Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
PLoS One. 2013 Oct 30;8(10):e77936. doi: 10.1371/journal.pone.0077936. eCollection 2013.
The levels of circulating microRNAs (miRNAs) in mice with experimental sepsis induced by cecal ligation and puncture (CLP) were determined using whole blood samples obtained from C57BL/6 mice at 4, 8, and 24 h after CLP; miRNA expression analysis was performed in these samples using an miRNA array. Microarray analysis revealed upregulation of 10 miRNA targets (miR-16, miR-17, miR-20a, miR-20b, miR-26a, miR-26b, miR-106a, miR-106b, miR-195, and miR-451). The expression of these miRNA targets in the whole blood, serum, and white blood cells (WBCs) of CLP mice was quantified using quantitative real-time PCR; these values were compared to those in sham-operated C57BL/6 mice, and the results indicated that these miRNA targets were significantly up-regulated in the whole blood and serum but not in the WBCs. In addition, the levels of these 10 miRNA targets in the serum of Tlr2-/-, Tlr4-/-, and NF-κB-/- mice at 8 h after CLP did not decrease significantly., which indicated that the transcription of these miRNAs was not directly mediated by the TLR2/NF-κB or TLR4/NF-κB pathway, and pathways induced by exposure to the gram-positive or gram-negative bacteria. Immunoprecipitation with the Argonaute 2 ribonucleoprotein complex revealed significantly increased expression of the 10 miRNA targets in the serum of mice after CLP, and the levels of 6 (miR-16, miR-17, miR-20a, miR-20b, miR-26a, and miR-26b) of these 10 miRNA targets increased significantly in exosomes isolated using ExoQuick precipitation solution. In this study, we identified circulating miRNAs that were up-regulated after CLP and determined the increase in the levels of these miRNAs, and our results suggest that circulating Ago2 complexes and exosomes may be responsible for the stability of miRNAs in the serum.
采用结扎和穿刺盲肠（CLP）法建立实验性脓毒症小鼠模型，于 CLP 后 4、8 和 24 h 从 C57BL/6 小鼠采集全血样本，采用 miRNA 芯片检测循环 microRNAs（miRNAs）水平；采用 miRNA 芯片对这些样本进行 miRNA 表达分析。微阵列分析显示 10 个 miRNA 靶标（miR-16、miR-17、miR-20a、miR-20b、miR-26a、miR-26b、miR-106a、miR-106b、miR-195 和 miR-451）上调。采用实时定量 PCR 检测 CLP 小鼠全血、血清和白细胞（WBC）中这些 miRNA 靶标的表达；与假手术 C57BL/6 小鼠比较，结果显示这些 miRNA 靶标在全血和血清中显著上调，但在 WBC 中未上调。此外，CLP 后 8 h TLR2-/-、TLR4-/-和 NF-κB-/-小鼠血清中这 10 个 miRNA 靶标水平未显著下降，提示这些 miRNA 的转录并非直接由 TLR2/NF-κB 或 TLR4/NF-κB 途径介导，而是由革兰阳性或革兰阴性细菌诱导的途径介导。用 Argonaute 2 核糖核蛋白复合物免疫沉淀发现 CLP 后小鼠血清中 10 个 miRNA 靶标表达显著增加，其中 6 个（miR-16、miR-17、miR-20a、miR-20b、miR-26a 和 miR-26b）的水平显著增加用 ExoQuick 沉淀溶液分离的外泌体中。本研究鉴定了 CLP 后上调的循环 miRNA，并确定了这些 miRNA 水平的增加，我们的结果提示循环 Ago2 复合物和外泌体可能负责血清中 miRNA 的稳定性。
