Application of circular RNA in forensic injury time inference

Abstract

The invention discloses application of circular RNA (Ribonucleic Acid) in forensic injury time inference, and belongs to the fields of genetic engineering technology and medicine. A circular RNA molecule Agl which does not exist in a circular RNA database is screened out for the first time through analysis of a tissue whole genome sequencing technology, and it is found that the expression quantity of the molecule is high, the expression quantity of the molecule is obviously increased along with prolonging of damage time within a certain period of time, and the molecule has significant correlation with the damage time; the damage time can be deduced according to the change of the content of the circular RNA in the damaged tissue, a reliable guarantee is provided for timely and accurately excluding or affirming suspicious persons, and the method has important forensic significance.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology and medicine, and in particular to the application of circular RNA in forensic injury time inference. Background Art

[0002] The inference of injury time is widely used in daily forensic work. Accurate inference of injury time is of great significance for delimiting the scope of investigation and identifying suspicious persons. Since the body is greatly affected by external environmental factors after injury, such as ambient temperature and humidity, there will be large errors in the inference of injury time, which brings certain difficulties to the accurate inference of injury time. Therefore, finding new detection indicators with high sensitivity, strong specificity and good stability is one of the problems that need to be solved urgently in the inference of injury time.

[0003] Circular RNA (circRNA) is a type of non-coding RNA molecule with a closed circular structure formed by covalent bonds. It does not have a 5' cap structure and a 3' polyA tail. RNaseR can recognize the free 3' end of linear RNA and degrade it. However, circRNA cannot be recognized by RNaseR due to its circular structure, so it has higher stability than traditional linear RNA. Recent studies have found that the types and contents of circular RNA in higher animals far exceed expectations. circRNA has many important regulatory functions. circRNA has conservative and tissue expression specificity in different species, and circRNA is insensitive to RNase, so it is more stable than linear RNA.

[0004] Whole transcriptome sequencing refers to the use of second-generation high-throughput sequencing technology to perform cDNA sequencing to quickly obtain sequence information of almost all transcripts of a specific organ or tissue of a species in a certain state. Whole transcriptome sequencing can simultaneously obtain three types of RNA information: mRNA, lncRNA, and circRNA. At present, there are few related research reports on the correlation between circRNA and the time of body injury at home and abroad. Therefore, obtaining new circRNA based on whole transcriptome sequencing analysis is of great significance for inferring the time of body injury in forensic medicine. Summary of the invention

[0005] The purpose of the present invention is to provide an application of circular RNA in forensic injury time inference to solve the problems existing in the above-mentioned prior art. The body injury time is inferred according to the change in the expression level of circular RNA Agl, which provides a reliable guarantee for timely and accurate exclusion or identification of suspicious persons, and has important forensic significance.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the application of circular RNA in forensic identification or inference of body injury time. The circular RNA is Agl, and the nucleotide sequence of Agl is shown in SEQ ID NO:1.

[0008] The present invention also provides the use of circular RNA in preparing molecular markers for forensic identification or inference of body injury time, wherein the circular RNA is Agl, and the nucleotide sequence of Agl is shown in SEQ ID NO:1.

[0009] The present invention also provides the use of a reagent for detecting the expression amount of circular RNA in preparing a kit for forensic identification or inferring the time of body damage, wherein the circular RNA is Agl, and the nucleotide sequence of Agl is shown in SEQ ID NO:1.

[0010] Optionally, 1-12 hours after the body is injured, the expression level of Agl is positively correlated with time.

[0011] The present invention also provides a method for forensic identification or inference of body injury time, comprising the step of determining the body injury time by detecting the expression of circular RNA in the body, wherein the circular RNA is Agl, and the nucleotide sequence of Agl is shown in SEQ ID NO: 1.

[0012] Optionally, 1-12 hours after the body is injured, the expression level of Agl is positively correlated with time.

[0013] The present invention discloses the following technical effects:

[0014] The present invention uses tissue whole genome sequencing technology to analyze a large number of circRNA molecules, and screens out a circRNA molecule Agl with a high expression level and an obvious increasing trend with the damage time.

[0015] The present invention discovered for the first time that there is a significant correlation between the circRNA molecule Agl and the injury time, providing a new method for forensic injury time inference research.

