MiRNAs as New Tools in Lesion Vitality Evaluation: A Systematic Review and Their Forensic Applications

Wound vitality demonstration is one of the most challenging fields in forensic pathology. In recent years, researchers focused on the application of histological and immunohistochemical staining in this sphere of study. It is based on the detection of inflammation, red cell infiltration, and tissue alterations at the histological examination, all of which are supposedly present in antemortem rather than post-mortem wounds. Nevertheless, some doubts about the reliability of those markers have arisen. Furthermore, the lack of a standardized protocol and the operator dependency of this approach make the proper interpretation of its results difficult. Moreover, a differential miRNAs expression has been demonstrated in antemortem and post-mortem wounds. Herein, a systematic review concerning the current knowledge about the use of miRNAs in lesion vitality evaluation is carried out, to encourage researchers to deepen this peculiar study area. A compendium about the potential miRNAs that may be further investigated as vitality markers is also provided. The aim is to collect all available data about this topic to direct further studies on this field and highlight the future applications of miRNAs in forensic pathology. We found 20 articles and a total of 51 miRNAs that are involved in inflammation and wound healing. Further studies are certainly needed to deepen the role of miRNAs in inflammatory processes in lesioned skin and to evaluate their reliability in distinguishing between antemortem and post-mortem lesions.


Introduction
Wound vitality demonstration is one of the most challenging fields in forensic pathology. It has been classically based on the presence of inflammation, red cell infiltration, and tissue alterations at the histological examination [1][2][3][4]. However, the differentiation between vital reactions and post-mortem changes is not always clear [5][6][7]. In recent years, researchers focused on the detection of inflammatory cells, cytokines, apoptosis mediators, and other markers that are supposedly present in antemortem rather than post-mortem wounds, through immunohistochemical stains [6][7][8][9][10][11]. Nevertheless, some doubts about the reliability of these methods have arisen, and the lack of a standardized protocol and the operator dependency of this approach makes it difficult to correctly interpret the results [12].
MicroRNAs (miRNAs) are short single-strand non-coding ribonucleic acids that have a fundamental role in the post-transcriptional regulation of gene expression, mainly inhibiting the mRNA translation [13][14][15]. Due to their ubiquity, they can be used as a diagnostic tool in different clinical areas, as well as in forensic pathology [16]. A differential miRNA expression in antemortem and post-mortem wounds has been observed [17][18][19]. This Biomedicines 2021, 9,1731 2 of 15 evidence opens new and promising possibilities to use them as lesion vitality markers. It is unlikely that a method alone could provide the trustworthiness needed in judicial cases, but the combination of histological, immunohistochemical, and genetic analysis may become a new tool in vitality diagnosis.

Materials and Methods
The present systematic review was carried out according to the Preferred Reporting Items for Systematic Review (PRISMA) standards [20]. A systematic literature search and a critical review of the collected studies were conducted. An electronic search of PubMed, Science Direct Scopus, Google Scholar, and Excerpta Medica Database (EMBASE) from database inception to November 2020 was performed. The search terms were "miRNAs", "wound vitality", and "lesions vitality" in the title, abstract, and keywords. The bibliographies of all located papers were examined and cross-referenced to further identify relevant literature. A methodological appraisal of each study was conducted according to the PRISMA standards, including an evaluation of bias. The data collection process included study selection and data extraction. Two researchers (A.C.M., E.M.) independently examined the papers with titles or abstracts that appeared to be relevant and selected those that analyzed miRNAs involved in wound vitality demonstration and miRNAs involved in wound healing that may be further investigated as vitality markers in post-mortem samples. Researchers resolved their disagreement concerning works eligibility by consensus. Only papers in English were included in the research. Two investigators performed data extraction (A.B., E.M.), and two other investigators verified them (A.B., E.M.), which were again verified by two other investigators (A.M., E.T.). This study was exempt from institutional review board approval, as it did not involve human subjects.

