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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 4  |  Issue : 3  |  Page : 135-141

Differentially expressed microRNAs as potential markers for vital reaction of burned skin


1 Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
2 Forensic Science Centre of Guangdong Provincial Public Security Department, Southern Medical University, Guangzhou, China
3 Department of Toxicology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China

Date of Web Publication28-Sep-2018

Correspondence Address:
Dr. Qi Wang
Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou, Guangdong
China
Dr. Xiao-Li Xie
Department of Toxicology, School of Public Health and Tropical Medicine, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou, Guangdong
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jfsm.jfsm_1_18

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  Abstract 


The identification of antemortem burns and postmortem burns is essential in forensic practice. In this study, microRNA (miRNA) microarray analysis was conducted to identify differentially expressed miRNAs in the skin of an experimental burn model. Microarray analysis revealed 24 differentially expressed miRNAs in antemortem burned mice skin, with 19 miRNAs significantly upregulated and 5 downregulated. Based on the intersection predicted using three databases (Targetscan, microRNA.org, and PITA), 293 potential miRNA targets were identified. These dysregulated miRNAs and their predicted targets were further analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. Several functional categories and signaling pathways were enriched, including the “fc epsilon ri signaling pathway,” “endometrial cancer,” and “mTOR signaling pathway.” Expression patterns of 10 differentially expressed miRNAs were verified by reverse transcription-quantitative polymerase chain reaction in mice skins. The results agreed with the results of microarray analysis. These findings suggest that differentially expressed miRNAs revealed by microarray are potential markers for forensic molecular diagnosis of antemortem burns.

Keywords: Forensic pathology, microRNA microarray, skin burns, vital reaction


How to cite this article:
Lyu HP, Cheng M, Liu JC, Ye MY, Xu D, He JT, Xie XL, Wang Q. Differentially expressed microRNAs as potential markers for vital reaction of burned skin. J Forensic Sci Med 2018;4:135-41

How to cite this URL:
Lyu HP, Cheng M, Liu JC, Ye MY, Xu D, He JT, Xie XL, Wang Q. Differentially expressed microRNAs as potential markers for vital reaction of burned skin. J Forensic Sci Med [serial online] 2018 [cited 2018 Dec 17];4:135-41. Available from: http://www.jfsmonline.com/text.asp?2018/4/3/135/242507

Hao-Pin Lyu, Ming Cheng and Jin-Cen Liu contributed equally to this work





  Introduction Top


In forensic practice, the detection of wound vitality is an important task, particularly during the examination of a burned body. When a corpse is found at a fire scene, a forensic pathologist must distinguish antemortem burns from postmortem burns. To determine whether death occurred due to thermal injury, it is necessary to identify whether the person was alive upon sustaining the burns. Conventional indicators of thermal injuries are skin erythema and blisters, soot deposits within the respiratory and/or digestive tracts, and elevated blood levels of carbon monoxide-hemoglobin. However, erythema and blister may occur with postmortem burns,[1] while soot deposits and elevated carbon monoxide-hemoglobin may be absent in cases of flash fire with no smoke or in open-air spaces.[2] Several studies suggested that immunohistochemical expression of heat-shock protein 70, fibronectin, P-selectin, von Willebrand factor, and PECAM-1 in the respiratory tract and lungs of fire victims support intravital reaction in fatal burns.[3],[4],[5] Furthermore, several functional categories and signaling pathways have been reported to be useful for determining wound vitality and wound age estimation.[6],[7],[8],[9],[10],[11],[12],[13]

MicroRNAs (miRNAs) are small noncoding RNA molecules with lengths of approximately 20–22 nucleotides and participate in posttranscriptional gene regulation.[14],[15] MiRNAs are stable and resistant to degradation.[16],[17] These characteristics make miRNAs potential biomarkers in the forensic practice of identifying antemortem and postmortem burns.

In this study, microarray analysis was conducted to determine gene expression profiles in the skin of an experimental burn model. The efficacy of this technique was evaluated to reveal differentially expressed miRNAs as potential markers for vital reactions.


