Identification of key molecular markers of acute coronary syndrome using peripheral blood transcriptome sequencing analysis and mRNA-lncRNA co-expression network construction

Figures & data

Table 1. Detailed information of sequencing samples

Group	Sample number	Gender	Age	Hypertension history	Diabetes history	Smoking history	Drinking history	Pressure conditions	Movement	Atherosclerosis history	Chest pain, chest tightness, arrhythmia, heart failure and other symptoms	Low Density Lipoprotein
Acute coronary syndrome (ACS) group	H1	Male	56	Yes	No	Yes	Yes	Big	Few	Yes	Yes	5.1 mmol/L
	H2	Female	70	No	Yes	No	No	Small	Normal	Yes	Yes	4.4 mmol/L
	H3	Male	67	Yes	Yes	Yes	Yes	Big	Few	Yes	Yes	3.8 mmol/L
Percutaneous coronary intervention_non-restenosis (PCI_NR) group	Z1	Female	60	Yes	Yes	No	No	Big	Few	Yes	No	4.6 mmol/L
	Z2	Male	75	Yes	No	Yes	No	Big	Few	No	Yes	5.7 mmol/L
	Z3	Male	59	No	Yes	Yes	Yes	Big	Few	No	Yes	4.1 mmol/L
PCI_restenosis (PCI_Re) group	X1	Female	63	No	No	No	No	Big	Normal	No	Yes	4.2 mmol/L
	X2	Female	71	No	Yes	No	No	Big	Few	Yes	No	5.4 mmol/L
	X3	Male	59	No	No	Yes	Yes	Small	Exercise	Yes	Yes	4.5 mmol/L
Normal controls (NC) group	C1	Female	61	No	No	No	No	Small	Normal	No	No	3.1 mmol/L
	C2	Male	78	No	No	Yes	No	Small	Normal	No	No	2.9 mmol/L
	C3	Female	79	No	No	No	No	Small	Normal	No	No	2.8 mmol/L
Group	High density lipoprotein	Total cholesterol	Triglyceride	Blood platelet count	Cardiactroponin (cTn)	Creatine kinase isoenzyme (CK-MB)	Electrocardiograph	Percutaneous coronary intervention (PCI) treatment	Time for stenosis after PCI	Coronary angiography results (percentage of restenosis in the original lesion)
Acute coronary syndrome (ACS) group	0.9 mmol/L	7.5 mmol/L	3.3 mmol/L	232	5.1 ug/L	189 U/L	ST-T changed
	1.1 mmol/L	6.9 mmol/L	4.4 mmol/L	274	0.2 ug/L	24 U/L	ST-T changed
	1.1 mmol/L	6.1 mmol/L	2.5 mmol/L	212	3.3 ug/L	131 U/L	ST-T changed
Percutaneous coronary intervention_non-restenosis (PCI_NR) group	1.2 mmol/L	6.8 mmol/L	3.6 mmol/L	178	0.04 ug/L	12 U/L	ST-T changed	1 diagonal bracket and 1 right crown bracket
	1.6 mmol/L	7.1 mmol/L	2.7 mmol/L	236	0.2 ug/L	13 U/L	ST-T changed	2 right crown stents
	1.5 mmol/L	7.2 mmol/L	2.9 mmol/L	208	0.1 ug/L	18 U/L	ST-T changed	2 roundabout support bracket
PCI_restenosis (PCI_Re) group	1.8 mmol/L	7.2 mmol/L	4.2 mmol/L	241	0.2 ug/L	15 U/L	ST-T changed	1 left main stent and 1 right proximal crown stent	12 months	100%
	1.0 mmol/L	8.2 mmol/L	3.4 mmol/L	203	0.2 ug/L	10 U/L	ST-T changed	2 anterior descending branches and 1 right crown	6 months	80%
	1.2 mmol/L	7.2 mmol/L	1.9 mmol/L	218	3.2 ug/L	61 U/L	ST-T changed	2 roundabout support bracket	1 month	99%
Normal controls (NC) group	1.6 mmol/L	5.3 mmol/L	2.3 mmol/L	189	0.03 ug/L	12 U/L	Normal
	1.4 mmol/L	5.4 mmol/L	2.2 mmol/L	230	0.01 ug/L	8 U/L	Normal
	1.8 mmol/L	5.6 mmol/L	1.9 mmol/L	285	0.01 ug/L	11 U/L	Normal

