A small RNA, microRNA as a potential biomolecular marker to estimate post mortem interval in forensic science: a systematic review

Background Post-mortem interval (PMI) is the cornerstone of the forensic field to investigate. The examination technique by seeing the changes in the body such as algor mortis, rigor mortis, and livor mortis is a traditional technique in which accuracy is influenced by many factors. A biomolecular technique that uses microRNA (miRNA) biomarkers is developing because miRNA has good stability than other RNA, so it meets the requirements to be used for PMI estimation. Method Following the PRISMA guidelines, journals were taken from 5 databases: Scopus, Science Direct, PubMed, Embase, and Springer. The review was carried out by two people. Inclusion criteria in this review are original research, published in the last 10 years, discussing miRNA as a biomarker for PMI estimation, and free full access. While exclusion criteria are not original research and not using English. Result Eighteen journals were reviewed in this study. The study was conducted using test animals (rats) and human samples with tissue sources taken from the liver, skeletal muscle, blood, bone, heart, skin, saliva, semen, brain, lung, vitreous humor, spleen, and kidney. miRNA expression levels after death showed different results based on miRNA target, tissue source, and others. Discussion The results of each study are different due to the use of different types of miRNA targets and tissue sources. miRNA has great potential to estimate PMI in forensic science, but it is necessary to control the influencing factors to obtain an accurate conclusion.

changes in protein, DNA, and RNA over time to improve the accuracy of PMI estimation [1,2,7,9].
Real-time quantitative polymerase chain reaction (RT-qPCR) is a development of tools and techniques in the biomolecular field and made it possible to study the variations of the forms and types of nucleic acids such as rRNA, mRNA, and miRNA [3]. Non-coding RNA has received attention because it has the potential as a biomarker [10]. One of the non-coding RNAs is microRNA (miRNA) [11].
MicroRNA (miRNA) was introduced by Lee et al. in 1993 [12][13][14]. miRNAs are a part of small non-coding RNAs. miRNAs regulate post-transcriptional gene expression by controlling messenger RNA (mRNA) translation [10,[14][15][16]. miRNAs are 20-25 nucleotide bases in length [11,13,17]. miRNAs have features that are attractive for analysis in the forensic field, such as high degradability resistance, specific tissue expression when compared to mRNAs, can be multiplied in large quantities, have increased sensitivity using the latest technology [15,18,19], and can be extracted together with the DNA [20].
Many studies have demonstrated that miRNAs have a significant role in biological processes such as cell proliferation, differentiation, pathogenesis, apoptosis, organ formation, defense against viruses, and metabolic control [17]. One of the essential potentials of miRNA is its use in forensic pathology [18].
Several studies have shown a continued expression of miRNAs at post-mortem intervals which offer stability to degradation under extreme conditions. This reason makes them very suitable for use in the forensic field [3,21]. Therefore, this systematic review aims to look at the development of miRNAs as a biomarker used to estimate post-mortem interval in animals and humans to be used in forensic science. In addition, we want to see the tissue targets specific to each miRNA that has been found and used.

Literature search technique
The review method used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol [22,23]. Journals are taken from 5 electronic data banks: Scopus, Embase, PubMed, Science Direct, and Springer. The journal that was used to be reviewed was published in the last 10 years. The keyword used were "microRNA or miRNA," "Post mortem interval or PMI," and "Forensic." It is carried out using the BOOLEAN system so that the result is "(((microRNA) OR (miRNA)) AND ((Post Mortem Interval) OR (PMI))) AND (Forensic)." The results are then entered into Zotero to check for duplication of journals found through search engines.

Article selection
In selecting articles or journals to be reviewed, an examination is carried out through the inclusion and exclusion criteria. The inclusion criteria were as follows: (1) journal is an original research article, (2) journal was published within the last 10 years, from January 2012 to December 2022; (3) journals discuss microRNA as a post-mortem interval marker in the forensic field; (5) journal can answer questions from PICO (Does miRNA have potential as a biomolecular marker to estimate post-mortem interval in forensics?) The exclusion criteria were as follows: (1) journal is not original research (literature/systematic review), (2) journals did not use English, (3) journals could not be accessed in free full text, (4) journal does not provide conclusions that discuss the topic of miRNA as a biomarker for estimating PMI.

Data synthesis
In this review, two reviewers synthesized data independently to avoid bias when conducting the review. Synthesized data include name of the first author of the journal, year of publication, location of study, sample, and the number of samples, treatment (euthanasia technique, temperature, cause of death), source of tissue used, RNA extraction technique, amplification technique, miRNA used or target, research results, conclusions, and research limitation.

