MicroRNA let-7i is a promising serum biomarker for blast-induced traumatic brain injury

Abstract

Blast-induced traumatic brain injury (TBI) is of significant concern in soldiers returning from the current conflicts in Iraq and Afghanistan. Incidents of TBI have increased significantly in the current conflicts compared to previous wars, and a majority of these injuries are caused by improvised explosive devices. Currently, no specific technique or biomarker is available for diagnosing TBI when no obvious clinical symptoms are present. Micro-RNAs are small RNA (~ 22nts) molecules that are expressed endogenously and play an important role in regulating gene expression. MicroRNAs have emerged as novel serum diagnostic biomarkers for various diseases. In this study, we studied the effect of blast overpressure injury on the microRNA signatures in the serum of rats. Rats were exposed to three serial 120-kPa blast overpressure exposures through a shockwave tube. Blood and cerebrospinal fluid were collected at various time points after injury, and microRNA modulation was analyzed using real-time PCR. Five microRNAs were significantly modulated in the serum samples of these animals at three time points post-injury. Further, we also found that the levels of microRNA let-7i are also elevated in cerebrospinal fluid post-blast wave exposure. The presence of microRNA in both serum and cerebrospinal fluid immediately after injury makes microRNA let-7i an ideal candidate for further studies of biomarkers in TBI.

FIG. 1.

Flow chart showing the experimental design of blast overpressure experiments in the rat (CSF, cerebrospinal fluid; IPA, Ingenuity Pathway Analysis).

FIG. 2.

Validation of miR-let 7i miRNA in the short interval (SII) and long interval (LII) groups. The levels of miRNA were normalized by the level of MammU6 endogenous control RNA, and all reactions were performed in triplicate. (A) Expression levels of let-7i were significantly upregulated in SII. (B) No significant upregulation of let-7i was observed in both subgroups of LII (*p<0.05).

FIG. 3.

Expression of miR-let-7i in both the short interval (SII) and long interval (LII) groups in cerebrospinal fluid (CSF) of rats exposed to blast overpressure (BOP). (A) A significant increase in the expression of miR-let-7i was observed in the SII groups post-BOP exposure. (B) No significant difference was observed in the expression of miR-let-7i in the LII groups (*p<0.05).

FIG. 4.

Functional interaction networks of known TBI-related protein biomarkers and inflammatory molecules predicted to be regulated targets of miRNA let-7i miRNA as predicted by the Ingenuity Pathway Analysis program (NMDA, N-methyl-D-aspartate; UCHL-1, ubiquitin C-terminal hydrolase-1; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-8, interleukin-8).