[0016] The present invention can infer the time of tissue damage according to the change of Agl expression in damaged tissue of the body at the crime scene, providing a reliable guarantee for timely and accurate exclusion or identification of suspicious persons, and has important forensic significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The histopathological test results of muscle tissue of mice in different groups at different injury times; A: control group mice; B: experimental group mice; C: control group under the microscope (HE staining, ×20); D: 1h under the microscope (HE staining, ×20); E: 2h under the microscope (HE staining, ×20); F: 12h under the microscope (HE staining, ×20); G: 24h under the microscope (HE staining, ×20); H: 3d under the microscope (HE staining, ×20); I: 7d under the microscope (HE staining, ×20);

[0019] Figure 2 Flowchart for whole transcriptome sequencing library construction;

[0020] Figure 3 is a heat map of differentially expressed genes;

[0021] Figure 4 is a scatter plot of differentially expressed genes;

[0022] Figure 5 The figure is a heat map of circRNA chromosome distribution; the length of the line indicates the relative size of the fold-change, red indicates upregulated circRNA, and blue indicates downregulated;

[0023] Figure 6 is the heat map of differentially expressed circRNAs;

[0024] Figure 7 The changes in Agl expression level with injury time. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Establishment of animal injury model

[0032] Twenty mice were divided into a control group and five groups with local thigh contusion at 2h, 6h, 12h, and 24h. Muscle tissues at the injured site were extracted and firstly subjected to histopathological examination to verify the success of the animal injury model (with injury-related histopathological changes) under a microscope.

[0033] The hair of the right lower limb and thigh of the mouse was cut and the skin was prepared. After anesthesia was performed by intraperitoneal injection of 10% chloral hydrate (0.35 ml / 100 g body weight), the quadriceps muscle of the right lower limb of the mouse was hit with a homemade hitter (weighing about 500 g). The skin at the hit site remained intact and there was no fracture. No treatment was given after the injury, and the mice were kept in separate cages and kept at room temperature.

[0034] The mice were killed by dislocating the neck at the time point of grouping, and the muscle tissue of the injured area was taken, and some fresh tissues were kept at -80℃ for freezing. The remaining tissues were fixed with 4% paraformaldehyde for 2 days, and then routinely dehydrated, transparent, wax-impregnated, embedded and sliced. The mice in the control group were not injured, and were killed by dislocating the cervical vertebra after 24 hours of routine feeding. The subsequent treatment was the same as the experimental group.

[0035] The results showed that the local tissue of the thigh injury in the skeletal muscle contusion group of mice was swollen, and bleeding and hematoma formation were observed. Under the microscope, a large number of red blood cells were observed at the injury site, and the tissue cell edema was obvious; a small amount of inflammatory cell infiltration was observed 2 hours after the injury, mainly neutrophils, and then the inflammatory cells gradually increased; a large number of inflammatory cells infiltrated from 24 hours to 2 days, mainly neutrophils; after 2 days, cellulose and fibroblasts increased, and inflammatory cells still existed in large numbers; on the 3rd day, fibroblasts and blood vessels increased significantly, inflammatory cells decreased, and granulation tissue was formed; on the 7th day, the number of fibroblasts began to increase, and a small number of inflammatory cells and a small number of new capillaries were seen, and scar tissue was formed, such as Figure 1 There were no abnormal changes such as bleeding and inflammatory cell infiltration in the skeletal muscle of mice in the control group.

[0036] 2. Whole genome sequencing of muscle tissue at different injury times

[0037] 2.1 RNA sample extraction

[0038] (1) From sample tissue, total RNA was extracted using Takara RNAiso Plus, and the extracted total RNA was sampled for quality inspection.

[0039] (2) RNA sample quality inspection method

[0040] a: Concentration detection A 260 / A 230 and A 260 / A 280 :The absorbance at different wavelengths of 260nm, 230nm and 280nm was measured by Nano Drop spectrophotometer to obtain A 260 / A 230 and A 260 / A 280 ratio.

[0041] b: 28S / 18S or 23S / 16S detection method: Agilent 2100 Bioanalyzer was used to detect the ratio of 28S / 18S or 23S / 16S by capillary electrophoresis.

