An Exploratory Study On The Estimation Of Skin Wound Age And The Identification Of Antemortem/postmortem Wounds Based On MicroRNA Microarray

ObjectiveWound age usually refers to the time interval experienced from organ tissue injury to death,which can be regarded as the survival time of an individual after physical injury.Wound age estimation has important practical significance and is always the focus and difficulty of forensic pathology research.With the development of molecular biology and research technology,it has become a hot topic to infer the wound age by using the temporal change of RNA in tissues.Many scholars have used real-time quantitative PCR to detect the expression changes of specific RNA in damaged tissues,and have made some progress in the prediction of the wound age.However,the existing research has some problems,such as the single RNA species and the oversimplified mathematical model,so that the practical application is limited.There have also been few studies on the identification of antemortem/postmortem wounds.In view of the above problems,this study intends to identify the expression changes of microRNA in antemortem/postmortem wounds and a period after wound,and screen the microRNA indicators related to the identification of antemortem/postmortem wounds and the estimation of wound age by microRNA microarray and qRT-PCR methods.Methods1.By referring to relevant literature and previous research of our team,we established a mouse skin incision model.We selected the skin tissue samples from the control group,the antemortem wound 1h,4h and 1d groups and the postmortem wound 1.5h group(n=3)for mi croRN A microarray detection.2.Bioinformatics analysis was performed on microRNA microarray data,including quantitative analysis,differential expression analysis,cluster analysis and functional enrichment analysis.3.Based on microarray data,candidate internal reference,wound time estimation and antemortem/postmortem wound identification markers were screened,which were detected at more time points by qRT-PCR method in this study.4.The RefFinder software was used to evaluate the expression stability of candidate internal reference markers in normal skin samples,antemortem and postmortem skin incision samples,aiming to screen suitable internal reference markers.The quantitative data(Cq value)of candidate wound time estimation and antemortem/postmortem wound identification markers were standardized,and the Δ Cq values were obtained for subsequent analysis.5.The relative expression of the experimental group and control group for candidate microRNA markers was calculated with 2-ΔΔCt method.The multiple comparison between control group and experimental groups was performed using ANOVA method,p<0.05 for the difference is statistically significant.6.The correlation between the expression quantity(Δ Cq value)of candidate markers and 0-240h wound time was analyzed using GraphPad Prism8.0 software.The p value indicates a significant relationship,and the correlation coefficient r indicates the direction and size of the relationship.7.The nonlinear regression analysis,i.e.,curve fitting,was conducted on the relevant microRNA markers,and nonlinear regression models were established using XLSTAT software.Residual graphs were drawn based on the actual measured values and the predicted values of the model to evaluate the effectiveness of each candidate marker in estimating wound time.Combining all relevant microRNA markers,multivariate nonlinear regression models were constructed using SPSS,and residual graphs were drawn.8.The range of expression quantity of candidate antemortem/postmortem wound identification markers in the control group and experimental groups was analyzed,and boxplot praphs were drawn.Results1.The microRNA microarray results showed that 1166(62.0%)microRNA could detect effective signals.Comparison was made among the control group,postmortem wound 1.5h group,antemortem wound 1h,4h and 1d groups.The differential expression analysis was performed based on the criteria of FC≥2.0 and P<0.05.49 microRNAs were differentially expressed between the control group and the postmortem wound 1.5h group,among which 33 were up-regulated and 16 were down-regulated.There were 64 microRNAs differentially expressed between the antemortem wound 1h group and the control group,among which 44 were up-regulated and 20 were down-regulated.There were 86 microRNAs differentially expressed between the antemortem wound 4h group and the control group,among which 40 were up-regulated and 46 were down-regulated.145 microRNAs were differentially expressed between the antemortem wound 1d group and the control group,among which 60 were upregulated and 85 were down-regulated.106 microRNAs were differentially expressed between the antemortem wound 4h group and the antemortem wound 1h group,among which 40 were up-regulated and 66 were