Human heart samples
For the purpose of this study, we collected four categories of human heart samples that were autopsied at the Department of Forensic Medicine, School of Basic Medical Sciences, Gannan Medical University over the period 2012-2019. A total of 34 cases of subjects who died for SCD were selected. These cases included 18 of AMI that presented none notable heart changes at autopsy and 16 of ASCVD with evidence of heart dysfunction. The causes of SCD were made after systemic autopsy. AMI was defined as sudden deaths without heart gross pathology, with or without mild coronary artery stenosis. ASCVD was defined as sudden deaths in clinic plus severe coronary artery stenosis at autopsy, often with gross or microscopic scarring surrounding myocardium. In addition, 14 cases of fatal injury and 14 cases of carbon monoxide (CO) intoxication were selected as the year-matched control cases. Patients who had pre-operative chemo- or radiotherapy history were excluded. Any case with heart implantation was also excluded. All of these cases undergone autopsy examination and were also excluded from influence of other potential substances such as alcohol, illicit drugs, or psychoactive drugs. Detailed information regarding the four categories of cases were documented in Table 1. The use of human heart samples for the purpose of research was approved by the Ethical Review Board at the School of Basic Medical Sciences, Gannan Medical University.
Histological evaluation
All histological evaluation was performed using the human heart samples dissected from the apex of each heart. Due to difficulty in obtaining fresh surgical heart samples, we used the autoptic FFPE samples for the purpose of this study. Unlike mRNAs or proteins that are prone to degradation and chemical modification, miRNAs are very small in size (~22nt), less likely to be degraded, and easier to recover from FFPE samples, all of which confer to miRNAs high forensic relevance[29]. The extraction of miRNAs from autoptic FFPE samples was commonly documented in publications[30]. After histological staining, independent pathologists were recruited to review the Hematoxylin & Eosin (H&E) staining sections and PicroSirius Red (PSR) staining sections using a multi-head microscope to result in consensus.
For the H&E staining, human heart tissues were immediately fixed in 10% neutral formaldehyde for 24 hours, followed by embedding in paraffin. Tissues were then sliced into 5 µm-thick heart slices. Then the slices were dewaxed and dehydrated in a gradient alcohol. Endogenous peroxidase activity was blocked using 3% H2O2 solution. The antigen retrieval was performed by steam heating in 10 mM citrate buffer (pH 6.0) for 10 minutes. Slices were then stained with hematoxylin solution for 10 min at room temperature and incubated in 1% hydrochloric-alcohol solution. Then, slices were washed using tap water, stained with eosin solution for 5 minutes, and separated by 75%, 85%, 90%, 95%, 100%, and 100% alcohol solutions for 2 minutes, respectively. Next, the slices were dried and sealed with neutral gums. After H&E staining, the morphological changes were observed and photographed under an optical microscope (Olympus, Tokyo, Japan).
For the PSR staining, human heart slices were deparaffinized to water and then immersed in 0.1% Sirius red staining solution dissolved in picric acid for 1 hour. Then slices were washed in acidified water containing 1.5% hydrogen chloride, dehydrated and mounted. Collagen and non-collagen components were red- and orange-stained, respectively.
Immunohistochemistry (IHC) staining
Heart slices from the four categories were also subject to IHC analysis. Briefly, the slides were incubated in 3% H2O2 solution for 10 minutes and antigen retrieval was performed by steam heating in 10 mM citrate buffer (pH 6.0) for 30 minutes. After epitope recovery, the slides were then treated with 10% of normal goat serum for 60 minutes, followed by incubation with active caspase 3 antibody (1:500, Cell Signaling Technology, Catlog #9664, MA, USA), CD31 antibody (1:1000, Cell Signaling Technology, Catlog #3528), and CD68 antibody (1:500, Cell Signaling Technology, Catlog #76437) incubation overnight at 4°C. The slides were washed and incubated with secondary horseradish peroxide (HRP)-linked secondary antibody (Vector Laboratories, MN, USA) at 1:500 dilutions for 1 hour. The samples were treated with the chromogen DAB for antigen detection and the final counterstaining was performed with hematoxylin.
RNA extraction and reverse Transcription
All paraffin was removed from the FFPE heart sections by treating with Deparaffinization Solution. The extraction of miRNAs from FFPE tissues was performed using a commercial miRNeasy FFPE kit (Qiagen, Catlog #217504, Hilden, Germany). Briefly, samples were incubated by heat treatment in an optimized lysis buffer, which contains proteinase K, to release RNAs from the sections. Supernatant was collected and treated with a DNase, followed by buffer RBC and ethanol treatments. The cleaned samples were then applied to an RNeasy MiniElute spin column, where the total RNAs including miRNAs, bound to the membrane and contaminants were efficiently washed away. Total RNAs including miRNAs were then eluted in a minimum of 14 μL of RNase-free water. Reverse transcription of the total RNA was performed using a Mir-X miRNA First-Strand Synthesis Kit (Takara, Tokyo, Japan) for RT-qPCR of miRNA. The synthesized cDNA was stored at -80 ℃ for later use.
Real-time quantitative polymerase chain reaction (RT-qPCR)
QuantiFast Multiplex RT-PCR Kit (Qiagen) was used for RT-qPCR analysis. According to the kit instructions, an aliquot of 25 μL reaction system was mixed and amplified using the Applied Biosystem 7500 fluorescence quantitative PCR instrument (Darmstadt, Germany). Primers used for this study were listed in Table 2. The 2-ΔCt method was used to calculate the relative expression of the target genes where ΔCtmiRNA=CtmiRNA−Cthousekeeping.
Statistical analysis
All data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using independent Student's t test. The diagnostic powers of miR-3113-5p, miR-223-3p, miR-133a-3p, and miR-499a-5p were evaluated by receiver operating characteristic (ROC) analysis. Areas under the curve (AUCs) were calculated. All statistical analyses were performed using GraphPad Prism 8.0 software (La Giolla, CA, USA). P < 0.05 was considered as statistically significant.