Human traumatic brain injury alters plasma microRNA levels

Abstract

Circulating microRNAs (miRNAs) present in the serum/plasma are characteristically altered in many pathological conditions, and have been employed as diagnostic markers for specific diseases. We examined if plasma miRNA levels are altered in patients with traumatic brain injury (TBI) relative to matched healthy volunteers, and explored their potential for use as diagnostic TBI biomarkers. The plasma miRNA profiles from severe TBI patients (Glasgow Coma Scale [GCS] score ≤8) and age-, gender-, and race-matched healthy volunteers were compared by microarray analysis. Of the 108 miRNAs identified in healthy volunteer plasma, 52 were altered after severe TBI, including 33 with decreased and 19 with increased relative abundance. An additional 8 miRNAs were detected only in the TBI plasma. We used quantitative RT-PCR to determine if plasma miRNAs could identify TBI patients within the first 24 h post-injury. Receiver operating characteristic curve analysis indicated that miR-16, miR-92a, and miR-765 were good markers of severe TBI (0.89, 0.82, and 0.86 AUC values, respectively). Multiple logistic regression analysis revealed that combining these miRNAs markedly increased diagnostic accuracy (100% specificity and 100% sensitivity), compared to either healthy volunteers or orthopedic injury patients. In mild TBI patients (GCS score > 12), miR-765 levels were unchanged, while the plasma levels of miR-92a and miR-16 were significantly increased within the first 24 h of injury compared to healthy volunteers, and had AUC values of 0.78 and 0.82, respectively. Our results demonstrate that circulating miRNA levels are altered after TBI, providing a rich new source of potential molecular biomarkers. Plasma-derived miRNA biomarkers, used in combination with established clinical practices such as imaging, neurocognitive, and motor examinations, have the potential to improve TBI patient classification and possibly management.

FIG. 1.

Temporal changes in plasma miRNA content after traumatic brain injury. Purified RNA from plasma collected at various time points after injury were assayed using miRNA-specific TaqMan probes directed against (A) miR-16, (B) miR-26a, (C) miR-92a, (D) miR-638, or (E) miR-765. Changes in threshold cycle are relative to the mean of the healthy volunteer (HV) values. Data are presented as the mean ± standard error of the mean of each group (*p < 0.05 by one-way ANOVA, or ANOVA on ranks; ANOVA, analysis of variance; miRNA, microRNA; TBI, traumatic brain injury).

FIG. 2.

Plasma miRNA levels are altered in severe TBI patients. RNA was purified from plasma samples collected within the first 24 h after injury, and assayed by TaqMan qRT-PCR. Dot histogram plots of the ΔC_T values for (A) miR-16, (B) miR-92a, (C) miR-765, and (D) area under the curve (AUC) values obtained from receiver operating characteristic (ROC) analysis for each miRNA are shown (healthy volunteer [HV] n = 8; severe TBI [sTBI] n = 7; miRNA, microRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; TBI, traumatic brain injury).

FIG. 3.

Comparison of relative plasma miRNA abundance in severe TBI versus orthopedic injury patients. Purified plasma RNA was assayed for (A) miR-16, (B) miR-92a, or (C) miR-765 relative abundance by TaqMan qRT-PCR assay. Dot histogram plots of the ΔC_T values are shown using the mean orthopedic C_T as the reference. (D) Area under the curve (AUC) values obtained from ROC analysis for each miRNA, indicating the diagnostic potential of each miRNA target (*p < 0.05 by two-tailed Student's t-test; orthopedic injury [Ortho], n = 8; severe TBI [sTBI], n = 7; miRNA, microRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; TBI, traumatic brain injury; ROC, receiver operating characteristic).

FIG. 4.

Diagnostic value of plasma miRNAs for identifying mild TBI patients. RNA was purified from plasma samples collected within the first 10 h after injury, and miRNA relative abundance was determined by qRT-PCR. Dot histogram plots of the ΔC_T values for (A) miR-16, (B) miR-26a, (C) miR-92a, (D) miR-638, or (E) miR-765. (F) Area under the curve (AUC) values obtained from ROC analysis for each miRNA (*p < 0.05 by two-tailed Student's t-test; healthy volunteer [HV], n = 8; mild TBI [mTBI], n = 11; miRNA, microRNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; TBI, traumatic brain injury; ROC, receiver operating characteristic).

FIG. 5.

Comparison of relative plasma miRNA levels in mild TBI versus orthopedic injury patients. TaqMan assays for (A) miR-16 or (B) miR-92a were performed using purified RNA from plasma collected within the first 10 h after trauma. Dot histogram plots of the ΔC_T values are shown using the mean orthopedic C_T as the reference (orthopedic injury [Ortho], n = 8; mild TBI [mTBI], n = 11; TBI, traumatic brain injury; miRNA, microRNA).