
Figure : Graphical abstract
Number |
Gender |
Age |
Diabetes |
Hypertension |
Renal inadequacy |
CTA |
Coronary angiography |
N1 |
M |
49 |
no |
no |
no |
- |
- |
N2 |
M |
55 |
no |
no |
no |
- |
- |
N3 |
M |
56 |
no |
no |
no |
- |
- |
N4 |
F |
78 |
no |
no |
no |
- |
- |
C1 |
M |
68 |
no |
no |
no |
++++ |
++++ |
C2 |
M |
72 |
no |
no |
no |
++++ |
++++ |
C3 |
M |
79 |
no |
no |
no |
++++ |
++++ |
C4 |
M |
65 |
no |
no |
no |
++++ |
++++ |
- : No calcification; ++++: Calcification with H>400. CTA: Computed tomography angiography
Note: All patients are enrolled during April-May, 2019. Patients with chronic diseases are excluded from the study.
Table S1 : Information of patients for RNA-sequencing
Normal control group |
Coronary calcification group |
|||||||
Pair number |
Gender |
Age |
CTA |
Coronary angiography |
Gender |
Age |
CTA |
Coronary angiography |
1 |
M |
66 |
- |
- |
M |
70 |
++++ |
++++ |
2 |
M |
70 |
- |
- |
M |
77 |
++++ |
++++ |
3 |
M |
61 |
- |
- |
M |
57 |
++++ |
++++ |
4 |
M |
76 |
- |
- |
M |
74 |
++++ |
++++ |
5 |
F |
76 |
- |
- |
F |
67 |
++++ |
++++ |
6 |
M |
57 |
- |
- |
M |
51 |
++++ |
++++ |
7 |
F |
68 |
- |
- |
F |
67 |
++++ |
++++ |
8 |
M |
73 |
- |
- |
M |
73 |
++++ |
++++ |
9 |
M |
62 |
- |
- |
M |
63 |
++++ |
++++ |
10 |
M |
65 |
- |
- |
M |
65 |
++++ |
++++ |
11 |
M |
61 |
- |
- |
M |
57 |
++++ |
++++ |
12 |
F |
56 |
- |
- |
F |
64 |
++++ |
++++ |
13 |
M |
69 |
- |
- |
M |
70 |
++++ |
++++ |
14 |
F |
52 |
- |
- |
F |
56 |
++++ |
++++ |
15 |
M |
55 |
- |
- |
M |
54 |
++++ |
++++ |
16 |
M |
63 |
- |
- |
M |
65 |
++++ |
++++ |
17 |
M |
72 |
- |
- |
M |
75 |
++++ |
++++ |
18 |
M |
68 |
- |
- |
M |
72 |
++++ |
++++ |
19 |
M |
73 |
- |
- |
M |
73 |
++++ |
++++ |
20 |
M |
63 |
- |
- |
M |
56 |
++++ |
++++ |
21 |
M |
79 |
- |
- |
M |
80 |
++++ |
++++ |
22 |
M |
64 |
- |
- |
M |
58 |
++++ |
++++ |
23 |
F |
70 |
- |
- |
F |
68 |
++++ |
++++ |
24 |
M |
72 |
- |
- |
M |
76 |
++++ |
++++ |
25 |
M |
62 |
- |
- |
M |
57 |
++++ |
++++ |
26 |
M |
78 |
- |
- |
M |
74 |
++++ |
++++ |
27 |
M |
64 |
- |
- |
M |
56 |
++++ |
++++ |
28 |
M |
77 |
- |
- |
M |
58 |
++++ |
++++ |
29 |
M |
78 |
- |
- |
M |
76 |
++++ |
++++ |
30 |
M |
72 |
- |
- |
M |
73 |
++++ |
++++ |
- : No calcification; ++++: Calcification with H>400. CTA: Computed tomography angiography
Note: All patients are enrolled during July-October, 2019, Patients with chronic diseases are excluded from the study.
