To make the results more convincing, we obtained heart specimen from 3 cases of deceased donars (case 1 is a 64-year-old man with intracranial mass lesion, case 2 is a 69-year-old man with diabetes, and case 3 is a 81-year-old man with cardiac arrest) at the department of human anatomy in Nanjing medical University in 2018. Fresh heart samples were collected and every specimen was analyzed by at least two experienced cardiac anatomists. After good exposure of the hearts, the coronary arteries were isolated and each coronary artery specimen was separated into 10 segments according to human anatomy: the left main trunk (LM), the proximal segment of the left anterior descending artery (LAD-P), the midsegment of the left anterior descending artery (LAD-M), the distal segment of the left anterior descending artery (LAD-D), the proximal segment of the left circumflex artery (LCX-P), the midsegment of the left circumflex artery (LCX-M), the distal segment of the left circumflex artery (LCX-D), the proximal segment of the right coronary artery (RCA-P), the midsegment of the right coronary artery (RCA-M), the distal segment of the right coronary artery (RCA-D). Every artery segments of case 1 and case 2 was divided into two groups in half: protein group and pathological group, and the artery segments of case 3 were divided into RNA group and pathological group likewise. Segments in the protein group and the RNA group were snap-frozen in liquid nitrogen and stored at -80˚C for backup purposes, and the segments in the pathological groups were fixed in 10% formalin overnight for further histological analysis.
Clinical and pathologic analysis
The segments in pathologic groups from 3 samples were fixed in 10% formalin overnight. Decalcification using EDTA (ethylene diamine tetraacetic acid) decalcifying solution (Solarbio® LIFE SCIENCES, Beijing, China) was conducted for about half a months subsequently. H&E (haematoxylin and eosin) staining was performed in the standardized laboratory in the pathology department of the First Affiliated Hospital of Nanjing Medical University. All of the pathological sections were observed under the optical microscope (Leica DM2500 Wien, Austria) and digitalized by the matched image analysis system (Leica LAS, Wetzlar, Germany). The rest of the samples were stored in wax blocks. The pathological staging of the slides was classified based on international standard. Lesions were scored as no lesion (NL) stage, lipid strain (LS) stage, fibrous plaque (FP) stage, atheromatous plaque (AP) stage and secondary lesion (SL) stage(8).
Protein exaction and quantification
In this study, label-free quantification was conducted for proteomics analysis. All of the samples from case 1 and case 2 were ground in liquid nitrogen and transferred to 1.5ml centrifuge tubes perspectively. 400 ul of L3 lysis buffer, 1 mM PMSF, 2 mM EDTA, 10 mM DTT were added into the tube and were mixed with a pipette. Put the centrifuge tubes to ice bath to ultrasound for 15 minutes, followed by centrifugation for 20 minutes at 13000 g, 4℃. The supernatant was precipitated with four-times the volume of cold acetone and 30 mM DTT at -20℃ for 2 hours. Afterwards, 4℃, 13000 g centrifugation for 20 minutes was performed and the supernatant was discarded, and 1 ml of cold acetone with 10 mM DTT was added into the precipitate. After fully mashing and concussion, the precipitate was left at -20℃ for 30 minutes and centrifuged at 13000 g for 20 min. The supernatant was discarded and the precipitate was added with 200 ul Μ2 lysis buffer after air-drying. At last, put the centrifuge tubes to ice bath to ultrasound for 5 minutes and then centrifuge at 4℃, 13000 g for 20 minutes. Take the supernatant and determine the protein concentration using the Bradford method (Bradford 1976). The protein identification and quantification was conducted with ProteinPilot software (version 4.5, SCIEX, Redwood City, California, USA).
RNA isolation and sequencing
Extraction of total RNA in the prepared samples from case 3 was conducted with Trizol (15596018，Invitrogen) according to the manufacturer’s instructions. The quality control was performed and the RNA integrity was checked with Agilent Bioanalyzer 2100 (Agilent technologies, US). RNA purity and concentration were checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). A total of 3 ug RNA was extracted from per sample for the further experiments. The ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. The sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The first strand cDNA was synthesized with random hexamer primer and M-MuLV Reverse Transcriptase, and the second strand cDNA was synthesized with DNA polymerase I and RNase H. In the reaction buffer, dNTPs and dTTP were replaced by dUTP. The overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified and the cDNA fragments with 150~200 bp in length were selected with AMPure XP system (Beckman Coulter, Beverly, USA). 3 ul USER Enzyme (NEB, USA) was used to react with size-selected, adaptor-ligated cDNA at 37℃ for 15 min, followed by 5 min at 95℃. The PCR was performed subsequently and the products were purified on AMPure XP system. The library quality was assessed on the Agilent Bioanalyzer 2100 system(8).
All the identified proteins from case 1 and case 2 were divided into 5 groups according to the pathological grading. The relative quantification of the proteins in LS group, FP group, AP group and SL group was compared to that in NL group. The proteins with no expression were removed. Differencial expression was defined as fold change > 2 or fold change < 0.5 and the significantly differential expression proteins (SDEPs) were defined as proteins showed differencial expression in all of the 4 comparison groups. The identified RNAs in the RNA sequencing included mRNAs (messager RNAs), lncRNAs (long non-coding RNAs) and circRNAs (circular RNAs). The quantification of circRNAs was performed and TPM (Transcripts per million reads) normalization was conducted for further analysis. The normalized expression quantities of the circRNAs in LAD-P, LAD-M, LAD-D, LCX-P, LCX-M, LCX-D, RCA-P, RCA-M and RCA-D groups were compared to that in LM group respctively (the lesions were mainly located in the left main trunk in case 3). The significantly differential expression circRNAs (SDECs) were screened by the method above. The gene-protein interaction analysis was conducted using Targetscan Human 7.2 software. Gene ontology (GO) functional classification was performed by Blast2GO software (http://www.blast2go.de), and contigs were divided into biological processes, cellular components and molecular functions according to GO terms. The target binding sites prediction between mir-155 and circRNAs was performed using circinteractomedatabase (https://circinteractome.nia.nih.gov).