All testing programs were performed according to protocols approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University. All sample providers were informed of their blood samples. Clinical data were used for this study, and informed consents were signed.
Sample collection
Total of 50 MI patients’ peripheral blood samples were collected before percutaneous coronary intervention surgery between March 2018 and January 2019 in the affiliated hospital of Qingdao University (Qingdao, China). All patients were not treated with heparin, radiotherapy, or chemotherapy. The matched healthy controls were collected from the volunteers of the physical examination center. Fresh peripheral blood samples (5 ml) form MI patients and healthy individuals were collected in EDTA tubes.
Total RNA extraction
The samples were used for RNA extraction via the Trizol method (Trizol-up reagents, TransGen, China). This experiment was performed according to the kit's recommendations. RNA samples were stored at −80°C.
RNA quantification and qualification
The purity of the extracted RNA was tested by a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The RNA concentration was detected by Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was measured by the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA degradation and contamination were monitored on 1% agarose gels.
Library preparation and circRNA sequencing
A total of 5 μg RNA per sample was used for the RNA library preparations. Firstly, rRNA was removed by Epicentre Ribozero™ rRNA Removal Kit (Epicentre, USA) and ethanol precipitation. Subsequently, the linear RNA was digested with RNaseR (Epicentre, USA). The sequencing libraries were produced by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA).
The cluster generation of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS. After that, the libraries were sequenced on an Illumina Hiseq 4000 platform. These experiments were performed following the manufacturer’s instruction and 150 bp paired-end reads were generated.
Data analysis and circRNA identification
Firstly, clean data were acquired by wiping off reads containing adapter and low-quality reads from raw data. The clean data with high quality were used for downstream analyses. Index of the reference genome was created using Bowtie2 v2.2.8 and paired-end clean reads were united to the reference genome by using Bowtie[19]. The circRNA was detected and verified by using find_circ[20] and CIRI2[21]. Circos software was used to construct the circos figure.
Divergent PCR
The junction site part of circRNAs was confirmed by PCR with divergent primers. Convergent primers were invoked as the control. The products of the PCR amplification were authenticated by 1% agarose gel electrophoresis. The primers for circRNA validation were listed in supplementary data1 table 1.
Variables, mean ± SD or n
|
ctrl, n = 50
|
MI,n=50
|
p-value
|
Age (years)
|
60.4±7.824
|
63.7±9.772
|
0.105
|
Gender (M/F)
|
27/23
|
30/20
|
0.545
|
high(cm)
|
166.71±8.564
|
164.86±9.969
|
0.394
|
weight(kg)
|
69.89±18.005
|
68.97±11.58
|
0.795
|
BMI
|
24.93±4.696
|
25.4±3.849
|
0.64
|
Systolic pressure
|
128.25±16.505
|
129.06±22.976
|
0.865
|
Diastolic pressure
|
72.64±8.735
|
76.06±14.695
|
0.236
|
Diabetes (yes/no)
|
4/46
|
10/40
|
0.084
|
Smoking history (yes/no)
|
16/34
|
24/26
|
0.102
|
Alcohol intake history (yes/no)
|
10/40
|
20/30
|
0.029
|
Family history of CHD (yes/no)
|
7/43
|
18/32
|
0.011
|
Other underlying diseases (yes/no)
|
12/38
|
15/35
|
0.499
|
Pharmacological therapy (yes/no)
|
11/39
|
16/34
|
0.26
|
TC (mmol/l)
|
4.92±1.142
|
4.21±1.063
|
0.104
|
LDL-C (mmol/l)
|
2.88±0.937
|
2.55±0.589
|
0.371
|
HDL-C (mmol/l)
|
1.35±0.378
|
0.99±1.82
|
0.003
|
Table 1
Detailed information of MI patient and control blood samples
Real time-quantitative PCR
Dysregulation circRNA reaffirmed was completed on a CFX96 Real-Time PCR Detection System (Bio-Rad). Total RNA was extracted using Trizol reagent. Reverse transcription reactions were performed by using the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, China) to make cDNA according to the manufacturer’s guide. TransStart Green qPCR SuperMix (TransGen) was used for Quantitative PCR (qPCR) analysis, the procedures were by the kit instructions. Levels of dysregulation circRNA analyzed by RT-qPCR were normalized to that of GAPDH[22]. The primers for RT-qPCR were listed in supplementary data1 table 2.