Figure 1. DEmRNAs analysis of the PCI_NR/PCI_Re group.

a: Volcano map of DEmRNAs in the PCI_NR/PCI_Re group; b: Heatmap of DEmRNAs in the PCI_NR/PCI_Re group; c: Top 15 significantly enriched biological process (BP); d: Top 15 significantly enriched cell composition (CC); e: Top 15 significantly enriched molecular function (MF); f: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heatmap shows result of a two-way hierarchical clustering of DEmRNAs (row) and samples (column). DEmRNAs clustering tree is displayed on the left. Red indicates above the reference channel. Green indicates below the reference channel.

Figure 2. DEmRNAs analysis of the NC/PCI_Re group.

a: Volcano map of DEmRNAs in the NC/PCI_Re group; b: Heatmap of DEmRNAs in the NC/PCI_Re group; c: Top 15 significantly enriched biological process (BP); d: Top 15 significantly enriched cell composition (CC); e: Top 15 significantly enriched molecular function (MF); f: Top 15 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heatmap shows result of a two-way hierarchical clustering of DEmRNAs (row) and samples (column). DEmRNAs clustering tree is displayed on the left. Red indicates above the reference channel. Green indicates below the reference channel.

Figure 3. DEmRNAs analysis of the NC/ACS group.

a: Volcano map of DEmRNAs in the NC/ACS group; b: Heatmap of DEmRNAs in the NC/ACS group; c: Top 15 significantly enriched biological process (BP); d: Top 15 significantly enriched cell composition (CC); e: Top 15 significantly enriched molecular function (MF); f: Top 15 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heatmap shows result of a two-way hierarchical clustering of DEmRNAs (row) and samples (column). DEmRNAs clustering tree is displayed on the left. Red indicates above the reference channel. Green indicates below the reference channel.

Figure 4. DEmRNAs analysis in ACS/PCI_NR group.

a: Volcano map of DEmRNAs in the ACS/PCI_NR group; b: Heatmap of DEmRNAs in the ACS/PCI_NR group; c: Top 15 significantly enriched biological process (BP); d: Cell composition (CC); e: Top 15 significantly enriched molecular function (MF); f: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heatmap shows result of a two-way hierarchical clustering of DEmRNAs (row) and samples (column). DEmRNAs clustering tree is displayed on the left. Red indicates above the reference channel. Green indicates below the reference.

Figure 5. Analysis of DEmRNAs in the intersection of the PCI_NR/PCI_Re and NC/PCI_Re groups.

a: Venn diagram of DEmRNAs in the PCI_NR/PCI_Re and NC/PCI_Re groups; b: Top 15 significantly enriched biological process (BP); c: Cell composition (CC); d: Molecular function (MF); E: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

Figure 6. Analysis of DEmRNAs in the intersection of the NC/ACS and ACS/PCI_NR groups.

a: Venn diagram of DEmRNAs in the NC/ACS and ACS/PCI_NR groups; b: Top 15 significantly enriched biological process (BP); c: Cell composition (CC); d: Molecular function (MF); e: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

Figure 7. Analysis of DElncRNAs in different groups.

a: Volcano map of DElncRNAs in the PCI_NR/PCI_Re group; b: Heatmap of DElncRNAs in the PCI_NR/PCI_Re group; c: Volcano map of DElncRNAs in the NC/PCI_Re group; d: Heatmap of DElncRNAs in the NC/PCI_Re group; e: Volcano map of DElncRNAs in the NC/ACS group; f: Heatmap of DElncRNAs in the NC/ACS group; g: Volcano map of DElncRNAs in the ACS/PCI_NR group; h: Heatmap of DElncRNAs in the ACS/PCI_NR group; i: Venn diagram of DElncRNAs in the PCI_NR/PCI_RE and NC/PCI_RE groups; J: Venn diagram of DElncRNAs in the NC/ACS and ACS/PCI_NR groups. Heatmap shows result of a two-way hierarchical clustering of DElncRNAs (row) and samples (column). DElncRNAs clustering tree is displayed on the left. Red indicates above the reference channel. Green indicates below the reference channel.