down-regulated.There were 71 microRNAs differentially expressed between the antemortem wound 1d group and the antemortem wound 1h group,among which 29 were up-regulated and 42 were down-regulated.There were 66 microRNAs differentially expressed between the antemortem wound 1d group and the antemortem wound 4h group,among which 50 were up-regulated and 16 were down-regulated.2.Considering the significance of functional analysis and the false-positive microarray data,we compared the postmortem wound 1.5h group,antemortem wound 1h group,antemortem wound 4h group and antemortem wound 1d group with the control group,and screened the differentially expressed microRNAs using the conditions of FC≥2.0,P<0.05 and the original signal>100.Ten differentially expressed microRNAs were screened between the postmortem wound 1.5h group and the control group.Three differentially expressed microRNAs were screened between the antemortem wound 1h group and the control group.Nine differentially expressed microRNAs were screened between the antemortem wound 4h group and the control group.Twenty-four microRNAs with differential expression between the antemortem wound 1d group and the control group were screened.3.TargetScan and miRDB database were used to predict the target genes of the differentially expressed microRNAs,and GO and KEGG function enrichment analysis were performed on the target gene sets of differentially expressed microRNAs.There were 1051 target genes for differentially expressed microRNAs between the postmortem wound 1.5h group and the control group.GO analysis showed that Binding,Protein Binding and Enzyme Binding were the most significant items at the molecular function level,Cell part,Cell and Intracellular components were the most significant items at the Cell component level,and Localization,Positive regulation of biological process and regulation of Localization were the most significant items at the biological process level.KEGG analysis showed that the differentially expressed microRNA target genes were mainly concentrated in the PI3K-Akt signaling pathway.The most significant pathway is the Hippo signaling pathway.There were 410 target genes for differentially expressed microRNAs between the antemortem wound 1h group and the control group.GO analysis results showed that Binding,Protein Binding and Enzyme Binding were the most significant items at the molecular function level,and Cell part,Cell and Neuron projection were the most significant items at the Cell component level.The most significant items in biological processes are Localization,Negative regulation of cellular process and regulation of biological process.KEGG analysis showed that target genes of differential expression microRNAs were mainly concentrated in Ras signaling pathway.The most prominent pathway is the Axon Guidance Route.There were 627 target genes for differentially expressed microRNAs between the antemortem wound 4h group and the control group.The GO analysis results show that the most significant items at the molecular function level are Binding,Protein Binding and Ion Binding,and the most significant items at the Cell component level are Intracellular,Intracellular part and Cell part/Cell.Positive regulation of biological process,Positive regulation of cellular process and Positive regulation of metabolic process were the most significant items in biological process.KEGG analysis results showed that target genes of differential expression microRNAs were mainly enriched in Endocytosis pathway,which was the most significant pathway.There were 1393 target genes for differentially expressed microRNAs between the antemortem wound 1d group and the control group.The GO analysis results show that the most significant items at the molecular function level are Binding,Protein Binding and Enzyme Binding,and the most significant items at the Cell component level are Intracellular,Intracellular part and Cell part/Cell.Regulation of biological process,Positive Regulation of biological process and Negative regulation of biological process were the most significant items in biological process.KEGG analysis showed that the differentially expressed microRNA target genes were mainly concentrated in the MAPK signaling pathway.The most significant pathway is the Human Immunodeficiency Virus 1 infection pathway.4.Five candidate internal reference markers were screened based on the microarray data and the stability was evaluated using Reffinder software.The results showed that the stability sort of the five candidate internal reference markers was miR-24-3p>5S rRNA>U6 snRNA>let-7a-5p>miR-203-3p。