Department of Pediatrics, Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA.
Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Dr., Rm 2031, Indianapolis, IN, 46202, USA.
Diabetologia. 2018 May;61(5):1124-1134. doi: 10.1007/s00125-018-4559-5. Epub 2018 Feb 14.
AIMS/HYPOTHESIS: Improved biomarkers are acutely needed for the detection of developing type 1 diabetes, prior to critical loss of beta cell mass. We previously demonstrated that elevated beta cell microRNA 21-5p (miR-21-5p) in rodent and human models of type 1 diabetes increased beta cell apoptosis. We hypothesised that the inflammatory milieu of developing diabetes may also increase miR-21-5p in beta cell extracellular vesicle (EV) cargo and that circulating EV miR-21-5p would be increased during type 1 diabetes development.
MIN6 and EndoC-βH1 beta cell lines and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the inflammatory milieu of early type 1 diabetes. Serum was collected weekly from 8-week-old female NOD mice until diabetes onset. Sera from a cross-section of 19 children at the time of type 1 diabetes diagnosis and 16 healthy children were also analysed. EVs were isolated from cell culture media or serum using sequential ultracentrifugation or ExoQuick precipitation and EV miRNAs were assayed.
Cytokine treatment in beta cell lines and human islets resulted in a 1.5- to threefold increase in miR-21-5p. However, corresponding EVs were further enriched for this miRNA, with a three- to sixfold EV miR-21-5p increase in response to cytokine treatment. This difference was only partially reduced by pre-treatment of beta cells with Z-VAD-FMK to inhibit cytokine-induced caspase activity. Nanoparticle tracking analysis showed cytokines to have no effect on the number of EVs, implicating specific changes within EV cargo as being responsible for the increase in beta cell EV miR-21-5p. Sequential ultracentrifugation to separate EVs by size suggested that this effect was mostly due to cytokine-induced increases in exosome miR-21-5p. Longitudinal serum collections from NOD mice showed that EVs displayed progressive increases in miR-21-5p beginning 3 weeks prior to diabetes onset. To validate the relevance to human diabetes, we assayed serum from children with new-onset type 1 diabetes compared with healthy children. While total serum miR-21-5p and total serum EVs were reduced in diabetic participants, serum EV miR-21-5p was increased threefold compared with non-diabetic individuals. By contrast, both serum and EV miR-375-5p were increased in parallel among diabetic participants.
CONCLUSIONS/INTERPRETATION: We propose that circulating EV miR-21-5p may be a promising marker of developing type 1 diabetes. Additionally, our findings highlight that, for certain miRNAs, total circulating miRNA levels are distinct from circulating EV miRNA content.
目的/假设：在β细胞大量丧失之前，迫切需要改进的生物标志物来检测 1 型糖尿病的发生。我们之前的研究表明，1 型糖尿病啮齿动物和人类模型中升高的β细胞 microRNA 21-5p（miR-21-5p）增加了β细胞凋亡。我们假设，在 1 型糖尿病发生过程中，发展中的糖尿病的炎症环境也可能增加β细胞细胞外囊泡（EV）货物中的 miR-21-5p，并且循环 EV miR-21-5p 在 1 型糖尿病发展过程中会增加。
用 IL-1β、IFN-γ 和 TNF-α处理 MIN6 和 EndoC-βH1 β细胞系和人胰岛，以模拟 1 型糖尿病早期的炎症环境。从 8 周龄雌性 NOD 小鼠中每周采集血清，直到糖尿病发病。还分析了 19 名儿童在 1 型糖尿病诊断时和 16 名健康儿童的血清。使用连续超速离心或 ExoQuick 沉淀从细胞培养物或血清中分离 EV，并测定 EV miRNAs。
细胞因子处理β细胞系和人胰岛导致 miR-21-5p 增加 1.5 至三倍。然而，相应的 EV 对此 miRNA 的富集程度更高，细胞因子处理后 EV miR-21-5p 增加了三至六倍。这种差异仅部分通过用 Z-VAD-FMK 预处理β细胞来抑制细胞因子诱导的半胱天冬酶活性而降低。纳米颗粒跟踪分析表明细胞因子对 EV 数量没有影响，这表明 EV 货物中的特定变化是导致β细胞 EV miR-21-5p 增加的原因。按大小分离 EV 的连续超速离心表明，这种影响主要是由于细胞因子诱导的外泌体 miR-21-5p 增加所致。从 NOD 小鼠的纵向血清采集表明，EV 从糖尿病发病前 3 周开始逐渐增加 miR-21-5p。为了验证与人类糖尿病的相关性，我们检测了新诊断为 1 型糖尿病的儿童与健康儿童的血清。与非糖尿病参与者相比，虽然糖尿病参与者的总血清 miR-21-5p 和总血清 EV 减少，但血清 EV miR-21-5p 增加了三倍。相比之下，糖尿病参与者的血清和 EV miR-375-5p 均同时增加。
结论/解释：我们提出循环 EV miR-21-5p 可能是 1 型糖尿病发展的有前途的标志物。此外，我们的研究结果表明，对于某些 miRNA，循环中的总 miRNA 水平与循环 EV miRNA 含量不同。