  Materials and Methods Top


Animal experimental protocol

Study 1: Six male BALB/c mice (7–9 weeks old; 25 ± 3 g) were obtained from the Laboratory Animal Centre of Southern Medical University. Three mice (labeled as M1, M2, and M3) were anesthetized by intraperitoneal injection of 0.3% pentobarbital sodium (50 mg/kg), and then shaved to expose the skin area with an electric razor. Dorsal skin was exposed to a heated (100°C) 30 mm × 10 mm sheet of copper for 4 s. This procedure generated a deep partial thickness burn in the back skin of the mice [18],[19] [Figure 1]. After 30 min, the mice were sacrificed under deep anesthesia with pentobarbital sodium (60 mg/kg intraperitoneal [IP]). The antemortem burned dorsal skin was excised from the three mice. Three unburned three mice (labeled as M4, M5, and M6) were also sacrificed under deep anesthesia with pentobarbital sodium (60 mg/kg IP). Group 1 included antemortem burned mice skin (burned M1, M2, and M3), while Group 2 included external control unburned mice skin (unburned M4, M5, and M6). All samples were immediately submerged in 1 mL of RNA stabilization solution (RNAlater, Ambion, Austin, TX, USA). Three RNA samples from each group were prepared and designated as internal Burned M1, M2, and M3 and external control M4, M5, and M6.
Figure 1: Gross (a and b) and microscopic (c and d) images of unburned (a and c) and antemortem burned (b and d) mouse skin

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Study 2: Sixteen male BALB/c mice were used for secondary evaluation by reverse transcription real-time (RT)-quantitative polymerase chain reaction (qPCR). The dorsal skin of 8 mice was first exposed to heated sheet of copper for 4 s as described above (antemortem burn skin specimen). After 30 min, the mice were sacrificed under deep anesthesia with pentobarbital sodium. Thirty minutes after sacrifice, the same mice with unburned ventral skin were exposed to the heated sheet of copper for 4 s again (postmortem burned skin specimen). The antemortem skins and postmortem burned skin were excised immediately. Eight unburned mice were shaved with an electric razor to expose the skin and sacrificed under deep anesthesia with pentobarbital sodium without the burn. All skin samples were immediately submerged in 1 mL of RNA stabilization solution (RNAlater) and stored at −80°C until further examination. This study was reviewed and approved by the Ethics Committee of Southern Medical University Institutional Board (Guangzhou, China).

Microarray and bioinformatics analysis

Total RNA was quantified using the NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was assessed using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Sample labeling, microarray hybridization, and washing were performed based on the manufacturer's standard protocols. Briefly, total RNA was dephosphorylated, denatured, and labeled with Cyanine-3-CTP. After purification, the labeled RNAs were hybridized onto the microarray. After washing, the arrays were scanned with the Agilent Scanner G2505C (Agilent Technologies, Santa Clara, CA, USA).

Feature Extraction software (version 10.7.1.1, Agilent Technologies, Santa Clara, CA, USA) was used to analyze array images to obtain raw data. Next, Genespring software (version 13.1, Agilent Technologies, Santa Clara, CA, USA) was employed to complete the basic analysis of the raw data. Initially, the raw data were normalized using the quantile algorithm. Differentially expressed miRNAs were then identified through their fold-changes and P values calculated using the t-test. The threshold for up- and down-regulated genes was a fold-change of ≥ 2.0 and P ≤ 0.05. To predict potential target genes, three databases (Targetscan, microRNAorg, PITA) were used, and target genes predicted by these three databases were selected for further analysis. Genes were selected for Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and Protein-Protein Interactions (PPI) analysis by using the DAVID (https://david.ncifcrf.gov)[20] and STRING databases (http://string-db.org).[21] The GO project is an international standardized gene functional classification system that describes gene attributes, including biological process, molecular function, and cellular component. The KEGG database is the major public pathway-related database. A critical assessment and integration of PPI can be conducted using the STRING database, which includes direct (physical) as well as indirect (functional) associations. The information stems from computational prediction, knowledge transfer between organisms, and interactions aggregated from primary databases.

Real-time quantitative polymerase chain reaction

Briefly, cDNA copies of total RNA were obtained using the PrimeScript RT reagent Kit (TaKaRa, Shiga, Japan). RT-qPCR was conducted in 48-well reaction plates with an Illumina Eco Real-Time PCR System (San Diego, CA, USA) and SYBR green kit (TaKaRa) according to the manufacturer's recommendations. U6 was used as an endogenous control for the RT-qPCR, and the relative expression levels were determined by the 2−ΔΔCt method.[22] The RT-qPCR conditions, thermal cycler parameters, and gene-specific primers used for amplification are listed in [Supplementary Table 1] and [Supplementary Table 2].