[0042] c: RIN value detection method: Agilent 2100 Bioanalyzer was used to detect the RIN value through RNA 6000 Nano analysis.

[0043] (3) Library construction

[0044] After the sample passes the quality inspection, use GENESEED ®RNA-seq Library Prep Kit for Illumina constructs a library. First, the designed DNA probe is hybridized with the RNA sample to remove rRNA from the total RNA. The RNA is then fragmented to synthesize the first-chain cDNA. During the synthesis of the second-chain cDNA, dUTP is incorporated into it using a chain-specific method to label it. At the same time, end repair is completed in this step. Then, A-tailing, linker ligation, ligation product purification, fragment size sorting, and library amplification are performed. Before PCR amplification, the second-chain template with dUTP is digested with UDG enzyme. After the amplification, the RNA-Seq library is purified and recovered with magnetic beads to obtain the RNA-Seq library.

[0045] The build process is as follows:

[0046] The whole transcriptome sequencing library construction flow chart is shown in Figure 2 .

[0047] (4) Library quality control

[0048] After the library is constructed, Qu bit3.0 is used for preliminary quantification, and then the library size range is detected using Agilent 2100 Bioanalyzer. After the size of the inserted target fragment is in line with expectations, the effective concentration of the library is accurately quantified using the Q-PCR method (library effective concentration > 3nM) to ensure the quality of the library.

[0049] (5) Sequencing

[0050] After the library inspection is qualified, different libraries are pooled according to the effective concentration and the target data volume requirements, and sequenced using the NovaSeqX Plus PE150 mode, with a sequencing data volume of 10G.

[0051] 2.2 Bioinformatics methods

[0052] First, the Raw reads were filtered to remove the adapter sequences, sequences containing more N, and low-quality reads to obtain high-quality data (Clean reads). Then the Clean reads were mapped to the reference genome (MM10 genome released by the International Mouse Genome Sequencing Consortium as the reference genome), and the mapping results were used for the next step of circRNA identification.

[0053] DCC software was used to comprehensively identify circ RNA and obtain highly reliable circ RNA. The identified circRNA was further sequenced, expressed, and analyzed for differential expression. Significantly differentially expressed circRNA was used for subsequent functional mining.

[0054] 2.3 Data Analysis

[0055] Abnormal components are removed through analysis such as quality assessment, comparison result control, and gene coverage.

[0056] 2.4 Gene expression analysis

[0057] ① Use RSEM (https: / / github.com / deweylab / RSEM) software to compare and analyze the results and obtain gene expression.

[0058] ② Results of differential expression analysis among groups

[0059] Finally, 320 up-regulated genes and 186 down-regulated genes were obtained, see Figure 3 and Figure 4 .

[0060] The differentially expressed genes were analyzed using R software clusterProfiler.

[0061] Here, GO (http: / / www.geneontology.org / ) / KEGG enrichment analysis was performed on the differentially expressed genes.

[0062] 2.5 circRNA analysis

[0063] ① circRNA prediction and identification

[0064] The software DCC (https: / / github.com / dieterich-lab / DCC%60) was used to identify circRNA and predict the expression of circRNA. The differentially expressed circRNA was identified using the edgeR tool. In addition, the distribution of circRNA on chromosomes was displayed, which can further explore the function of circRNA to some extent. In addition, the present invention displays the distribution of circRNA on chromosomes (see Figure 5 ), to some extent, the function of circ RNA can be further explored.

[0065] ②Analysis of differential expression among groups

[0066] We filtered out circRNAs with low TMM expression (retaining circRNAs with CPM > 0.1 in at least half of the samples), and then used edgeR to perform differential expression analysis of circ RNAs based on the grouping information. The heat map of differentially expressed circRNAs is shown in Figure 6 .

[0067] The following circRNA molecule Agl with high expression and obvious decreasing trend with damage time was obtained, which was not recorded in the relevant database. The specific information of the molecule is shown in Table 1 below.

[0068] Table 1 Information of circular RNA molecules

[0069]

[0070] Verification Example

[0071] 1. Experimental methods

[0072] (1) Animal validation test: 45 healthy adult (9 weeks) male C57BL / 6J mice (provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were randomly divided into a normal control group and groups 1h, 2h, 6h, 12h, 24h, 2d, 3d, and 7d after injury, with 5 mice in each group. The animal injury model was made as described above, and muscle tissue at the injury site was extracted.

