Coronary artery disease (CAD) is one of the most common heart diseases, characterized by the hardening and narrowing of arteries, resisting blood supply to cardiac muscle. Despite extensive research, the pathogenesis and therapeutic options for CAD remain limited. Epigenetic regulation plays a critical role in CAD progression. Here, we report a unique DNA methylation-miRNA-mRNA regulatory network for CAD, delineated through DNA methylation assays, miRNA and mRNA sequencing, bioinformatics analyses. We also identified key signaling pathways in this network, including the miR194 promoter-miR194-MAPK signaling pathway by pyrosequencing, methylation PCR, qRT-PCR. This pathway could play a role in CAD by apoptosis. Our findings suggested that this signaling pathway may be a potential therapeutic target for CAD. We believe that our study significantly contributes to an improved understanding of the role of specific miRNAs methylation, miRNA, and mRNAs in CAD pathogenesis.
Coronary artery disease (CAD) is one of the most common types of heart disease characterized by the hardening and narrowing of arteries, resisting blood supply to cardiac muscle. Despite extensive research, the pathogenesis and therapeutic options for CAD remain limited. Interestingly, recent research has highlighted the role of epigenetics in cardiovascular development and disease . Epigenetic regulation includes DNA methylation, histone modifications, microRNAs (miRNAs), long non-coding RNAs, and circular RNAs . Several studies have illustrated that miRNAs have great potential as cardiovascular biomarkers .
miRNAs are small endogenous non-coding RNA molecules composed of 19–22 nucleotides that affect various biological processes, such as development, differentiation, apoptosis, and cell proliferation, by regulating gene expression. Cardiac-specific miRNAs, including miR-1, miR-133, miR-208, and miR-30b, exhibit some critical diagnostic value [4,5,6,7]. DNA methylation refers to the addition of a methyl group on the fifth carbon atom of 5ʹ- CpG (cytosine-phosphate-guanine)-3ʹ through the action of DNA methyltransferase (DNMT). DNA methylation is involved in various pathophysiological activities, such as time- and space-specific gene expression, X-chromosome inactivity, aging, cancer, and cardiovascular disease [8, 9]. It is stable and can be influenced by environmental and genetic factors. DNA methylation is often localized to the promoter region, regulating gene expression . DNA hypermethylation on the promoter of the gene inhibits the gene expression, whereas hypomethylation activates it. A systematic review involving 32 DNA methylation studies on CAD  and a large-sample cohort with 13,356 individuals was conducted to ascertain the correlation of coronary heart disease events with DNA methylation . The same effect of DNA methylation may be observed in miRNAs. miR-223 can influence the development of atherosclerosis and ischemic stroke, with hypomethylation of the promoter being related to atherosclerotic cerebral infarctions . Therefore, we hypothesized that the epigenetic regulation of DNA methylation and miRNAs occurs together in the development of CAD, and that they may form a regulatory network involving transcriptional and epigenetic control of gene expression.
Here, we report the epigenetic regulation of DNA methylation and miRNAs that occur in CAD and may form a regulatory network involving transcriptional and epigenetic genes.
Data preprocessing and differentially methylated regions (DMRs) and differentially expressed genes (DEGs)
We collected 88 blood samples from CAD patients (n = 53) and healthy controls (n = 35). First, we randomly chose 5 blood samples in two groups for DNA methylation-Seq, miRNA-Seq, and RNA-Seq. We obtained massive data of CpGs and transcripts in patient peripheral blood with CAD and healthy controls. Each gene expression profile revealed 8710 and 8580 known and novel miRNAs, respectively, and 32,300 mRNAs were detected. PCA also revealed distinct expression signatures of miRNA and mRNA between CAD and healthy control (Fig. 1a, b). Following analyses, we obtained 295 DE miRNAs, of which 171 and 124 were downregulated and upregulated, respectively (Additional file 1 and 2). We also acquired 470 DEGs, of which 220 and 250 were downregulated and upregulated, respectively. The respective DEG heat maps and volcano plots are presented in Fig. 1c, d, e, and f.