Statistics

All RT-qPCR experiments were performed in triplicate, and the results were reported as the mean ± standard error of the mean Correlation analyses between miRNA microarray and RT-qPCR data were performed using linear regression (Pearson correlation analysis). The Student's t-test (two-tailed) was used to compare groups. Statistical analyses were performed using GraphPad Prism version 5.01 (GraphPad, Inc., La Jolla, CA, USA). P < 0.05 was considered to indicate statistical significance.


  Result Top


Significant expression changes in microRNAs from burned skin

Antemortem burned skins (Burned M1, M2, and M3) were analyzed by computational approaches and compared to skins from unburned mice (external control M4, M5, and M6). Expression differences are shown in a volcano map in [Figure 2]. Microarray analysis revealed 24 differentially expressed miRNAs in antemortem burned mice skin compared to those in unburned skins, with 19 miRNAs significantly upregulated and 5 downregulated [Table 1].
Figure 2: The difference expressed in the comparison is reflected in the map of the volcano. Red dots represent up-regulated miRNAs, and blue dots represent down-regulated miRNAs

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Table 1: 24 differentially expressed miRNAs in burned mice skins, which involved 19 miRNAs that were significantly up-regulated and 5 that were down-regulated

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Function and pathway analysis

To further determine the biological functions of these 24 differentially expressed miRNAs, intersections predicted using three databases (Targetscan, microRNA. org, and PITA) were used to predict the target genes, which revealed 293 putative target genes [Figure 3] and [Supplementary Table 3]. The functions of these target genes were determined by GO analysis. Significant GO terms corresponding to molecular functions included “Protein binding,” “Protein kinase binding,” “DNA binding,” and “Metal ion binding” [Figure 4]a. The main GO terms for a biological process included “Transcription, DNA-templated,” “Regulation of transcription, DNA-templated,” “Positive regulation of transcription from RNA polymerase II promoter,” and “Positive regulation of gluconeogenesis” [Figure 4]b. The significant GO categories corresponding to cellular components included “Membrane,” “Cytoplasm,” “Plasma membrane,” and “Nucleus” [Figure 4]c. KEGG pathway analysis demonstrated that “Fc epsilon RI signaling pathway,” “Endometrial cancer,” “mTOR signaling pathway,” and “Prostate cancer” were the most enriched pathways containing differentially expressed genes (DEGs) [Figure 5]. STRING software was used to analyze the PPI networks of the DEGs [Supplementary Figure 1].
Figure 3: Using TargetScan, PITA, microRNAorg database, the target gene prediction of the difference miRNA was predicted, and the target gene of the three databases was intersecting

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Figure 4: Gene Ontology enrichment analysis of targets of selected microRNAs covers 3 domains: molecular function (a), biological process (b), and cellular component (c)

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Figure 5: The 20 most significantly enriched pathways are shown that were mapped with Kyoto Encyclopedia of Genes and Genomes pathway analysis

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Microarray results were verified by real-time quantitative polymerase chain reaction

All qPCRs yielded a single peak on the dissociation curve, indicating specific amplification using the primers. To evaluate the microarray results, 10 miRNAs (upregulated miRNAs: mmu-miR-6969-5p, mmu-miR-7005-5p, mmu-miR-183-3p, mmu-miR-711, mmu-miR-135a-1-3p, mmu-miR-710, mmu-miR-7668-3p, mmu-miR-8090; downregulated miRNAs: mmu-miR-6924-3p, mmu-miR-5132-5p) were selected randomly for RT-qPCR confirmation [Table 2]. The results of RT-qPCR were consistent with the microarray data for these genes [Figure 6]a. The regression equation between microarray (x) and RT-qPCR (y) data (Log2 Ratio) was y = 0.8851x + 0.3134, with an r2 of 0.9074, confirming the microarray data obtained in the present study [Figure 6]b. These results were also validated when the number of mice was increased to 8 in each group. The expression levels of mmu-miR-6969-5p, mmu-miR-7005-5p, mmu-miR-183-3p, mmu-miR-711, mmu-miR-135a-1-3p, mmu-miR-710, mmu-miR-7668-3p, and mmu-miR-8090 were higher in antemortem burned skins than in postmortem burned and unburned skins. The expression levels of mmu-miR-6924-3p and mmu-miR-5132-5p were lower in antemortem burned skins than in postmortem burned and unburned skins. However, differences were not observed between unburned skins and postmortem burned skins [Figure 6]c.
Table 2: 10 significantly differentially expressed miRNAs were selected for further study

