BackgroundLipopolysaccharide (LPS) is recognized as the most potent microbial mediator presaging the threat of invasion of Gram-negative bacteria that implicated in the pathogenesis of sepsis and septic shock. This study was designed to examine the microRNA (miRNA) expression in whole blood from mice injected with intraperitoneal LPS.MethodsC57BL/6 mice received intraperitoneal injections of varying concentrations (range, 10–1000 μg) of LPS from different bacteria, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, and Serratia marcescens and were killed 2, 6, 24, and 72 h after LPS injection. Whole blood samples were obtained and tissues, including lung, brain, liver, and spleen, were harvested for miRNA expression analysis using an miRNA array (Phalanx miRNA OneArray® 1.0). Upregulated expression of miRNA targets in the whole blood of C57BL/6 and Tlr4−/− mice injected with LPS was quantified using real-time RT-PCR and compared with that in the whole blood of C57BL/6 mice injected with lipoteichoic acid (LTA) from Staphylococcus aureus.ResultsFollowing LPS injection, a significant increase of 15 miRNAs was observed in the whole blood. Among them, only 3 miRNAs showed up-regulated expression in the lung, but no miRNAs showed a high expression level in the other examined tissues. Upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on real-time RT-PCR was dose- and time-dependent. miRNA induction occurred after 2 h and persisted for at least 6 h. Exposure to LPS from different bacteria did not induce significantly different expression of these miRNA targets. Additionally, significantly lower expression levels of let-7d, miR-25, miR-92a, miR-103, and miR-107 were observed in whole blood of Tlr4−/− mice. In contrast, LTA exposure induced moderate expression of miR-451 but not of the other 7 miRNA targets.ConclusionsWe identified a specific whole blood–derived miRNA signature in mice exposed to LPS, but not to LTA, from different gram-negative bacteria. These whole blood-derived miRNAs are promising as biomarkers for LPS exposure.

MicroRNAs (miRNAs) are a class of highly conserved, non-coding RNAs involved in post-transcriptional gene regulation. The muscle-specific miRNAs, miR-1, miR-133a, and miR-206, are expressed in skeletal muscles and have been shown to contribute to muscle development. To profile their expression after sciatic nerve denervation and reinnervation, the soleus muscles of the rats were analyzed with quantitative real-time PCR at 1 week, 1 month, 2 months, and 4 months after the experiments. In addition, a combined approach using computational prediction by the miRanda website and the Agilent Whole Rat Genome 4 x 44 k oligo microarray experiment was performed to investigate the potential target genes of these three miRNAs in the denervated and reinnervated muscles. The results revealed that with the first downregulation of miR-1 and miR-133a within 1 month in the denervated muscle, the expression of miR-1 and miR-133 increased by approximately 2-fold at 4 months after denervation and reinnervation; on the other hand, the expression of miR-206 was significantly increased to approximately 3-fold 1 month later only following reinnervation but not following denervation, and lasted at least for 4 months. The expression pattern of miR-206 was different from that of miR-1 and miR-133a. Notably, two genes (Hnrpu and Npy) and one gene (Ptprd) were potentially regulated both in the denervated and reinnervated muscle by miR-1 and miR-133a, respectively. There were six potential target genes (Hnrpu, Lsamp, MGC108776, Mef2, Npy, and Ppfibp2) of the upregulated miR-206 in the reinnervated muscle. Among these, three (Hnrpu, Npy, and MGC108776) were potentially regulated by both miR-1 and miR-206. Because the Mef2 transcription factor was reported to promote the transformation of type II fast glycolytic fibers into type I slow oxidative fibers, the upregulation of miR-206 with decreased expression of the Mef2 transcript in the 4 month reinnervated muscle, which presented type II fiber predominance 4 months after nerve microanastomosis, might indicate the role of miR-206 in determining the fiber type after peripheral nerve regeneration.

BackgroundWe profiled the expression of circulating microRNAs (miRNAs) in mice using Illumina small RNA deep sequencing in order to identify the miRNAs that may potentially be used as biomarkers to distinguish between gram-negative and gram-positive bacterial infections.ResultsRecombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14 + Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection.ConclusionsThis study identified mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p as potential circulating miRNAs for gram-positive bacterial infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-014-0106-y) contains supplementary material, which is available to authorized users.

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.