Following quality control, we detected 2.02 million CpGs in each sample. Using the screening criterion (coverage ≥ 5; 2. P value ≤ 0.05), we obtained a total of 28,461 DMRs, of which 4498 and 23,963 were hypermethylated and hypomethylated, respectively. When DMRs were matched to the differentially expressed miRNA promoter, 64 miRNAs and the corresponding promoter sites in the DMRs were selected. Besides, 203 DEGs in RNA-Sequencing matched the potential targets of 64 miRNAs. The information on the control and CAD groups in sequencing is shown in Table 1. There are differences in the two groups, such as hypertension, LDL, TG, aspirin administration, statins administration, Gensini scores.
Immune-related molecular functions and biological processes of DE RNAs play a role in CAD
We identified CAD-related genes by performing functional and pathway enrichment analyses using DAVID 6.8 . We identified the gene ontology (GO) functions of significantly different downregulated genes, 20 biological processes, 15 cellular components, and five molecular functions. The top 25 GO terms are shown in Fig. 2a. For upregulated genes, 27 biological processes, 5 cellular components, and 5 molecular functions were identified. The top 25 GO terms are shown in Fig. 2b. The top GO enrichment terms in downregulated mRNAs were associated with antigen processing and presentation, immune response, and T cell costimulation. The top GO enrichment terms in upregulated mRNAs were related to nucleosome assembly, signal transduction, defense response to a virus, histone H3-K27 trimethylation. Meanwhile, biological processes related to immunity and inflammation also play a role in GO enrichment, such as antigen processing and presentation, immune response, and T cell costimulation, and so on.
Through the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment [15,16,17], we obtained 26 and 19 pathways that had upregulated and downregulated mRNAs, respectively. The tops KEGG pathways in downregulated mRNAs comprised B cell receptor signaling pathway, hematopoietic cell lineage, tuberculosis, HTLV-I infection, lysosome, phagosome, cell adhesion molecules, primary immunodeficiency, antigen processing and presentation, rheumatoid arthritis. The tops KEGG pathways in upregulated mRNAs comprised systemic lupus erythematosus, viral carcinogenesis, mitogen-activated protein kinase (MAPK) signaling pathway, hepatitis B, HTLV-1 infection, inflammatory bowel disease, rheumatoid arthritis, osteoclast differentiation (Fig. 2c, d). The main pathways were related to immunity, such as the systemic lupus erythematosus, MAPK signaling, and B cell receptor signaling pathways.
DMRs, DE miRNAs, and mRNAs formed gene–gene networks by mutual interaction
Methylation of miRNA promoters controls the miRNA production, influencing miRNA expression, consequently impacting the downstream mRNA expression. After obtaining the differentially expressed miRNA and mRNA, according to the regulatory relationship between genes, the UCSC database and Miranda miRNA target gene database was used (https://genome.ucsc.edu/), (https://omictools.com/miranda-tool/). The gene regulatory network was screened. If the DNA methylation miRNA and miRNA mRNA regulatory relationships obtained by the two methods are verified in the sequencing results, the gene pairs that are both potentially related and differentially expressed in the sequencing results are retained, that is, the gene pairs are differentially expressed and there is an intergenic regulatory relationship.
We used the Pearson correlation coefficients and starBase to construct the gene–gene interaction network between DMR, miRNA, and mRNA. The network included 32 DMRs (five hypomethylated and 27 hypermethylated regions), 32 miRNAs (similar to DMR, 13 downregulated and 19 upregulated miRNAs), and 180 mRNAs (60 downregulated and 120 upregulated miRNAs; Fig. 3). We inferred that 28 DMRs-regulated miRNAs and that this subsequently changed the target expression.