Table S2 : Information of patients for RNA qPCR validation
human circRNA detection primers |
|
|
Name |
Forward primer 5'-3' |
Reverse primer 5'-3' |
circTOP1 |
CCCAGAGTTGGATGGTCAGG |
CGCCGAGCAGTCTCGTATTT |
circDTL |
AGGTCATCAATGCAGCCTCA |
CCTTCTTCATTGGCAACTGCT |
circHAGH |
CCTTCAGCGGATTTGGGGAT |
TGGGCCTCACCTCACTCTC |
circLZIC |
GCAGGTGCTATACTCAGCCA |
AACCTTGTCCGAAGCTGACC |
circMCU |
TTCACGCAGGGGAAACTTGA |
AGCAGCTAAGATGTCACTGGC |
circPHF7 |
CTTCCAGTTTGCTGGCTATGC |
TGAGGACAATACAGGCTCTTCC |
circKIAA2026 |
GCAAAACACAAGAAGCACAAATCT |
CGACGCCTTGTTGAGGTACA |
AL133477.2 |
GCCTCTGACATTATGGGCTGT |
TTCCAGGAGCAAAGCTGAGG |
AL136526.1 |
TTCCGGGGATTCAACTGCAA |
GAACCTCGGTCGTCGGTAAG |
AC007686.4 |
GGTAGCAGAAGGGAAGGGTG |
TTATCCCAGCATGAGGCGTG |
CAV1 |
GTCAACCGCGACCCTAAACA |
ATGCCAAAGAGGGCAGACAG |
CCDC152 |
AGCAAACTTTATCAGGACATGCAG |
TCTTAGCTTTGCATTTAACTCTGAA |
MEST |
TCGGGTGATTGCCCTTGATT |
GTTGATCCTGCGGTTCTGGA |
TSPAN13 |
TTGCGCTTGTTTAGCCCTGA |
CTTCGGAACCCACAGCAGTT |
GMPR |
GCTCAAGCTCTTCTACGGGA |
TGAGCTCCTTGAGTTTGGCG |
MKRN1 |
CAGAGGACTGGGTGAATGCTA |
CACACAGTTCTCCCCGTATC |
TNS1 |
TACCCCTGAGGAACGGGAC |
ACGGGAAACTCCCCACTGA |
YBX3 |
AAGCACCAACTGAGAACCCT |
GTGTTGGTTAAGGTTGTGGCCT |
PROSER2 |
TCATCGACTTAGTGCAGCCA |
GGTGGAGGAGTCTCAGCATC |
TRIM58 |
GCAGAGAACATCCCCATGGAA |
CCTCCATGGTTCTCCATCCTG |
FECH |
CAGTACCGCAGGATTGGAGG |
GCAATAGCCCTTTCTAGGCCA |
GAPDH |
CTTCATTGACCTCAACTACATGG |
CTCGCTCCTGGAAGATGGTGAT |
Table S3 : List of qRT-PCR primer pairs used in this study
Figure : Graphical abstract
Figure 1: RNA-seq analysis of differentially expressed RNAs from peripheral blood of coronary artery calcification (CAC) patients. a, The workflow of CAC RNA-seq and in-depth investigations in this study. The bioinformatics software were also indicated. b, Heatmap of correlation between calcification (C) or normal samples (N) by Pearson’s correlation coefficient analysis
Figure 2: Bioinformatics analysis of differentially expressed mRNAs and lncRNAs in CAC. a, Hierarchical cluster heatmap of the differentially expressed mRNA in CAC. b, Hierarchical cluster heatmap of the differentially expressed lncRNAs in CAC. c, Representation of the lncRNA- mRNA co-expression network (correlation coefficient absolute value >0.99). The boxes represent mRNA, and the circles represent lncRNA. Red, upregulated; blue, downregulated
Figure 3: Gene Set Enrichment Analysis (GSEA) analysis of differentially expressed mRNAs in CAC RNA-seq. a, GSEA bar plot of the up-regulated or down-regulated gene ontology (GO) term in CAC. b, Representation of enrichment plot of the six RNA process-related GO ontology
Figure 4: Validation of mRNAs and lncRNAs identified from RNA-seq by RT-qPCR in clinical CAC samples. a, The RT-qPCR validation results of 8 dysregulated mRNAs. b, The RT-qPCR validation results of 3 dysregulated lncRNAs. The patients information of the CAC samples for validation are included in the Table S3. Data are median SD. *p < 0.05, **p < 0.01, ***p < 0.001 are based on the Student’s t-test
Figure 5: The bioinformatics analysis and RT-qPCR validation of the differentially expressed circRNAs and miRNAs. a, Hierarchical cluster heatmap of differentially expressed circRNA. b, Hierarchical cluster heatmap of differentially expressed miRNA. c, RT-qPCR validation of circRNAs identified from RNA-seq in clinical CAC samples. The patients information of the CAC samples for validation are included in the Table S3. Data are median SD. *p < 0.05, **p < 0.01, ***p < 0.001 are based on the Student’s t-test
Figure 6: Construction of circRNA-miRNA-mRNA network. a, Predicted functional mechanisms of the validated six circRNAs in the RNAseq. MRE, miRNA response elements. RBP, RNA binding protein. ORF, open reading frame. b, circRNA-miRNA-mRNA interacting network for the validated circRNAs (correlation coefficient absolute value 0.99) . Boxes represent differentially expressed mRNA, triangles represent differentially expressed miRNA, circles represent differentially expressed circRNAs. Red line, circRNA-miRNA interaction; grey line mRNA- miRNA interaction. Pink, upregulated; blue, down-regulated
Figure S1: Expression profiling analysis for circRNAs in CAC. (A) Venn plot of the circRNAs identified in CAC and control group. C: CAC; N: normal. (B) Box plot of all circRNAs expression level in CAC and control group.(C) Box plot of all circRNAs spliced sequence length distribution in CAC and control group. (D-E) The distribution of circRNAs spliced sequence length in CAC and control group
Tables at a glance
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