Literature search and description
The search was conducted on five electronic data banks: Scopus (""n = 29), Science Direct (n = 174), PubMed (n = 26), Embase (n = 28), and Springer (n = 274) using the BOOLEAN system to obtain journals for a total number of 535. From 535 journals, 77 were automatically excluded by the system for journals published under 2012, and 74 were duplicated, bringing the total to 384. The automatic screening was carried out to exclude 208 journals that were not research journals, bringing the total journal to 176. Title and abstract screening was carried out to look for the journal that matched the inclusion criteria, as many as 139 journals, so the number of journals becomes 37. Thirty-seven journals were read entirely, and 19 journals did not meet the criteria or did not answer PICO, so the final journals that will be reviewed are 18. The PRISMA flow chart results are in Fig. 1.

Data synthesis and results
Synthesized data include the first author's name, year of publication, study location, sample, and the number of samples, Treatment (euthanasia technique, temperature storage, cause of death), tissue source used, RNA extraction technique, amplification technique, miRNA target, results, conclusions, and research limitations (Table 1).
From 18 journals, 8 journals found results in the form of changes in miRNA expression levels after an interval of death [4,6,15,19,21,25,26,33]. One journal showed a negative correlation between miRNA expression levels and post-mortem interval, namely miR-16 and let-7e [19]. The results of other studies say that further research is needed for miR-3381-3p because it has great potential. In addition, 10 journals found that miRNA expression level values were stable throughout the post-mortem interval [9, 24, 27-32, 34, 35].
In addition, several different treatments were carried out. Derakhshanf et al gave different treatment when preparing the sample. They use CO 2 gas and ketamine/xylazine injection [4]. Kim SY et al. gave different treatment by taking blood samples from different places such as peripheral blood, heart blood, and coronary sinus blood with the results