Hypomethylation of miR194 regulates cell death and inflammation pathways
Three miRNAs were selected which has close relation with DMRs using formed gene–gene network. Using qRT-PCR, we detected miR-194, miR-200a, and let-7b in a validation cohort in Tab. 2 (n = 78, CAD 48 blood samples and healthy control 30 blood samples). All three miRNAs were differentially expressed in CAD. Similarly, we measured RAS, MAPK1, FOS, and FAS using qRT-PCR in the validation cohort. We found that RAS, MAPK1, FOS, and FAS were differentially expressed in CAD. The expression patterns of these three miRNAs and four mRNAs in the validation cohort were similar to those in the sequencing cohort (Fig. 4a-g).
Pyrosequencing verified hypomethylation of miR194
Three DMRs corresponding to unique miRNAs were verified using pyrosequencing, including miR-194-cg64658184, miR-200a-cg1095596, and let-7b-cg46507614. First, we analyzed the methylation of these three miRNAs in CAD and healthy controls. miR-194-cg64658184 and miR-200a-cg1095596 were hypomethylated and upregulated in CAD, whereas let-7b-cg46507614 was hypermethylated and downregulated. Thereafter, we conducted pyrosequencing to identify 11 CpGs from these three DMRs. We obtained two CpGs, miR-194 -cg64660076, and miR-200a -cg1167297 (P = 0.002, 0.044) between CAD and healthy controls (Fig. 4h).
Methylation of miR-194 and miR-200a in oxidative HUVECs with Sequenom MassARRAY
The Sequenom MassARRAY system was used to detect the methylation of miR-194 and miR-200a in oxidative and normal HUVECs (Human Umbilical Vein Endothelial Cells). It was observed that miR-194 promoters were hypomethylated in oxidative HUVECs in pyrosequencing, which matched the results of patients with CAD by sequencing (Fig. 5a). The methylation levels of miR-200a were inconsistent, whereas miR200a-CpG7, CpG8, and CpG9 were hypomethylated, and miR200a-CpG3.4 and CpG5.6 hypermethylated. Nevertheless, the mean of miR-200a methylation appeared hypomethylated in pyrosequencing as sequencing in CAD. (Fig. 5b).
5aza and miR-194 inhibitor validated gene–gene interactions using qRT-PCR
In oxidative HUVECs, the level of miR-194 increased with qRT-PCR (P < 0.05). 5-Aza is a DNA-hypomethylating agent. When HUVECs were hypomethylated, the expression of miR-194 was enhanced (Fig. 5c). We then inhibited miR-194 in HUVECs, the levels of RAS, MAPK1, and FAS were downregulated in oxidative HUVECs. When treated with miR194 inhibitor, these three mRNAs were upregulated compared with oxidative HUVECs (P < 0.05; Fig. 5d-g).
Pearson correlation between CpG island methylation and clinical characteristics of the study population
We performed the Pearson correlation analyses between miR194 promoter methylation, miR194 and clinical characteristics of the study population (n = 10) in Table 3. The result indicated miR194 promoter methylation was significantly associated with Gensini scores (r = − 0.772, p = 0.009), and miR194 was significantly related to LDL-C (r = − 0.716, p = 0.02), HDL-C (r = 0.667, p = 0.035), and Gensini scores (r = 0.834, p = 0.003).
Pearson correlation between miR194 promoter methylation and miR194. Data are summarized for binary variables. SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglyceride; FBG, fasting blood glucose.
Using an AnnexinV/propidium iodide (PI) apoptosis assay, we detected the level of damaged early and late apoptotic and normal endothelial cells with oxidative damage (Fig. 6a-d), indicating that H2O2 treatment and miR194 inhibitor induction triggered apoptotic cell death of HUVECs. However, miR194 inhibitor didn’t decrease apoptosis rate.
