Analysis of micro-RNA expression profiles in human islets and islet-derived exosomes during cytokine mediated β cell stress and death. Analysis of micro-RNA expression profiles in human islets and islet-derived exosomes during cytokine mediated β cell stress and death

Biomarkers capable of monitoring β cell stress during the evolution of type 1 diabetes (T1D) are currently lacking. MicroRNAs (miRNAs) are a class of small non-coding RNAs ~22 nucleotides in length that modulate gene expression by binding to the 3’untranslated region of target mRNAs. Given their stability in biological fluids and enrichment in cell-derived EVs, we hypothesized that miRNAs from human islet and islet-derived EVs could identify β-cell stress/death and be leveraged in T1D biomarker strategies. To test this, human islets were obtained from 10 cadaveric donors (5 male/5 female) and treated with or without cytokines (IL-1β and IFN-γ) for 24 hrs, as an ex vivo model of T1D. Small RNA sequencing was performed and identified 1110 and 890 miRNAs in total and 20 and 14 differentially expressed (DE) miRNAs (fold change≥1.5 and p<0.05) from islets and EVs, respectively. These findings were validated in an independent set of cytokine-treated islets and islet-derived EVs (7M and 5F donors). Interestingly, miRNA expression pattern was strikingly different between male and female donors at baseline and under cytokine stress with < 10% overlap among the DE miRNAs. miR-155-5p and miR-146a-5p were the only two miRNAs that were upregulated in cytokine-treated islets and EVs in both the sexes. Functional enrichment analysis of DE miRNAs identified pathways such as insulin signaling, ER stress and apoptosis. Taken together, these data suggest that miRNA expression patterns change dynamically in both human islets and islet-derived EVs in response to pro-inflammatory cytokine stress. EV miRNAs were largely distinct from those in the islet fraction, suggesting that miRNAs are selectively packaged into EVs in response to extrinsic cues. Finally, these data highlight the importance of considering sex as a biological variable when defining T1D biomarkers. Overall design: Identification of differentially expressed miRNAs from human islets and islet-derived exosomes treated with/without proinflammatory cytokines (IL-1B + IFN-g)

目前尚缺乏可用于监测1型糖尿病（type 1 diabetes，T1D）进展过程中β细胞应激状态的生物标志物。微小RNA（microRNAs，miRNAs）是一类长度约22个核苷酸的非编码小RNA，通过结合靶mRNA的3'非翻译区调控基因表达。鉴于其在生物体液中具有稳定性，且在细胞来源的细胞外囊泡（extracellular vesicles，EVs）中富集，我们推测源自人类胰岛及胰岛来源EVs的miRNAs可用于识别β细胞应激/死亡状态，进而应用于T1D生物标志物研究策略。
为验证这一假说，我们从10名遗体捐献者（5名男性、5名女性）体内获取人类胰岛，以是否添加细胞因子（IL-1β与IFN-γ）处理24小时，构建T1D的离体模型。随后进行小RNA测序，分别在胰岛与EVs中总计鉴定出1110种和890种miRNAs，其中差异表达（differentially expressed，DE）miRNAs分别为20种和14种（倍数变化≥1.5且p<0.05）。
上述研究结果在另一组独立样本中得到验证，该组样本来自7名男性、5名女性捐献者的细胞因子处理胰岛及胰岛来源EVs。有趣的是，无论在基线状态还是细胞因子应激条件下，男性与女性捐献者的miRNA表达模式均存在显著差异，其差异表达miRNAs的重叠率不足10%。miR-155-5p与miR-146a-5p是仅有的两种在两种性别捐献者的细胞因子处理胰岛及EVs中均上调的miRNAs。
对差异表达miRNAs进行功能富集分析后，发现了胰岛素信号通路、内质网应激（ER stress）及细胞凋亡等相关通路。综上，本研究数据表明，在促炎细胞因子应激条件下，人类胰岛及胰岛来源EVs中的miRNA表达模式均会发生动态变化。EVs中的miRNAs与胰岛组分中的miRNAs存在显著差异，这提示miRNAs会根据外界信号被选择性包装进入EVs中。最后，本研究数据凸显了在定义T1D生物标志物时，将性别作为生物学变量纳入考量的重要性。
实验整体设计：针对经/未经促炎细胞因子（IL-1β + IFN-γ）处理的人类胰岛及胰岛来源外泌体，鉴定其中的差异表达miRNAs。