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Figure 6: Real-time quantitative polymerase chain reaction validation of 10 microRNAs.(a) The expression levels in real-time quantitative polymerase chain reaction of eight up-regulated microRNAs. All were consistent with the microarray data. (b) The regression equation between microarray (x) and real-time quantitative polymerase chain reaction (y) data (Log2Ratio) is y = 0.8851x + 0.3134, while the r2 is 0.9074.(c) mmu-miR-6969-5p, mmu-miR-7005-5p, mmu-miR-183-3p, mmu-miR-711, mmu-miR-135a-1-3p, mmu-miR-710, mmu-miR-7668-3p, and mmu-miR-8090 were up-regulated while mmu-miR-6924-3p and mmu-miR-5132-5p were down-regulated in antemortem burned mice skin samples when compared with unburned and postmortem burned groups (n = 8)

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  Discussion Top


When a fire results in fatality, the possibility of attempted concealment of a homicide should be evaluated. However, conventional external and/or internal indicators are sometimes unspecific, unremarkable, or even absent. Therefore, novel markers for wound vitality are urgently needed in forensic practice. Previous studies indicated that some functional categories and signaling pathways are useful for determining wound vitality.[9],[10],[11],[23] Gauchotte reported that CD15 and tryptase were useful markers for the vital reaction.[24] Hernández-Cueto suggested that cathepsin D is a useful marker for diagnosing wound vitality.[25] Few studies have examined fire death. In several studies increased heat-shock protein 70, fibronectin, P-selectin, von Willebrand factor, and PECAM-1 were observed in the respiratory tract and lungs of fire victims.[3],[4],[5] Hidemichi reported that the mRNA expression of AQP3 was increased in antemortem burn skin.[26]

MiRNAs are small noncoding RNA molecules. Extracted miRNAs are stable at room temperature for 1 year and remain detectable in storage at −20°C for 10 years.[27] Thus, they are useful for forensic practice. In this study, miRNA microarray analysis was conducted to determine the miRNA expression profiles in the skin of an experimental burn model. A total of 19 upregulated and 5 downregulated miRNAs were detected in burned mice skins compared to that in unburned skins. In addition, 10 differentially expressed miRNAs were selected randomly, and their expression levels were examined as a secondary check (n = 8) by RT-qPCR. The expression levels of these miRNAs in antemortem burned and unburned groups were consistent with the microarray data, confirming that our microarray data is reliable. In the present study, postmortem burned specimens obtained from antemortem burned mice were also examined. The 10 differentially expressed miRNAs listed above showed significant differences between antemortem burned skins and postmortem burned skins and between unburned skins. However, no difference was detected between postmortem burned specimens and unburned skins.

These results indicate that the differentially expressed miRNAs detected by microarray are not affected by postmortem burn and thus are potential markers for vital reaction to burns, although further validation is needed.

Using three databases (Targetscan, miRNA.org, PITA), 293 target genes were predicted. GO analysis of these 293 genes revealed that “transcription, DNA-templated,” “protein binding,” and “membrane” were enriched GO terms containing DEGs belonging to biological process, molecular function, and cellular component, respectively. KEGG pathway analysis demonstrated that “Fc epsilon RI signaling pathway,” “Endometrial cancer,” and “mTOR signaling pathway” were the most enriched pathways. These bioinformatic analyses may improve the understanding of the process of wound healing, particularly in the early phase.

In our previous study, we used Illumina RNA-seq technology to determine gene expression profiles in contused mouse skin.[28] Genes from different functional categories and signaling pathways were enriched, including immune system process, immune response, defense response, cytokine − cytokine receptor interaction, complement and coagulation cascades, and chemokine signaling pathway. The results were quite different from those of this study. These differences may be because of the different models of injury used. Exposure to heat may seriously affect the expressions of miRNAs and differ from mechanical injury. Further studies of human samples and various types of wound samples are needed to clarify the underlying mechanism.

In summary, this is the first study to use miRNA microarray analysis to analyze an experimental skin burn model. A total of 19 upregulated and 5 downregulated miRNAs were detected in burned mice skins. Our findings indicate that miRNA microarray is a powerful tool for revealing differentially expressed miRNAs as potential markers for vital reaction to burns, although further investigations are required to confirm these results.

Financial support and sponsorship

This research was supported by the National Natural Science Foundation of China (Grant No. 81401556 and 81601641), the Natural Science Foundation of Guangdong Province (No. 2014A030310504 and 2014A030310293), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (No. 2015-311), and the Special Foundation of President of School of Public Health of Southern Medical University (Grant No.GW201619).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2]



 

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