circ_ID
|
chr
|
full_ length
|
splice_ length
|
gene_ name
|
MI_ readcount
|
Control_ readcount
|
hsa_circ_0000826
|
chr18
|
39618
|
994
|
ANKRD12
|
816.599493
|
79.5731602
|
hsa_circ_0000896
|
chr19
|
506
|
338
|
FARSA
|
446.180.0157
|
72.0326233
|
hsa_circ_0000116
|
chr1
|
3460
|
353
|
MAN1A2
|
1933.94798
|
167.154168
|
hsa_circ_0000994
|
chr2
|
1832
|
1832
|
SLC8A1
|
313.178477
|
0
|
hsa_circ_0001445
|
chr4
|
464
|
269
|
SMARCA5
|
19590.5155
|
2907.27544
|
hsa_circ_0001414
|
chr4
|
6372
|
585
|
TMEM165
|
720.396765
|
102.724399
|
hsa_circ_0030720
|
chr13
|
6198
|
358
|
UBAC2
|
328.578223
|
24.0160633
|
hsa_circ_0001062
|
chr2
|
181
|
181
|
ZC3H6
|
516.545138
|
118.380163
|
hsa_circ_0000615
|
chr15
|
874
|
874
|
ZNF609
|
509.23034
|
0
|
Table 2
The general information of circRNA
Creation ROC curves
ROC curves were created to estimate the diagnostic value of circRNAs for MI. A ROC curve was calculated and the specificity and sensitivity of predictive power were measured by the area under the curve (AUC), AUC was used to assess the diagnostic value of the circRNAs in the peripheral blood of MI patients[23]. SPSS24.0 and Graphpad prism7 were used for ROC curve calculating and drawing.
GO and KEGG enrichment analysis
Gene Ontology (GO) enrichment analysis for host genes of differentially expressed circRNAs were carried out by the GO seq R package, gene length bias was corrected during this procedure [24]. GO terms with a corrected P value less than 0.05 were considered to be significantly enriched by differential expressed genes. As a database resource for understanding high-level functions and utilities of the biological system[25], KEGG is used to explore information from molecular-level (such as the cell, the organism, and the ecosystem). Especially largescale molecular datasets generated by genome sequencing and other high throughput experimental technologies (http://www.genome.jp/kegg/). We used KOBAS software to test the statistical enrichment of differential expression genes or circRNA host genes in KEGG pathways[26].
Prediction the network of circRNAs-miRNAs-mRNAs
circRNA can regulate the translation of mRNA via binding to a miRNA[10]. MiRNA target site in exons of circRNA loci was verified by using miRanda. The binding sites of miRNA and mRNA were predicted by RNAhybrid. Cytoscape was used to construct the circRNA-miRNA- mRNA networks.
Cell culture and treatment
AC16 cells were cultured in DMEM (Gibco) with 10% fetal bovine serum (TransGen), and 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen) in a humidified 5% CO2 incubator at 37°C. When the cultured cells reached approximately 70% confluently, they were treated with H2O2 (100μM) incubated at 37°C for 12 hours in the complete culture medium. The cells were collected for RNA extraction and dysregulated circRNA reconfirmation.
Transfection
We used Lipofectamine 3000 (Thermo Fisher) for transfection cirSLC8A1 siRNA (BGI) and overexpression vector (GENE Chem). The procedures were in accordance with the kit instructions.
Mitochondrial staining and analysis of mitochondrial fission
Cells were plated onto the poly-L-lysine coated coverslips. After treatment, they were stained for 30 minutes with 0.02 µM MitoTracker Red at 37°C. Mitochondria were imaged using a laser-scanning confocal microscope (Zeiss LSM510 META).
Apoptosis assays
Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) using a kit from TransGen. The detection procedures were in accordance with the kit instructions.
Immunoblotting
Cells were lysed for 20 minutes on ice in RIPA lysis buffer containing a protease inhibitor cocktail and DMSF. The samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with primary antibodies anti-Caspase3 (Abcom, 1:1000), anti-Cyto c (Abclone, 1:1000) anti-Tubulin (Affinity, 1:1000) anti-β-actin (TransGen, 1:2000) at 4°C overnight with gently shaking. After three times washing with PBS, the horseradish peroxidase (HRP)-conjugated secondary antibodies were added. Antigen antibody complexes were visualized by enhanced chemiluminescence. Enhanced ECL TM prime detection reagent (GE) used to visualize antigen-antibody complexes, the density quantified by Image J.
Statistical analysis
The results are expressed as the mean ± SEM of at least three independent experiments. The statistical comparison between different groups was completed by one-way analysis of variance (ANOVA) for multiple comparisons or t‐test with Welch's correction for two groups. Statistical analyses were completed with GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA). P < 0.05 was regarded as statistically significant.