Using PI cell cycle detection, H2O2 intervention can inhibit the proliferation of HUVECs. The S- and G2 phases of cell proliferation were reduced, with the S-phase at 8.03% and the G2 phase at 12.02% (Fig. 7a–d). The proportion of G0-G1 cells in the proliferation resting phase increased to 79.95%. Transfection with a miR194 inhibitor improved the cell cycle inhibition, and the S-phase increased to 12.72%.
Despite extensive research on CAD-related methylation, DNA methylation and the associated miRNA-mRNA CAD regulation remain poorly understood. We screened 88 subjects for DNA methylation sites, miRNAs, and mRNAs in the CAD and normal control groups. High-throughput sequencing was used to randomly select five cases from each group to detect the level of DNA methylation, miRNA, and mRNA expression in peripheral blood mononuclear cells and to screen differential genes between the CAD and control groups. Correlation analysis was performed to construct a DNA methylation-miRNA-mRNA gene regulatory network. We screened and identified a total of 28,461, 295, and 470 differentially expressed methylation sites, miRNAs, and mRNAs, respectively, in CAD.
Functional and signal pathway analyses of differential genes revealed that immunity and inflammation-related biological processes played a role in GO enrichment. Using the University of California Santa Cruz (UCSC) database, MiRanda, and gene correlation analyses, we demonstrated that the CAD-related DNA methylation-miRNA-mRNA regulatory network involved 46 DNA methylation sites, 45 miRNAs, and 109 mRNAs. In vitro experiments demonstrated that oxidative damage altered the cell cycle and promoted apoptosis. Interestingly, miR-194 transfection inhibited apoptosis, restored the cell cycle, and changed the gene expression in the MAPK signaling pathway. Demethylation altered miR194 expression (Fig. 8).
Some studies have investigated methylation in patients with Myocardial infarction (MI). A genomic DNA methylation (DNAm) profile of DNA isolated from whole blood was obtained by analysis with Infinium HumanMethylation450 BeadChip. Three DNAm loci were shown to be significantly correlated between the whole genome and MI . In 2018, the Infinium HumanMethylation450 assay was used to examine the genome-wide DNA methylation profiles in three pairs of samples from patients with acute coronary syndrome and control samples. Methylation-specific polymerase chain reaction (MSP) was also used to validate Sequenom MassARRAY analysis in ACS (acute coronary syndrome), stable coronary artery disease, and control samples. A total of 11,342 differentially methylated CpG sites were identified. Compared with the control sample, the ACS group included 8,865 hypomethylated and 2,477 hypermethylated CpG sites. MSP analysis and Sequenom MassARRAY analysis from 81 ACS samples, 74 stable coronary artery disease samples, and 53 healthy samples confirmed that the reference results of the HumanMethylation450 array significantly corrected the differential CpG methylation of SMAD3. These data identified ACS-specific DNA methylation profiles with several novel DM CpG sites, some of which could be candidate markers for early diagnosis of ACS . Other studies have screened and verified the genome in the gene bank and analyzed methylation and related targets to determine the correlation between DNA methylation and gene expression [20,21,22,23]. However, DNA methylation changes and validation of ex vivo cells have not been reported in CAD patient screening to validation.
In an in vitro experiment, we showed that miR-194 transfection inhibited apoptosis, restored the normal cell cycle, and changed gene expression in the MAPK signaling pathway. In patients with CAD, the MAPK signaling pathway changed accordingly. The inhibition of miR-194 significantly increased the expression of the related pathways, indicating that miR-194 may be an important inhibitor of the MAPK signaling pathway. miR-194 is a tumor suppressor similar to p53 as a typical p53-responsive miRNA. p53 plays a role in the aging process, and miR-194 may significantly promote the development of cellular senescence . It has been suggested that miR-194-5p is closely related to hypothermia oxygen–glucose deprivation, which is an essential process in ischemic diseases such as CAD . Alongside a middle cerebral artery occlusion Sprague–Dawley rat model, mir-194–1 was downregulated compared with the sham group (0.20 and 0.99) . However, MI downregulates plasma levels of miR194-5p in obese and non-obese animals, associated with cardiac function, mitochondrial lipids, and myocardial fibrosis . The expression in MI was 0.60 relative to 1.0 in the control group. In addition, MAPK signal pathway such as p38 and c-jun N-terminal kinase (JNK) which cause inflammation and apoptotic cell death and ERK 1/2 which regulates myocyte differentiation and proliferation, promotes cell survival, and confers tissue protection .