5.We compared the antemortem wound 1h group,antemortem wound 4h group and antemortem wound 1d group with the control group,and screened the candidate wound time estimation microRNAs using the conditions of FC≥2.0,P<0.05 and the original signal>100.Nine microRNAs were screened between the antemortem wound 1h group and the control group.24 microRNAs were screened between the antemortem wound 4h group and the control group.53 microRNAs were screened between the antemortem wound 1d group and the control group.Finally,15 microRNAs(let-7c-5p,miR-19b-3p,miR-20a-5p,miR-26a-5p,miR-96-5p,miR-127-3p,miR-142A-3p,miR-146a-5p,miR-188-5p,miR-199a-5p,miR-223-3p,miR-497a-5p,miR-1982-5p,miR-6360 and miR-7040-5p)were screened as candidate estimation markers.6.The expression levels of 15 candidate microRNA markers were compared among groups.When U6 snRNA was used as internal reference,the expression levels of seven microRNAs(miR-20a-5p,miR-26a-5p,miR-127-3p,miR-146a-5p,miR-199a-5p,miR-223-3p and miR-497a-5p)showed differences between groups.When 5S rRNA was used as internal reference,the expression levels of eight microRNAs(miR-19b-3p,miR-20a-5p,miR-96-5p,miR-127-3p,miR-142A-3p,miR-199a-5p,miR-223-3p,and miR-6360)showed differences between groups.7.We analyzed the correlation between the expression levels of 15 candidate microRNA markers and 0-240h wound time.When U6 snRNA was used as internal reference,the expression levels of seven microRNAs(let 7c-5p,miR-19b-3p,miR-20a-5p,miR-26a-5p,miR-223-3p,miR-6360 and miR-7040-5p)showed a temporal characteristic.When 5S rRNA was used as the internal reference,the expression levels of six microRNAs(let-7C-5p,miR-19b-3p,miR-20a-5p,miR-199a-5p,miR-497a-5p and miR-7040-5p)showed a temporal characteristic.8.U6 snRNA was used as internal reference and seven related microRNAs were combined to estimate the wound time.The multivariate nonlinear regression model was established as follows:Y=-48.267*X22-6.961*X32-83.589*X1+587.528*X2-107.414*X3+181.874*X4-22.566*X5+165.431*X6-155.886*X7-1046.562.The R2 value of goodness of fit for this model is 0.999.Taking 5S rRNA as internal reference,and combining six relevant microRNAs,the multivariate nonlinear regression model was established as follows:Y=23.005*X22+12.358*X32-15.159*X1-591.741*X2-189.137*X3+5.075*X4-35.745*X5+43.293*X6+4386.386.The goodness of fit R2 value for the model was 0.976.9.Among the 15 microRNA markers that were differentially expressed in the antemortem wound group vs the control group,four markers(let-7c-5p,miR-26a-5p,miR-223-3p and miR-497a-5p)showed no expression difference in the postmortem wound group vs the control group(P>0.05,and the coefficient of variation in the control group and 1.5h postmortem wound group was lower than 0.01),which were selected as candidate identification markers.10.The expression levels of four candidate microRNA markers were compared among groups.When U6 snRNA was used as internal reference,the expression levels of two microRNAs(miR-223-3p and miR-497a-5p)showed differences between groups.When 5S rRNA was used as internal reference,the expression levels of two microRNAs(let-7c-5p and miR-223-3p)showed differences between groups.11.Box-plot was used to show the expression levels(ΔCq values)of four candidate identification markers in the control group,11 antemortem wound groups and three postmortem wound groups.When U6 snRNA was used as internal reference,three microRNAs(let-7c-5p,miR-26a-5p and miR-223-3p)could distinguish antemortem/postmortem wounds at specific time points(segments);When 5S rRNA was used as internal reference,three microRNAs(let-7c-5p,miR-223-3p and miR-497a-5p)could distinguish antemortem/postmortem wounds at specific time points(segments).Conclusion1.In this study,microRNA microarray data were obtained from normal mouse skin samples and incised skin samples of antemortem wound 1h/4h/1d and postmortem wound 1.5h.2.Inter-group differential expression analysis was performed for microarray data,and target gene prediction and function enrichment analysis were conducted for differentially expressed microRNAs.3.U6 snRNA and 5S rRNA were stably expressed in normal skin samples and incised skin samples of antemortem wound 0-240h and postmortem wound 0.5-3h,indicating they could be used as internal reference markers for quantitative microRNA detection.4.When U6 snRNA was used as internal reference,seven microRNAs(let-7C-5p,miR-19b-3p,miR-20a-5p,miR-26a-5p,miR-223-3p,miR-6360 and miR-7040-5p)were correlated with 0-240h skin wound time.When 5S rRNA was used as internal reference,six microRNAs(let-7c-5p,miR-19b-3p,miR-20a-5p,miR-199a-5p,miR-497a-5p and miR-7040-5p)were correlated with 0-240h skin wound time.5.In this study,the multivariate nonlinear regression models were established for skin wound time estimation using U6 snRNA and 5S rRNA as internal reference markers,and the goodness of fit was 0.999 and 0.976,respectively.6.When 5S rRNA was used as internal reference,the expression level of let-7C-5p was significantly differentiated between antemortem and postmortem wounds,which is expected to be used for the identification of antemortem/postmortem wounds of skin incisions.