Cellular and extracellular miRNAs are blood-compartment-specific diagnostic targets in sepsis

Abstract

Septic shock is a common medical condition with a mortality approaching 50% where early diagnosis and treatment are of particular importance for patient survival. Novel biomarkers that serve as prompt indicators of sepsis are urgently needed. High-throughput technologies assessing circulating microRNAs represent an important tool for biomarker identification, but the blood-compartment specificity of these miRNAs has not yet been investigated. We characterized miRNA profiles from serum exosomes, total serum and blood cells (leukocytes, erythrocytes, platelets) of sepsis patients by next-generation sequencing and RT-qPCR (n = 3 × 22) and established differences in miRNA expression between blood compartments. In silico analysis was used to identify compartment-specific signalling functions of differentially regulated miRNAs in sepsis-relevant pathways. In septic shock, a total of 77 and 103 miRNAs were down- and up-regulated, respectively. A majority of these regulated miRNAs (14 in serum, 32 in exosomes and 73 in blood cells) had not been previously associated with sepsis. We found a distinctly compartment-specific regulation of miRNAs between sepsis patients and healthy volunteers. Blood cellular miR-199b-5p was identified as a potential early indicator for sepsis and septic shock. miR-125b-5p and miR-26b-5p were uniquely regulated in exosomes and serum, respectively, while one miRNA (miR-27b-3p) was present in all three compartments. The expression of sepsis-associated miRNAs is compartment-specific. Exosome-derived miRNAs contribute significant information regarding sepsis diagnosis and survival prediction and could serve as newly identified targets for the development of novel sepsis biomarkers.

Keywords: biomarker; blood compartment; exosome; liquid biopsy; miRNA; sepsis.

Figure 1

Exosome characterization by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and WB. TEM revealed vesicles with an average size range of about 100–150 nm (A). NTA showed a sharp size distribution with a mean particle diameter of 154.4 ± 40.2 nm for sepsis patients (black) and 225.2 ± 24.3 nm for volunteers (grey) (B). Exosomes were detected by WB using the exosomal markers CD81, TSG101 and syntenin‐1 (C).

Figure 2

Venn diagram of differential miRNA expression profiles from septic shock patients validated by RT‐qPCR.

Figure 3

Sepsis detection by miRNAs: Only cellular miR‐199b‐5p detected both sepsis and septic shock with high confidence (n = 6).

Figure 4

Relationship between miRNA expression level and disease severity. Eleven miRNAs in at least one compartment display stringent correlation of normalized Cq values and disease severity in septic patients upon admission to the ICU.

Figure 5

Survival prediction by miRNAs. Exosomal miR‐30a‐5p and miR‐125b‐5p and serum miR‐193a‐5p predict survival of septic patients with high confidence.

Figure 6

Significantly different miRNA expression levels between days 0 and 4. Samples on day 4 were available from eight of the patients with sepsis or septic shock previously used in RT‐qPCR expression analysis at day 0. Grey bars indicate notable changes in expression within a group of patients. Tested miRNAs were exosomal miR21‐5p, miR27b‐3p, miR100‐5p, miR193a‐5p, miR199b‐5p, serum miR26b‐5p and miR193a‐5p, as well as blood cellular miR27b‐3p, miR143‐3p, miR150‐5p, miR199b‐5p, miR223‐3p and let‐7b‐5p.