The present study has some limitations. First, our sample size for the validation was small. A larger cohort is required to verify the entire network. Although we conducted gene sequencing and validation, all our samples were from the same Hospital. Therefore, it is unclear whether influence would be observed in patients from different areas and races. The validity of the data should be verified in more cohorts. DNA methylation was associated with CAD. We found that a network could connect DNA methylation-miRNA-mRNA. The level of methylation of mRNA could change directly, but not through miRNAs. The detailed mechanism need exploring in future.
In conclusion, we identified differential gene expression profiles of DNA methylation sites, miRNAs, and mRNAs were found in CAD (28,461 methylation sites, 295 miRNAs, and 470 mRNAs). Gene function involved immunity and the inflammatory response pathway. We characterized a specific DNA methylation-miRNA-mRNA regulatory network for CAD and screened the key signaling pathways of the miR194 promoter-miR194-MAPK signaling pathway. Through in vitro experiments, we demonstrated the relationship between the miR194 promoter methylation and miR194-MAPK regulation. Our findings suggest that the miR194 promoter and miR194-MAPK signaling pathway may be related to CAD and may be a potential therapeutic target.
Materials and methods
Participant recruitment and sample collection
Our study consisted of 35 patients with CAD and 35 controls from the Guang Anmen Hospital, Beijing, China. The inclusion criteria are as follows: (i) the coronary angiography was to estimate the extent of CAD for all subjects according to the criteria defined by the American Heart Association. At least one major epicardial vessel with > 50% stenosis was defined as CAD, whereas subjects with < 50% stenosis were defined as controls [29, 30] The age is between 45 and 75 y; (ii) exclusion criteria were index event due to uncontrolled hypertension and/or blood pressure remaining ≥ 180/110 mm Hg despite treatment, New York Heart Association class III or IV congestive heart failure irrespective of ejection fraction, or New York Heart Association class II heart failure with left ventricular ejection fraction ≤ 40%, persisting at the end of the run-in period despite treatment, severe valvular heart disease, arrhythmia, cardiomyopathy, and stable angina pectoris, clinically apparent liver, kidney, hematological system, nervous system and mental disease, malignancy within the preceding 3 y, inability to provide informed consent, or comply with study requirements .
Risk factors such as smoking history, hypertension, and diabetes: A standardized questionnaire was applied to assess smoking history, hypertension, and diabetes in this study subjects. Smoking history was classified as either “never smoking” or “smoking” (including both former and current smokers). Hypertension was classified as either “non-hypertension” or “hypertension” (including diagnosed as hypertension before and SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg currently). Diabetes was classified as either “non-diabetes” or “diabetes” (including diagnosed as diabetes before and fasting blood glucose ≥ 7.0 mmol/L or postprandial blood glucose ≥ 11.1 mmol/ L currently) . The severity of CAD was evaluated by Gensini score 
The study protocol was approved by the institutional ethics committee of Guang Anmen Hospital, Beijing. The patients volunteered for the research with written consent. All patients signed an informed consent approved by the Institutional Review Board. The patients’ blood samples on an empty stomach were drawn in the morning within 24 h after admission. The blood of the outpatients and the controls were drawn on the morning of the second day after admission. 4 ml venous blood was collected and placed in an EDTA tube. Peripheral blood mononuclear cells (PBNCs) were centrifuged within 6 h.
