自发性1型糖尿病NOD小鼠胰腺微小RNA表达谱分析

Micro-RNA expression profile in insulitis of type 1 diabetes model NOD mouse

目的 分析1型糖尿病（T1DM）小鼠胰腺中微小RNA表达谱,筛查T1DM相关微小RNA.方法 20只雌性6～8周龄T1 DM小鼠,体重（21 ±3）g,15周始测定血糖.于18、25周分期处死,18周处死的3只小鼠采用芯片方法分析T1DM小鼠胰腺中微小RNA表达谱；所有小鼠胰腺病苏木精-伊红染色和胰岛炎分级标准分级（胰岛炎0级；胰岛炎2级；胰岛炎3级＋胰岛萎缩＋显性糖尿病）.胰腺提取RNA,经定量逆转录PCR（qRT-PCR）验证.结果 微小RNA芯片结果在聚类分析中,无胰岛炎归为一类,中度和重度胰岛炎归为一类.20只小鼠中有13只小鼠发生显性糖尿病,并存在0～3级的胰岛炎,2级、3级胰岛炎分别有21、31个微小RNA显著变化.对已报道的7个免疫相关微小RNA单独分析,6种无明显变化,但凋亡相关微小RNAmiR-125b存在下调趋势,miR-125b-5p下降倍数在NOD007、NOD006中分别为0.42和0.60倍,miR-125b＊和miR-125b-3p均在2级胰岛炎变化最大,分别为0.05和0.36倍.定量PCR发现miR-125b表达在无胰岛炎组和胰岛炎组存在差异.结论 微小RNA表达随胰岛炎进展而变化,提示微小RNA在T1DM的发病中起重要作用.miR-125b参与了调节T1DM胰岛炎中胰岛细胞凋亡.

Objectives To analyze the micro-RNA（miRNA） expression profile in pancreas of type 1 diabetes （T1DM） model of NOD mouse and elucidate the association between miRNA and type 1 diabetes. Methods Twenty NOD mice were raised, and 3 mice sacrificed on 18 weeks, remaining were killed on 25 weeks. Pancreas sample were collected for pathological analysis to classify the insulitis. MicroRNA array were used to analyze miRNA expression profile in 3 samples with different grade of insulitis （0 grade, 2＂d grade, and 3r~ grade insulitis）, results were validated by quantitative reverse transcription-PCR in pancreas sample. Results 13/20 NOD mice developed diabetes by 25 weeks, there were 0-3 grades of insulitis. By microarray analysis, clustering analysis can distinguish insulitis with no insulitis; indicate a different miRNA profile in insulitis. Compared with no insulitis, 2＂d grade insulitis had 2 upregulated miRNA and 19 downregulated miRNA, while 3rd grade insulitis had 31 downregulated miRNA. For the 7 immune associated miRNA, miR-125b had significant downregulation, miR-125b-Sp downregulated for 0.42 and O. 60 folds, miR-125b ＊ and miR-125b-3p downregulated for 0. 05 and O. 36 folds, which was validated by qRT-PCR. Conclusion Distinct miRNA expression profile is found during insulitis progression, indicating a potential role of miRNA in T1DM development, miR-125b has significant down-regulation effect in T1DM, which indicates its role for islet cell apoptosis.