The study was approved by the ethics committee of Huadong Hospital, affiliated with Fudan University (2017K001). All procedures conformed to the principles outlined in the Declaration of Helsinki. All patients provided written informed consent before enrollment in the study.
Data source and preprocessing
ExoRBase is a repository of human blood exosome-derived long RNA species, including circRNAs, lncRNAs and mRNAs. Following detection by normalized RNA-sequencing data analysis, exoRBase collected and integrated RNA expression profiles involving healthy individuals and patients with corresponding diseases. The RNA expression profiles of normal individuals (n=32) and patients with CHD (n=6) were downloaded from the exoRBase database followed by background correction and data normalization in R software (version 4.0.2).
Screening of differentially expressed genes, circRNAs and lncRNAs
The limma and sva packages were applied to the RNA-sequencing data analysis to remove batch effects and filter differentially expressed genes, circRNAs and lncRNAs by comparing the CHD group to the control group. Long RNA species with P-values of less than 0.05 were considered to be differentially expressed genes, circRNAs and lncRNAs. The volcano plot and heatmap were created by GraphPad Prism 8.0 and TBTools, respectively.
Prediction of potential miRNAs for differentially expressed genes, circRNAs and lncRNAs
ENCORI, the encyclopedia of RNA interactomes (http://starbase.sysu.edu.cn/), was applied to the prediction of potential miRNAs for differentially expressed genes and circRNAs. The predicted miRNAs of genes were further validated by the TargetScan and miRanda databases. Only the intersecting miRNAs were selected for subsequent analysis. The miRcode database (http://www.mircode.org/), which provides transcriptome-wide microRNA target prediction, was used to study lncRNA-miRNA interactions. LncRNAs/circRNAs and mRNAs with the same targeted miRNAs were selected to construct an interaction network, and the results were visualized with Cytoscape 3.8.1.
Functional and pathway enrichment analysis
The Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/home.jsp) v6.8 was applied to function and pathway enrichment analysis, including Gene Ontology (GO) terms in the molecular function (MF), biological process (BP) and cellular component (CC) categories for target genes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.
Patient sample collection
Patients with lower limb PAD (PAD Group) were recruited between August 2020 and November 2020. Patients with PAD all satisfied the diagnostic criteria, and they all had an intermittent claudication of <200 m, an ABI of <0.9 and an occluded superficial femoral artery of ³10 cm based on computed tomography angiography. Age-matched healthy individuals were recruited as the control group (without cardiovascular disease, diabetes or hyperlipidemia), and all participants had a normal ABI of approximately 1.2. The exclusion criteria included (1) history of tumor or connective tissue disease, (2) steroid use within 3 months, (3) recent infection, (4) failure to sign the informed consent form, and (5) history of CHD.
Blood samples were collected from both groups at 8:00 am after fasting for at least 8 hours. Fresh blood samples were subjected to centrifugation at a speed of 3000 g for 10 min at 4°C. The supernatant was subsequently transferred to another tube, and serum exosomes were isolated using a Hieff™ Quick exosome isolation kit (for Serum/Plasma, Yeasen Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. The morphological characteristics and size distribution of serum-derived exosomes were further assessed by transmission electron microscopy and nanoparticle tracking analysis as previously described.
Total RNA was extracted from serum exosomes with TRIzol reagent (Invitrogen, Carlsbad, Calif). Subsequent reverse transcription of circRNA was conducted with Random Primers N6 from Hifair® III 1st Strand cDNA Synthesis Kit (gDNA digester plus, Yeasen Biotech Co., Ltd.) following the instructions of the manufacturer. The comparison of the expression level of target circRNA was performed by RT-qPCR based on the ABI QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), with GAPDH as an internal reference. Corresponding primers were designed with circPrimer 1.2 (http://www.bioinf.com.cn/, Supplementary Table I).
Statistical analysis was performed with Student’s t-test or the c2 test as appropriate using SPSS (version 24.0). A P-value of less than 0.05 was considered to be significant.