A miRNome analysis at the early postmortem interval

Abstract

The postmortem interval (PMI) is the time elapsing since the death of an individual until the body is examined. Different molecules have been analyzed to better estimate the PMI with variable results. The miRNAs draw attention in the forensic field to estimate the PMI as they can better support degradation. In the present work, we analyzed the miRNome at early PMI in rats' skeletal muscle using the Affymetrix GeneChip™ miRNA 4.0 microarrays. We found 156 dysregulated miRNAs in rats' skeletal muscle at 24 h of PMI, out of which 84 were downregulated, and 72 upregulated. The miRNA most significantly downregulated was miR-139-5p (FC = -160, p = 9.97 × 10^-11), while the most upregulated was rno-miR-92b-5p (FC = 241.18, p = 2.39 × 10^-6). Regarding the targets of these dysregulated miRNAs, the rno-miR-125b-5p and rno-miR-138-5p were the miRNAs with more mRNA targets. The mRNA targets that we found in the present study participate in several biological processes such as interleukin secretion regulation, translation regulation, cell growth, or low oxygen response. In addition, we found a downregulation of SIRT1 mRNA and an upregulation of TGFBR2 mRNA at 24 h of PMI. These results suggest there is an active participation of miRNAs at early PMI which could be further explored to identify potential biomarkers for PMI estimation.

Keywords: Microarrays; Non-coding RNA; Postmortem interval; miRNA.

Figure 1. Volcano plot of miRNAs expression at 24 h of PMI.

In this graph the p-value (−log₁₀) is plotted against the fold change (FC) of the 1,218 rat miRNAs included in the microarray. Red dots indicate upregulated miRNAs, while blue dots the downregulated. The hightlighted dots indicate the top most significant up and down regulated miRNAs.

Figure 2. Hierarchical clustering of the 156 differentially expressed miRNAs.

The fold changes of the 156 differentially expressed miRNAs found at 24 h of PMI compared with controls were hierarchically clustered and represented as a dendrogram. Red color represents upregulated miRNAs and blue color downregulated miRNAs, and its intensity is related with the grade of miRNA expression.

Figure 3. Network of the mRNA targets of downregulated miRNAs found at 24 h of PMI.

The targets of the miRNAs rno-miR-125b-5p, rno-miR-138-5p, rno-miR-92a-3p, rno-miR-133a-3p, rno-miR-133b-3p, rno-miR-93-5p, rno-miR-22-3p, rno-miR-26b-5p, rno-miR-181c-5p, rno-miR-181d-5p, rno-miR-221-3p, rno-miR-222-3p, rno-miR-140-5p, rno-miR-191a-5p, and rno-miR-455-3p are shown. The green circles represent the miRNA, and blue squares their mRNA target.

Figure 4. Gene ontology enrichment analysis.

The main biological pathways where the target genes of rno-miR-125b-5p/rno-miR-351-5p, rno-miR-92a-3p, rno-miR-22-3p, and rno-miR-291a-3p participate are shown. The x-axis corresponds to the enrichment ratio.

Figure 5. Gene expression analysis of TGFBR2, SIRT1 and BMF genes in rats’ skeletal muscle at early post-mortem interval.

The fold change (FC) of these mRNA was analyzed in rats’ skeletal muscle at 24 h of PMI relative to the 0 h-PMI group, using quantitative RT-qPCR. The fold change was calculated with the 2^−∆∆CT method using GAPDH as internal control. The black squares represent the mean FC from each group; the whisker corresponds to the 95% confidence interval, and the dots are the jittered FC of each sample. Comparisons between the PMI were done with the Wilcoxon rank sum test.