DNA and RNA extraction from PBNCs
DNA extraction from PBNCs: 300 μl blood sample was put into a centrifuge tube. We added red blood cell lysate and placed it at room temperature for 5 min. During this period, the mixture was reversed several times and centrifuged. The supernatant was aspirated and discarded. We added 200 μl buffer solution and shook it until completely mixed. 4 μl RNase A solution was added, oscillated for 15 s, and placed at room temperature for 5 min to remove RNA. Then we added 20 μl Proteinase K solution, 200 μl buffer solution GB, 200 μl absolute ethanol. We added the previously mixed liquid into the adsorption column CB3, centrifuged, and poured out the waste liquid. We added 500 μl buffer solution GD to the adsorption column CB3, fully shook for 15 s. Then we added 600 μl rinsing solution PW into the adsorption column CB3, centrifuged, and repeat the operation once. We centrifuged it for 2 min, opened the cover, and put it in the centrifuge tube. Placed it at room temperature for 10 min. Dropped 60 μl ddH2O into the middle of the adsorption membrane and place it at room temperature for 5 min. After centrifugation, the solution was added to the adsorption column CB3, placed at room temperature for 2 min, and centrifuged to obtain the extracted DNA solution.
Total RNA extraction from PBNCs: The EDTA tube was inverted and evenly mixed. We placed it in a 15 ml tube and added 3 times the volume of red blood cell lysate, thoroughly mixed it. We placed it at room temperature for 5 min, during which, mixed it several times to ensure complete reaction of reagents. Then we centrifugated it and suction the supernatant. We added red blood cell lysate to 4 ml, thoroughly mixed, and placed it at room temperature for 5 min. After centrifugation, the supernatant was carefully removed, and the white precipitate was retained as PBNCs. 1 ml Trizol was added and mixed with PBNCs, and then transferred to 2 ml centrifuge tube and stored in—80 ℃ refrigerator.
DNA methylation high-throughput sequencing
We used the Roche Seqcap EPI methylation enrichment kit, following the manufacturer’s instructions . We sequenced the captured bisulfite transformed DNA samples with Illumina hiseq2500. The sequence read lengths were 2 × 150 bp. For data processing, the original data was evaluated by fastqc (FastQC http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/version0.10.1); clean data were compared to the reference genome using the bsmap software (https://code.google.com/archive/p/bsmap/version2.90). Sam files were sorted according to the chromosomes and loci using the Picard software (http://broadinstitute.github.io/picard/version1.140). Duplicates were marked and removed using the Picard software. The bamtools software (https://github.com/pezmaster31/bamtools/version2.4.0) was used to filter and remove reads that could not be compared. The Bamutil software (https://github.com/statgen/bamUtil/version1.0.14) removed the overlapped area in the middle of the paired reads. Following these steps, we statistically analyzed the optimal comparisons. Based on the methylation detection results of bsmap, the frequency of the methylated C and the unmethylated C at each site was tested using two distribution tests to identify whether the site was a real methylated site. According to the two conditions of sequencing depth ≥ 5 and P ≤ 0.05, the C locus was considered the methylation site. The test results were statistically analyzed. The DMR between the samples was identified using the swDMR software.
miRNA high-throughput sequencing
We used the NEBNext Small RNA Library Prep Set for Illumina kit (New England Biolabs) to process and build a small RNA library, according to the manufacturer’s instructions . We used the Illumina Hiseq2500 for single-terminal sequencing. We processed data and used FastQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/version0.10.1) to detect the original sequence. We compared our BLAST results with the RFam database to annotate the miRNA sequence. Clean reads were compared with human precursor/mature miRNA in miRBase (http://www.mirbase.org/version20.0) to screen for known miRNAs. We compared the uncommented sequence with the human genome to analyze its expression distribution. We used Mireap (http://mireap.sourceforge.net/version0.2) to predict potential new miRNAs. The number of sequences of each known miRNA was standardized according to the total number of sequences paired into the miRBase 20.0 database. The number of sequences per million paired sequences (reads per million mapped reads, RPM) was used as the expression amount. FDR correction was applied to p value to obtain Q value as follows: The screening criteria for differential miRNA were as follows: | log2 (fold change) |≥ 1, P value < 0.05.
