Analysis of miRNA Expression Profiles in Traumatic Brain Injury (TBI) and Their Correlation with Survival and Severity of Injury

Abstract

Traumatic brain injury (TBI) is the leading cause of traumatic death worldwide and is a public health problem associated with high mortality and morbidity rates, with a significant socioeconomic burden. The diagnosis of brain injury may be difficult in some cases or may leave diagnostic doubts, especially in mild trauma with insignificant pathological brain changes or in cases where instrumental tests are negative. Therefore, in recent years, an important area of research has been directed towards the study of new biomarkers, such as micro-RNAs (miRNAs), which can assist clinicians in the diagnosis, staging, and prognostic evaluation of TBI, as well as forensic pathologists in the assessment of TBI and in the estimation of additional relevant data, such as survival time. The aim of this study is to investigate the expression profiles (down- and upregulation) of a panel of miRNAs in subjects deceased with TBI in order to assess, verify, and define the role played by non-coding RNA molecules in the different pathophysiological mechanisms of brain damage. This study also aims to correlate the detected expression profiles with survival time, defined as the time elapsed between the traumatic event and death, and with the severity of the trauma. This study was conducted on 40 cases of subjects deceased with TBI (study group) and 10 cases of subjects deceased suddenly from non-traumatic causes (control group). The study group was stratified according to the survival time and the severity of the trauma. The selection of miRNAs to be examined was based on a thorough literature review. Analyses were performed on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples, with a first step of total RNA extraction and a second step of quantification of the selected miRNAs of interest. This study showed higher expression levels in cases compared to controls for miR-16, miR-21, miR-130a, and miR-155. In contrast, lower expression levels were found in cases compared to controls for miR-23a-3p. There were no statistically significant differences in the expression levels between cases and controls for miR-19a. In cases with short survival, the expression levels of miR-16-5p and miR-21-5p were significantly higher. In cases with long survival, miR-21-5p was significantly lower. The expression levels of miR-130a were significantly higher in TBI cases with short and middle survival. In relation to TBI severity, miR-16-5p and miR-21-5p expression levels were significantly higher in the critical-fatal TBI subgroup. Conclusions: This study provides evidence for the potential of the investigated miRNAs as predictive biomarkers to discriminate between TBI cases and controls. These miRNAs could improve the postmortem diagnosis of TBI and also offer the possibility to define the survival time and the severity of the trauma. The analysis of miRNAs could become a key tool in forensic investigations, providing more precise and detailed information on the nature and extent of TBI and helping to define the circumstances of death.

Keywords: miR-130a; miR-155; miR-16; miR-21; miR-23a; miRNA; molecular diagnostics; molecular pathology; molecular pathology of traumatic injuries; traumatic brain injury.

Figure 1

Distribution of study group by survival time expressed in days.

Figure 2

Distribution of study group by severity of trauma, as defined by AIS score parameters.

Figure 3

Box plot showing miRNA expression levels analysed in cases and controls: (A) hsa-miR-16-5p, (B) hsa-miR-19a-3p, (C) hsa-miR-21-5p, (D) hsa-miR-23a-3p, (E) hsa-miR-130a-3p, and (F) hsa-miR-155-5p.

Figure 4

Clustered heat map showing the differential expression of the 5 miRNAs in the 3 subgroups defined on the basis of survival time (left figure) and on the basis of trauma severity (right figure). The coloured boxes represent the relative expression of the group (measured by Pearson’s distance metric), and the miRNAs are clustered in the heat map using a comprehensive clustering algorithm.

Figure 5

Expressivity (up- and downregulation) of the miRNAs examined in this study and their correlation with pathways, pathophysiological effects, and target cell populations.