Results
The search performed as described above identified 197 articles, which were screened to exclude duplicates. The resulting 163 reference lists were then screened based on their title and abstract, which left 87 articles for further consideration. Non-English papers were excluded. The following inclusion criteria were used: (1) original research articles, (2) reviews and mini-reviews, and (3) case reports/series. These publications were carefully evaluated, considering the main aims of the review. This evaluation left 20 scientific papers comprising original research articles, case reports, and case series. Figure 1 illustrates our search strategy. Table 1 shows the studies included in this review with a brief description. As shown in Table 2, which summarizes the main characteristics of the articles included in this review, most of the founded studies are about miRNAs involved in wound healing and therefore they are based on in vitro and/or in vivo experiments [17,18,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37].
Three studies used human skin lesion samples collected post-mortem (2) and antemortem (1) [19,29,37]. Animal studies were based on antemortem samples, mainly collected within a precise interval after wounding (days), while antemortem human skin samples were hypertrophic skin scars specimens collected during surgical procedures. In vitro and antemortem studies investigated the role of miRNAs in wound healing and, as previously mentioned, they were included in this review because they could suggest new miRNAs to study for wound vitality demonstration in post-mortem samples. Moreover, antemortem studies provide new knowledge about the expression of miRNAs in wound healing in different timeframes, and consequently, they may be used in further studies to determine the age of a lesion. Concerning the type of skin lesion, the articles investigated miRNAs expression in incisional [18] and excisional wound [21][22][23]26,27,30,31,[33][34][35], ligature mark [19], burned skin [37], chronic ulcer [32], and hypertrophic scar [29]. There is heterogeneity among researchers concerning the type of adopted investigation. Real-time polymerase chain reaction (RT-PCR) was performed in 17 studies out of 20. In 11 studies, Western blot was used for protein analysis [21,22,[24][25][26][27][28]30,32,33,36]. Histological examination of the wounded skin was performed in 13 out of 20 studies, but only in 8 studies, this eval-uation was supplemented by immunohistochemical analysis and in 5 by hybridization in situ [17][18][19][21][22][23][24][25][27][28][29][32][33][34][35][36]. Immunofluorescence analysis was performed in two studies, while a morphometric analysis appeared in the work of Long and coll [31]. Flow cytometry analysis was used by Lin et al. to isolate fat cells [28]. Phase-contrast morphometry was performed in one study [36]. In all the studies included in this review, a statistical analysis on obtained results was performed, except for Viticchiè et al., Yang et al.,and Yu et al. [33,35,36].  Table 1 shows the studies included in this review with a brief description.  As a result of our research, we found that 51 miRNAs are implied in wound healing and wound vitality demonstration. To understand the molecular pathways in which these miRNAs are involved, we collected their target genes and/or proteins, when specified by the included papers, as shown in Table 3.

Discussion
MiRNAs have attracted the attention of researchers in various fields [38][39][40][41]. They influence many signaling pathways involved in inflammation [42][43][44][45]. Due to this property, miRNAs could be used in vitality wound demonstration. In this review, we present many miRNAs that have a role in wound healing and, therefore, may be investigated as post-mortem vitality markers.
In conclusion, by comparing the difference in the expression of miRNAs in the wound healing process, these biomarkers could become useful for the chronological diagnosis of the lesions [65], as shown in Figure 2. Only those miRNAs that are expressed according to a precise timeframe were considered. Only the first day of increase is shown. * miR-183-3p shows a different behavior between humans and rats; it was detected overexpressed in humans over 2 days and in rats over the course of 120 days. ↑ indicates up regulation, ↓ indicates down regulation.
Nevertheless, the reader should bear in mind that our work has some limitations. The pool of papers is heterogeneous, and it includes different kinds of studies, counting in vitro, in vivo, and human samples. Moreover, the small sample size (only 20 papers included) could interfere with the reliability of our findings. Certainly, more studies are needed in this field. Once the role of miRNAs in the wound healing process has been completely clarified, we hope to implement this knowledge in forensic applications soon thereafter. For future applications, it is conceivable to combine miRNAs analysis and histological and immunohistochemical tissue investigation, which have already been widely Only those miRNAs that are expressed according to a precise timeframe were considered. Only the first day of increase is shown. * miR-183-3p shows a different behavior between humans and rats; it was detected overexpressed in humans over 2 days and in rats over the course of 120 days. ↑ indicates up regulation, ↓ indicates down regulation.
Nevertheless, the reader should bear in mind that our work has some limitations. The pool of papers is heterogeneous, and it includes different kinds of studies, counting in vitro, in vivo, and human samples. Moreover, the small sample size (only 20 papers included) could interfere with the reliability of our findings. Certainly, more studies are needed in this field. Once the role of miRNAs in the wound healing process has been completely clarified, we hope to implement this knowledge in forensic applications soon thereafter. For future applications, it is conceivable to combine miRNAs analysis and histological and immunohistochemical tissue investigation, which have already been widely investigated in the forensic literature [1][2][3][4][6][7][8][9][10][11]. As in many other fields, miRNAs investigation carries great potential in forensic science [66][67][68]. A possible project could be an interlaboratory study to investigate the miRNAs differential expression in the same lesion and with the same analytical procedure, in addition to histological and immunohistochemical evaluation. The aim is to create a standardized protocol and start implementing these analyses since the reliability and reproducibility of analytical methodologies are the foundation for acceptance in legal proceedings.

Conclusions
Even if in the last years, some studies have provided new evidence about the forensic applications of miRNAs, much evidence still needs to be disclosed. MiRNAs are involved in many molecular pathways, and, owing to their ubiquity, they have been considered as markers in several fields. Herein, a compendium of the potential miRNAs that should be further investigated as wound vitality and age markers was provided. We found 20 articles on miRNAs involvement in skin wound and healing, but only 2 of them specifically referred to vitality demonstration in the forensic field. Further studies are certainly needed to deepen the role of miRNAs in inflammatory processes in wounded skin and to evaluate their reliability in distinguishing between antemortem and post-mortem lesions.