MicroRNA-19b predicts widespread pain and posttraumatic stress symptom risk in a sex-dependent manner following trauma exposure

Abstract

Posttraumatic widespread pain (PTWP) and posttraumatic stress symptoms (PTSS) are frequent comorbid sequelae of trauma that occur at different rates in women and men. We sought to identify microRNA (miRNA) that may contribute to sex-dependent differences in vulnerability to these outcomes. Monte Carlo simulations (x10,000) identified miRNA in which predicted targeting of PTWP or PTSS genes was most enriched. Expression of the leading candidate miRNA to target PTWP/PTSS-related genes, miR-19b, has been shown to be influenced by estrogen and stress exposure. We evaluated whether peritraumatic miR-19b blood expression levels predicted PTWP and PTSS development in women and men experiencing trauma of motor vehicle collision (n = 179) and in women experiencing sexual assault trauma (n = 74). A sex-dependent relationship was observed between miR-19b expression levels and both PTWP (β = -2.41, P = 0.034) and PTSS (β = -3.01, P = 0.008) development 6 months after motor vehicle collision. The relationship between miR-19b and PTSS (but not PTWP) was validated in sexual assault survivors (β = -0.91, P = 0.013). Sex-dependent expression of miR-19b was also observed in blood and nervous tissue from 2 relevant animal models. Furthermore, in support of increasing evidence indicating a role for the circadian rhythm (CR) in PTWP and PTSS pathogenesis, miR-19b targets were enriched in CR gene transcripts. Human cohort and in vitro analyses assessing miR-19b regulation of key CR transcripts, CLOCK and RORA, supported the potential importance of miR-19b to regulating the CR pathway. Together, these results highlight the potential role that sex-dependent expression of miR-19b might play in PTWP and PTSS development after trauma/stress exposure.

Fig 1.

miR-19 is a strong candidate regulatory hub for pain and PTSS. In silico analyses, using Monte Carlo cross validation approaches to estimate microRNA with the largest number of pain and PTSS associated mRNA targets, identified top candidate regulatory hubs for pain and PTSS. Individual microRNA (represented by circles) with the largest –Log p-value represent microRNA with the highest level of enrichment of predicted target sites in pain and PTSS related genes. A significance threshold corresponding to an empirical p value of p=0.05 is indicated by a green horizontal dotted line and microRNA with p values that are more significant than this threshold are shaded green. The most significant such microRNA and thus the strongest candidate regulatory hub for pain and PTSS is miR-19.

Fig 2.

miR-19b is differentially expressed in women and men who develop posttraumatic widespread pain (PTWP) and/or posttraumatic stress symptoms (PTSS) six months following trauma exposure. (a) Simplified study design schematic showing that women and men were enrolled in the emergency department following motor vehicle collision or at sexual assault nurse examiner sites following sexual assault, blood samples were obtained in the ED, and PTWP and PTSS outcomes were assessed six months following trauma exposure. (b and c) miR-19b expression levels (n=153) were negatively associated with the probability of developing PTWP and PTSS in women (n=95, 62.5%, red dots) and were positively associated with the probability of developing PTWP and PTSS in men (n=57, 37.5%, blue dots) following motor vehicle collision. miR-19b expression levels were measured via small RNA-seq. (d) RT-qPCR data showing relative miR-19b expression levels in an additional subset of women and men (n=26, 69% women) who developed co-morbid PTWP/PTSS or recovered following motor vehicle collision trauma. Data are represented as mean ± SEM. *p < 0.05. (e) miR-19b expression levels (measured via small RNA-seq) from women sexual assault survivors plotted relative to their probability of developing PTSS six months following trauma exposure.

Fig 3.

miR-19b is expressed differently in male and female rats following stress exposure. (a) Schematic diagram of the animal protocol used to assess miR-19b expression levels in blood and brain of animal models of PTWP and PTSS. Male and female Sprague Dawley rats were either unstressed or exposed to unpredictable sound stress (USS) or single prolonged stress (SPS). Blood and brain tissue were isolated 7 (SPS) or 14 (USS) days following stress exposure, as these timepoints have been well-validated as the period in which stress-induced fear learning and PGE₂-responsive pain behaviors are observed (see text for details and references). (b) Circulating miR-19b expression levels in male (blue, n=10) and female (red, n=12) rats unstressed (Control) or exposed to USS. (c) Circulating miR-19b expression levels in male (blue, n=23) and female (red, n=17) rats unstressed (Control) or exposed to SPS. (d) miR-19b expression levels in the amygdala of male (blue, n=10) and female (red, n=12) rats unstressed (Control) or exposed to USS. (e) miR-19b expression levels in the hippocampus of male (blue, n=10) and female (red, n=12) rats unstressed or exposed to USS. Data are represented as mean ± standard deviation and were analyzed by two-way ANOVA. Significant post-hoc differences between control and stress groups were determined with Tukey’s multiple comparison test. *p < 0.05, **p<0.001

Fig 4.

Preliminary evidence suggesting that miR-19b regulates three genes involved in rhythmic processing. (a - c) Correlation between circulating miR-19b expression levels and circulating mRNA expression levels in men (blue circles, n=16 PTSS and n=18 Recovered) and women (red circles, n=22 PTSS and n=34 Recovered) in the early aftermath of MVC (n=90). The relationship between miR-19b and RORA (panel a), CLOCK (panel b), and NPAS2 (panel c) mRNA is shown for men and women who developed PTSS six months following trauma exposure (filled circles) and in those who recovered (open circles). Expression levels of miR-19b and each mRNA transcript represent log transformed RNA seq reads. Pearson correlation coefficients (R) and p values are presented for each subgroup. (d – e) Dual luciferase reporter assays examining direct binding of miR-19b to the (d) RORA 3’UTR, (e) CLOCK 3’UTR, and (f) NPAS2 3’UTR. Black bars indicate binding of miR-19b mimic or control mimic to wild type 3’UTRs while gray bars represent binding of miR-19b or control mimic to 3’UTRs with miR-19b seed sites mutated in each of the predicted binding sites as indicated. Relative luciferase activity (y axis) refers to firefly luciferase activity/renilla luciferase activity (i.e. the firefly construct containing the 3’UTR is normalized to the vector-only renilla construct). Predicted miR-19b – target hybrids are shown below each graph; RORA and CLOCK are predicted to have two miR-19b binding sites each and NPAS2 is predicted to have one miR-19b binding site. p values were determined for panels (d - f) using the Mann-Whitney non-parametric test. *, p<0.05; #,p<0.001.