Murine platelet miRNA pattern and its regulation by a MK/platelet-specific genetic ablation of Dicer.

Purpose: To assess the platelet miRNA pattern which is regulated in a Dicer-dependent manner, we conducted a global screen of platelet miRNA expression of murine platelets from Dicerlox/lox and DicerPf4delta/Pf4delta mice using Next-generation deep sequencing analysis. Methods: Platelets were isolated from 10- to 12-week-old Dicerlox/lox and DicerPf4delta/Pf4delta mice of male sex. Total RNA including miRNA was isolated from murine platelets using the QIAsymphony RNA Kit. The sequencing library was prepared using the QIAseq miRNA Library Kit with 10 to 20 ng input. The libraries were pooled in an equimolar fashion and sequenced on a NovaSeq6000 as paired-end reads with a sequencing depth of >12 Mio clusters (i.e. read-pairs) per sample. Results: we were able to detect a total of 427 miRNAs in these platelets, of which 81 were significantly regulated (> |0.5| logFC threshold) in a Dicer-dependent manner. The majority of miRNAs were significantly downregulated, but a smaller proportion of miRNAs was slightly, but significantly upregulated in platelets lacking Dicer. Conclusions: this study discloses differential expression patterns of further platelet miRNAs that potentially link multiple intercellular processes and pathways implicated in several stages of the pathogenesis of myocardial I/R injury研究目的：为评估以Dicer依赖方式调控的血小板微小RNA（miRNA）表达谱，本研究采用下一代深度测序技术，对来自Dicerlox/lox与DicerPf4delta/Pf4delta小鼠的血小板进行全基因组miRNA表达筛选。 实验方法：我们从10至12周龄的雄性Dicerlox/lox及DicerPf4delta/Pf4delta小鼠体内分离血小板。使用QIAsymphony RNA提取试剂盒从小鼠血小板中提取包含miRNA在内的总RNA。采用QIAseq miRNA文库制备试剂盒，以10~20 ng的起始样本量构建测序文库。将文库以等摩尔浓度混合后，在NovaSeq6000测序平台上进行双端测序，每个样本的测序深度均超过12百万个簇（即读对）。 实验结果：本研究共在上述血小板中检测到427种miRNA，其中81种以Dicer依赖方式发生显著表达调控（logFC阈值> |0.5|）。绝大多数miRNA在Dicer缺失的血小板中显著下调，仅少数miRNA呈现轻度但显著的上调趋势。 研究结论：本研究揭示了更多血小板miRNA的差异表达模式，这些模式可能将多种细胞间过程与通路，与心肌缺血再灌注（I/R）损伤发病机制的多个阶段相关联。