[0073] (2) Total RNA extraction and reverse transcription

[0074] Fresh contused muscle tissue was ground with a Fijet grinder, and the tissue lysis kit (Shanghai Fijet Biotechnology Co., Ltd.) was used to lyse the sample tissue to obtain total RNA, and the purity and concentration of total RNA were detected by Qubit fluorescence quantification meter (Life Technologies). 5X All-In-One RT MasterMix kit was configured with 20 μL of reverse transcription system, and the reaction conditions were: 42℃ for 15 minutes, 85℃ for 5 minutes to complete the reverse transcription.

[0075] (3) RT-qPCR

[0076] The selected primers of Agl gene were designed and synthesized by Guangzhou Jisai Biotechnology Co., Ltd. In the experiment, β-actin was used as the internal reference gene, and its information is shown in Table 2. Real-time fluorescence quantitative PCR was performed with the internal reference and the target gene.

[0077] Table 2 Primer sequences of Agl

[0078]

[0079] The sample was placed in the ABI PRISM 7500 HT instrument and qPCR was performed using the SYBR Green Pro Taq HS premixed qPCR kit. The total reaction system was 20 μL. The reaction conditions were 95°C for 30 seconds in the first step; 95°C for 5 seconds and 60°C for 30 seconds in the second step, for a total of 40 cycles. The fluorescence intensity was detected at 60°C for each cycle. After the reaction was completed, the computer software automatically analyzed the fluorescence signal and converted it into a Ct value. -ΔΔCt The relative expression level of the target gene was calculated by this method.

[0080] 2. Experimental results

[0081] (1) A large number of red blood cells were observed in the contused skeletal muscle of mice, and histiocytic edema was obvious; a small amount of inflammatory cell infiltration was observed 2 hours after injury, mainly neutrophils, and then the number of inflammatory cells gradually increased; a large number of inflammatory cells infiltrated from 24 hours to 2 days, mainly neutrophils; after 2 days, cellulose and fibroblasts increased, and inflammatory cells still existed in large numbers; on the third day, fibroblasts and blood vessels increased significantly, inflammatory cells decreased, and granulation tissue was formed; on the seventh day, the number of fibrocytes began to increase, a small number of inflammatory cells and a small number of new capillaries were seen, and scar tissue was formed. No abnormal changes such as bleeding and inflammatory cell infiltration were observed in the skeletal muscles of mice in the control group.

[0082] (3) Real-time fluorescence quantitative PCR test results

[0083] As shown in Table 3 and Figure 7 As shown in the figure, Agl gradually increased with the injury time 1 hour after skeletal muscle injury in mice, reached a peak at 12 hours, and then gradually decreased to normal at 24 hours. Compared with the normal control group, the expression of circular RNA molecule Agl changed significantly between 1 hour and 12 hours, with statistical significance, p<0.005.

[0084] Table 3

[0085]

[0086] Agl: Chr_Start_End_Strand is chr3_116776529_116782643_-, and the sequence is (SEQ ID NO: 1):

[0087]

[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of circular RNA in forensic injury time inference, characterized in that: The circular RNA is Agl, and the nucleotide sequence of Agl is shown in SEQ ID NO:

1.

2. The use of circular RNA in the preparation of molecular markers for forensic injury time estimation, characterized in that: The circular RNA is Agl, and the nucleotide sequence of Agl is shown in SEQ ID NO:

1.

3. Use of a reagent for detecting the expression of circular RNA in the preparation of a kit for forensic injury time estimation, characterized in that: The circular RNA is Agl, and the nucleotide sequence of Agl is shown in SEQ ID NO:

1.

4. The use according to any one of claims 1 to 3, characterized in that: 1-12 hours after the body is injured, the expression level of Agl is positively correlated with time.

5. A method for forensic identification or inference of the time of body injury, characterized in that: The method comprises the step of judging the time of body damage by detecting the expression amount of circular RNA in the body, wherein the circular RNA is Agl, and the nucleotide sequence of Agl is shown in SEQ ID NO:

1.

6. The method according to claim 5, characterized in that 1-12 hours after the body is injured, the expression level of Agl is positively correlated with time.

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