. A total of 670 predicted targets for these altered miRNAs were extracted from PicTar and TargetScan and functional characterisation mapped these targets to several biological processess related to varied metabolic pathways. The Wnt signaling pathway that has been shown to be linked to diabetes emerged as the most prominent pathway from these sets of target genes. Overall design: Four animals each from control db/+ and diabetic db/db mice were taken for the experiment. There was a significant increase in the body weight and plasma glucose levels in the diabetic group as compared to the control group. The livers were excised and total RNA was isolated, labelled and hybridized to mouse miRNA Microarray (V1) microarrays from Agilent. After hybridization, the slides were washed and scanned on an Agilent microarray scanner (model G2565BA) at 100 and 5% XDR settings. Agilent Feature Extraction software version 9.3.5 was used to extract the raw data. Microarray Data Analysis: The raw data were global median normalized and log transformed and analyzed by Significance Analysis of Microarray (SAM). SAM calculates a score for each gene as a change of expression relative to the standard deviation of all measurements and therefore identifies genes that are significantly associated with an outcome variable such as the disease stage. In SAM tests, a false Discovery Rate (FDR) of less than 5% was selected and parameters were set as default.

本分析发现，对照db/+小鼠与糖尿病db/db小鼠的肝脏组织中，共有13种微小RNA（microRNAs）的表达存在显著差异。其中，miR-34a、miR-107、miR-378、miR-378*、miR-31、miR-31*、miR-151-5p、miR-676、miR-22、miR-93及let-7b呈表达上调，而let-7e与miR-227则呈表达下调。我们从PicTar和TargetScan数据库中提取了上述差异表达微小RNA的共计670个预测靶基因，并通过功能注释将这些靶基因富集至多条与不同代谢通路相关的生物学过程中。既往研究证实与糖尿病相关的Wnt信号通路（Wnt signaling pathway）是该靶基因集最显著富集的通路。

实验设计：本实验分别选取4只对照db/+小鼠与4只糖尿病db/db小鼠。与对照组相比，糖尿病组小鼠的体重与血浆葡萄糖水平均显著升高。处死后采集肝脏组织，提取总RNA并进行标记，随后与安捷伦（Agilent）公司的小鼠微小RNA微阵列（V1版）进行杂交反应。杂交完成后，对芯片玻片进行洗涤，随后使用安捷伦微阵列扫描仪（型号G2565BA），以100%与5% XDR参数进行扫描。采用安捷伦Feature Extraction软件V9.3.5提取原始数据。

微阵列数据分析：对原始数据进行全局中位数归一化与对数转换后，采用微阵列显著性分析（Significance Analysis of Microarray, SAM）进行差异分析。SAM通过计算每个基因的表达变化相对于所有测量值的标准差的得分，从而筛选出与疾病分期等结局变量显著相关的基因。本次SAM分析设定错误发现率（false Discovery Rate, FDR）小于5%，其余参数均采用默认设置。