Discussion
PMI estimation has always been an exciting and vital challenge in forensics [36,37]. Especially when the corpse is already severely damaged, it is more difficult to assess the lesions that show the dynamic process of death [37]. miR-NAs have characteristics such as being more stable compared to longer RNAs such as mRNA [18]. Eighteen journals that discussed miRNA as one of the biomarkers to estimate post-mortem interval were reviewed. Some miRNAs decrease expression levels at the time of death increases, and some miRNAs have stable expression levels until sometime after death. All studies used RT-qPCR techniques to determine expression levels. RT-qPCR is an assay that uses fluorogenic primers to combine transfer energy by resonance fluorescence techniques with traditional RT-PCR [38,39]. RT-qPCR is widely used because it has sensitivity and specifications in time-lapse studies of death, and RT-qPCR has reliability and accuracy in analyzing RNA levels in specimens [30].
From 18 journals, there are five journals that used the heart as a target to see miRNA expression [9,26,27,31,32]. miRNAs's targets are miR-3185, miR-122, miR-133a, and miR-1. miR-3185 showed increasing expression levels but has no significant correlation with post-mortem interval (40H) [26]. miR-122, miR-133a, and miR-1 have stable expression throughout the post-mortem interval [9,27]. Stability of miR-122, miR-133a, and miR-1 makes these miR-NAs can be used as reference genes in the heart [9,27]. Lv Y et al. also found that miR-1 and miR-133a in the heart showed high and stable expression levels up to 144 h [9]. Tu C et al. reported that miR-133a was also stable up to 8 days after death in cardiac and skeletal muscle [27]. Lv Y et al. found that miR-1 had stable expression levels up to 144 h after death in the femoral quadriceps muscle; this is because miR-1 has a task in the physiological formation of muscle tissue [28]. In contrast, Li We et al. found that miR-1 is stable during post-mortem intervals up to 168 h with samples from the heart [31].
Four journals used the liver as the target organ to look at the expression of miR-206, miR-150, miR-12, miR-195, and miR-133a [4,9,21,27]. miR-133a and miR-206 showed a decrease in expression levels at 24 h after death while miR-195 increased in the first 24 h after death [21]. However, the expression levels of the five markers do not correlate with PMI [21]. miR-122 is a miRNA expressed in the liver and has been studied widely. Wang et al. said that miR-122 did not correlate with expression levels and PMI in the early 24 h [21]. Lv Y et al. found that miR-122 degraded in line with postmortem interval, especially at high temperatures, but at low temperatures, it was stable until 144 h, so it was concluded that it did not meet the criteria as a reference gene [9]. In contrast, Tu C et al. research showed that miR-122 showed a stable expression level at 8 days of death in samples taken from the heart and liver [27]. Derakhshanfar A et al. said that miR-122 increased expression levels at an interval of 6, 8, and 48 h after death [4].
This condition can be different because some studies use a special treatment such as sample preparation techniques by euthanasia using CO 2 and ketamine injection. These differences in preparation methods give different levels of expression in the liver 4. Samples were stored at 4 °C, 15 °C, 25 °C, and 35 °C. There were differences in miR-122 expression levels. At 35 °C, miR-122 decreased due to faster degradation. So, it is concluded that higher temperatures will accelerate the degradation of miR-122 in the liver [9]. miR-23-3p, miR-381-3p, and miR-114-3p were tested and showed different results in skeletal muscle. miR-382-3p expression increased throughout from 0 to 24 h. In contrast, miR-23-3p showed a decrease in expression levels from 0 to 24 h, while miR-114-3p decreased at hours 0 to 6 but not significantly [6].
Kim SY et al. researched miR-1, miR-16, miR-208b, and let-7e with sample sources taken from different blood vessels. miR-1 and miR-208b demonstrated different expression levels according to tissue source, while let-7e and miR-16 expressed similar levels. However, the four markers were not significantly correlated with post-mortem intervals [24]. In contrast, Na J et al. took samples from bone, found differences in miRNA expression levels along with post-mortem interval increased, and showed a negative correlation between expression levels and PMI [19].
It was concluded that miR-16 and let-7e showed different expression results depending on the target organ/tissue. In samples taken from blood, miR-16 and let-7e did not significantly correlate between PMI and expression levels [24]. Whereas in samples taken from Pattela bone, miR-16 and let-7e showed a decrease in expression levels as the time of death increased with correlation coefficient values R 2 = − 0.3329 and R 2 = − 0.3025 [19].
In a study using skin samples, Zhang H et al. found miR-203 showed stable expression levels at < 10 h [32]. Meanwhile, Ibrahim SF et al. used miR-205 and miR-21 as target miRNAs in the skin. They found that miR-205 and miR-21 had increased expression levels 24 h after death and had a drastic decrease at 48 h [33]. Alshehhi S et al. found that miR-205 showed stable expression levels for 360 days after death and became one of the specific markers in saliva [34].
In human samples, the cause of death and the source/ location of sampling affect the expression levels. Blood samples from different origins give different expression levels, such as peripheral blood, cardiac blood, and coronary sinus blood. For example, miR-208 showed greater expression levels in blood sourced from coronary sinus blood [24]. Deaths caused by mechanical asphyxia led to increased expression levels of miR-3185 in the heart compared to hemorrhagic shock and craniocerebral injury [26]. Odriozola et al. reported insignificant levels in miRNA expression (miR-541, miR-484, miR-34c, mir-142-5p, miR-20a, miR-888, and miR-671-3p) after death between who dies at night and during the day in vitreous humor samples [15]. Different with Corradini et al., they found significant differences in miRNA expression levels (miR-106b, miR-96, miR,142-5p, and miR-219) after death between samples that died during the day and at night in the first ≤ 24 h in the vitreous humor and ≤ 48 h in blood [35]. This difference in results could be due to differences in miRNA targets used in each study. Zhang H et al. conducted a survey on miR-1 which is considered a reference marker in the heart that showed a stable expression level in samples with a death interval of < 10 h in cases of death due to trauma, while in cases of asphyxia, it was stable in 10-22 h [32].
With this review, miRNAs are one of the biomolecular markers with excellent potential to estimate post-mortem interval. miRNA shows different expression levels when tested in other tissues, so it is necessary to choose the right miRNA target according to the target tissue. Storage of samples at different temperatures will also affect the expression level so that it can be considered during the study. The cause of death of the samples will also affect the expression level. It is necessary to conduct more in-depth research on miRNA by controlling any shortcomings in previous research studies so accurate results can be obtained using miRNA as a marker.
From this review, each study has different limitations, such as inadequate samples in each group of representatives.
The target of miRNA is still limited to each tissue target. In addition, information about the condition of the sample was not obtained, such as the previous disease, the duration of the diseases. and the cause of death in some sample.
Recommendations: (1) Research is carried out by adding and selecting miRNAs specific to the target tissue. (2) The researcher must obtain all the sample's data, including the cause of death and disease. (3) Samples are placed at room temperature to simulate corpses found in the open area. (4) Each group needs to add more sample.

Conclusion
From this review, we can say that miRNA has enormous potential as one of the biomarkers o estimate PMI in forensic science. One reason is the stability and robustness of miRNA compared to others RNAs. Many factors affect miRNA expression levels, such as tissue source, time of death, cause of death, and temperature. Each miRNA has a target gene that will express according to its target region. The results of this review show the existence of miRNAs that have the potential as reference genes. In future research, it is necessary to pay attention to the number of adequate samples and the selection of specific miRNAs. Control of all influencing factors can reduce research bias.
Funding Open Access funding provided by Aalto University. The authors gratefully acknowledge the support of the University of Indonesia for the study.

Data Availability
The reviewed data are available in the referenced publications and summarized for synthesis in Table 1.

Declarations
Ethical approval This study obtained the data from online database and did not involve human participants and/or animals. The Ethics Committee of the Faculty of Dentistry, University of Indonesia, has confirmed that no ethical approval is required.

Conflict of interest The authors declare no competing interests
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