mRNA high-throughput sequencing
According to the manufacturer’s instructions, we used the Ribo-Zero Magnetic Gold Kit (Illumina, San Diego, CA, USA) and the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) to construct the full transcriptome library. We used the Bioanalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA, USA) for quality inspection of the library. We sequenced using the Illumina Hiseq2000/2500 and produced read lengths of 2 × 100/150 bp. The original data was evaluated using fastqc (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/version0.10.1), and compared with the reference genome by the TopHat 2.0 program (http://tophat.cbcb.umd.edu/version2.0.10). After the transcripts of each sample were assembled separately, the transcripts of all samples were summarized and merged using the command of cuffmerge. Then, the ensembl transcript database was used as the annotation reference for mRNA, and the number of sequences in each transcript was standardized according to the total length and samples. The number of sequences in every 1 million pairs to every 1000 bases in exon was used as the expression amount. FDR correction was applied to p value to obtain Q value as follows: The screening criteria for differential mRNA were as follows: P value < 0.05 and Q value ≤ 0.05.
Construction of a regulatory network among genes
After obtaining the differential expression of DNA methylation, miRNA, and mRNA among the groups according to the regulatory relationships between genes, the gene regulatory network was screened using the UCSC database (https://genome.ucsc.edu/) and the MiRanda miRNA target gene database (http://www.microrna.org/microrna/version3.3a). Then, we chose miRNAs as follows: | log2 (fold change) |≥ 1, P value < 0.05 and Q value ≤ 0.05. The criteria for differential mRNA were as follows: P value < 0.05 and Q value ≤ 0.05.
Verification of the methylation level of related sites using pyrosequencing
The reaction system was set up for bisulfite transformation: the transformation was carried out on a 9700 type PCR instrument (with a hot cover), and the DNA was frozen and preserved after treatment. We used the pyromark PCR kit as follows: First, the single strain PCR products were purified, and the primers were annealed. PCR products were retained for pyrosequencing on the pyromark ID instrument. The assay and run were established in the pyromark CpG software. We prepared the related items and sequences. Sequencing results were analyzed.
Detection of the methylation level of miR-194 and miR-200 promoter using sequencing
The genomic DNA of tissue or peripheral blood was extracted using the phenol–chloroform method. The DNA concentration, OD 260, OD 280, and OD 260/280 were measured using a Nanodrop 1000 spectrophotometer. The DNA sample OD 260/280 used in this experiment was between 1.8–1.9, regarded as qualified DNA. The DNA samples were treated with bisulfite, amplified by PCR, and reacted with SAP. The 384 pore plates were placed into the sample adding instrument. After the parameter setting is completed, the sample was spotted on the chip. We then entered information about the target sequence in the epicity software. After sampling, the chip was placed in the mass spectrometer, and methylation analysis was performed according to the molecular weight difference of C and T bases in the fragment. The methylation ratio of MS was obtained from Epityper software version 1.0 (https://www.cd-genomics.com/EpiTYPER-DNA-Methylation-Analysis.html Sequenom, San Diego, CA) (Additional files 1, 2).
The cells were collected and treated 72 h later. Total RNA was extracted by the Trizol method and then reverse transcribed into cDNA for PCR. PCR reaction system: cDNA (diluted 10 times) 4 μL, SYBR Green 5 μL, primer 0.2 μL + primer (10 μmol/L), 0.2 μL each, water 0.6. μL, total 10 μL of PCR conditions: 95 °C for 5 min; 95 °C for 10 s, 60 °C for 20 s, a total of 50 cycles; 5 °C for 10 s, 60 °C for 10 s, and 40 °C for 30 s. The CT values of the target and control genes were automatically collected by the quantitative fluorescence analyzer, and the relative mRNA expression was calculated using the 2−ΔΔCT.
