Serum microRNA signatures identified by Solexa sequencing predict sepsis patients' mortality: a prospective observational study

Abstract

Background: Sepsis is the leading cause of death in Intensive Care Units. Novel sepsis biomarkers and targets for treatment are needed to improve mortality from sepsis. MicroRNAs (miRNAs) have recently been used as finger prints for sepsis, and our goal in this prospective study was to investigate if serum miRNAs identified in genome-wide scans could predict sepsis mortality.

Methodology/principal findings: We enrolled 214 sepsis patients (117 survivors and 97 non-survivors based on 28-day mortality). Solexa sequencing followed by quantitative reverse transcriptase polymerase chain reaction assays was used to test for differences in the levels of miRNAs between survivors and non-survivors. miR-223, miR-15a, miR-16, miR-122, miR-193*, and miR-483-5p were significantly differentially expressed. Receiver operating characteristic curves were generated and the areas under the curve (AUC) for these six miRNAs for predicting sepsis mortality ranged from 0.610 (95%CI: 0.523-0.697) to 0.790 (95%CI: 0.719-0.861). Logistic regression analysis showed that sepsis stage, Sequential Organ Failure Assessment scores, Acute Physiology and Chronic Health Evaluation II scores, miR-15a, miR-16, miR-193b*, and miR-483-5p were associated with death from sepsis. An analysis was done using these seven variables combined. The AUC for these combined variables' predictive probability was 0.953 (95% CI: 0.923-0.983), which was much higher than the AUCs for Acute Physiology and Chronic Health Evaluation II scores (0.782; 95% CI: 0.712-0.851), Sequential Organ Failure Assessment scores (0.752; 95% CI: 0.672-0.832), and procalcitonin levels (0.689; 95% CI: 0.611-0.784). With a cut-off point of 0.550, the predictive value of the seven variables had a sensitivity of 88.5% and a specificity of 90.4%. Additionally, miR-193b* had the highest odds ratio for sepsis mortality of 9.23 (95% CI: 1.20-71.16).

Conclusion/significance: Six serum miRNA's were identified as prognostic predictors for sepsis patients.

Trial registration: ClinicalTrials.gov NCT01207531.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Comparison of 12 miRNAs expression levels between sepsis non-survivors and survivors in a validation set.

Differentially expressed miRNAs identified by Solexa sequencing were validated by qRT-PCR in sepsis survivors (S; N = 15) compared to non-survivors (D; N = 15). MiR-16 (p = 0.005), miR-15a (p = 0.021), miR-223 (p = 0.015), miR-483-5p (p<0.001), miR-193b* (p<0.001), miR-122 (p<0.001), and miR-499-5p (p = 0.006) were significantly different after validation with qRT-PCR. Expression levels of the 12 miRNAs were normalized to U6 snRNA above normal controls and given as fold-changes (2–ΔΔCt ). △△Ct  =  (CtmiRNA-CtU6 snRNA) patients-(CtmiRNA-CtU6 snRNA)controls. Mann-Whitney U-test was used for statistical comparisons.

Figure 2. Expression levels of 8 miRNAs in sepsis non-survivors and survivors in a confirmation set.

These 8 miRNAs were significantly differentially expressed between sepsis non-survivors and survivors after qRT-PCR validation in a smaller study sample. Then, these were checked by qRT-PCR in a larger study sample size (Non-survivors = D, n = 73; Survivors = S, n = 93). Only 6 of the 8 miRNAs remained as significantly different between the D group and S groups. Expression levels of these 8 miRNAs were normalized to U6 snRNA U6 snRNA above normal controls and given as fold-changes (2–ΔΔCt ), △△Ct =  (CtmiRNA-CtU6 snRNA)patients-(CtmiRNA-CtU6 snRNA)controls. Mann-Whitney U-test or student t-test was used for statistical comparisons.

Figure 3. Receiver operating characteristic (ROC) curves for serum miRNAs for sepsis non-survivors (n = 73) and survivors (n = 93).

These six miRNAs were finally confirmed by qRT-PCR. Areas under the ROC curves are also shown.

Figure 4. Receiver operating characteristic (ROC) curves for serum miRNAs and clinically used indicators for sepsis non-survivors (n = 73) and survivors (n = 93).

Serum levels of miRNAs were quantified using real time qPCR. Each qPCR was done in triplicate in 96-well plates. Expression levels of the selected miRNAs were normalized to U6 snRNA and presented as fold-changes (2-ΔΔCt) above normal controls.