Cell culture and grouping
HUVECs were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/L streptomycin. The medium was cultured in a 5% CO2 incubator at 37 °C and changed daily. The third to eighth-generation cells were used in all experiments. HUVECs grew to about 80% fusion degree. After centrifugation, a 1.5 mL DMEM medium (4 °C precooling) containing 10% DMSO was added to the cell precipitate. After blowing and mixing, the cell suspension was transferred to a 2 mL cryopreservation tube and stored at—80 °C.
HUVECs were divided into four groups: blank, H2O2 model, 5-aza, and transfection groups. The H2O2 model group was treated with 100 μL of H2O2 for 3 h. The 5-aza group was treated with 50 μM 5-aza for 24 h. Cell samples were collected 24 h after treatment with H2O2 for 3 h.
The cells were transfected in an 80% fusion state. The Lipofectamine RNAiMAX liposomes were mixed in the proportion of 6 μL:100 μL in the Opti-MEM solution; the siRNA was mixed in the proportion of 2 μL:100 μL in the Opti-MEM solution; the siRNA was mixed in the proportion of 1:1 in the Lipofectamine RNAiMAX liposome mixture. The mixture was stored at room temperature for 5 min, added to the cells, placed in the incubator for 48 h, and collected.
Annexin V/PI determination of apoptosis
The HUVECs were divided into groups of 11. Forty-eight hours after infection with the lentiviral vector control and lentiviral-Bmi1-shRNA and 24 h after treatment with cisplatin, cells were centrifuged for 5 min. We added 100 μL of binding buffer to cells (1 × 106) in 1.5 mL centrifuge tubes. Annexin V was incubated at 5 μL, following which, we added 3 μL PI. Apoptosis was analyzed using flow cytometry with 400 μL binding buffer.
We used SPSS19.0 for all statistical analyses. Data were expressed as mean ± standard deviation. We used independent sample t-tests to analyze the normal distribution between groups. We used the rank-sum test to analyze the non-normal distribution. For multiple comparisons, the quantitative data of normal distributions were analyzed by variance analysis and the Q test. The quantitative data of non-normal distribution were analyzed by nonparametric tests.
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Thank you, Dr. Wang Li from Beijing Children's Hospital for your technical guidance in MSP, Xin Qi from the pathology department of Guang'anmen Hospital for your guidance in AV/PI, and the Ouyi company for your technical guidance in pyrosequencing, Zhaoling Li from Guang'anmen Hospital for your help in revising the manuscript. The authors apologize to colleagues whose work was not cited due to space limitations or our oversight.
This work was supported by the National Natural Science Foundation of China (No. 81473561 and No. 81904185) and Excellent young scientific and technological talents of China Academy of Chinese Medicine Sciences (No. ZZ14-YQ-015).
Lian Duan, Yongmei Liu, Jun Li: Contributed equally to this work.
Authors and Affiliations
Department of Cardiology, Guang Anmen Hospital, No. 5 Beixiange, Xicheng District, Beijing, China
Lian Duan, Yongmei Liu, Jun Li, Yun Zhang, Yan Dong & Wang Jie
Department of Cardiology, China-Japan Friendship Hospital, No. 2 Yinghuayuan East Street, Chaoyang District, Beijing, China
LD, YML, and JW contributed to the conception of the study. The research protocol was drafted by LD, YML, and JW. The manuscript was revised by JL. The samples were collected by LD, YML and JQL. The in vitro experiments were conducted by LD, and YD. JW and LD analyzed the results and performed experimental quality checks. LD revised the manuscript with reviewers’ comments. All authors have read and agreed to the publication of this version of the manuscript.
The study protocol was approved by the institutional ethics committee of Guang Anmen Hospital, Beijing. All patients were volunteers and provided signed, informed consent prior to participation in the study. All methods were performed in accordance with the relevant guidelines and regulations.
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