Figure 8. Co-expression analysis of DEmRNA-DElncRNA.

a: DEmRNA-DElncRNA co-expression network in the PCI_NR/PCI_Re and NC/PCI_Re groups; b: Biological process (BP) enrichment of co-expressed mRNA in PCI_NR/PCI_Re and NC/PCI_Re intersection group; c: Cell composition (CC) enrichment of co-expressed mRNA in PCI_NR/PCI_Re and NC/PCI_Re intersection group; d: Molecular function (MF) enrichment of co-expressed mRNA in PCI_NR/PCI_Re and NC/PCI_Re intersection group; e: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of co-expressed mRNA in PCI_NR/PCI_Re and NC/PCI_Re intersection group. f: DEmRNAs-DElncRNAs co-expression network in the NC/ACS; g: DEmRNAs-DElncRNAs co-expression network in the ACS/PCI_NR; h: BP enrichment of co-expressed mRNA in the NC/ACS and ACS/PCI_NR intersection group; i: CC enrichment of co-expressed mRNA in the NC/ACS and ACS/PCI_NR intersection group; j: MF enrichment of co-expressed mRNA in the NC/ACS and ACS/PCI_NR intersection group; k: KEGG pathways enrichment of co-expressed mRNA in the NC/ACS and ACS/PCI_NR intersection group. In the DEmRNAs-DElncRNAs co-expression network, the circular, V-shaped, red, green, red lines, and blue lines represent mRNA, lncRNA, up-regulation, down-regulation, positive correlation and negative correlation, respectively.

Figure 9. Diagnostic analysis of PDZK1IP1 (a), PROK2 (b) and LAMP3 (c).

AUC: area under curve

Table 2. The information of 14 individuals for RT-PCR validation

Group	Sample number	Gender	Age	Hypertension history	Diabetes history	Smoking history	Drinking history	Pressure conditions	Atherosclerosis history	Chest pain, chest tightness, arrhythmia, heart failure and other symptoms	Low Density Lipoprotein
Normal controls (NC) group	1	Female	73	No	No	No	No	Small	No	No	2.9 mmol/L
	2	Female	66	No	No	No	No	Small	No	No	3.2 mmol/L
	3	Female	71	No	No	No	No	Small	No	No	2.8 mmol/L
	4	Male	46	No	No	No	No	Small	No	No	2.3 mmol/L
Acute coronary syndrome (ACS) group	1	Male	55	Yes	Yes	Yes	Yes	Big	Yes	No	4.6 mmol/L
	2	Male	58	Yes	No	Yes	Yes	Big	Yes	Yes	3.7 mmol/L
	3	Female	72	No	Yes	No	No	Small	Yes	Yes	4.8 mmol/L
	4	Male	70	Yes	No	Yes	Yes	Big	No	Yes	3.9 mmol/L
	5	Female	69	Yes	No	No	No	Big	Yes	Yes	4.1 mmol/L
Percutaneous coronary intervention_restenosis (PCI_Re) group	1	Male	75	Yes	Yes	No	Yes	Small	Yes	Yes	4.2 mmol/L
	2	Male	77	Yes	No	Yes	Yes	Big	Yes	No	3.2 mmol/L
	3	Female	70	Yes	Yes	No	No	Small	Yes	Yes	4.3 mmol/L
	4	Male	72	Yes	Yes	Yes	Yes	Small	Yes	Yes	4.8 mmol/L
	5	Male	59	No	Yes	Yes	Yes	Big	No	Yes	4.1 mmol/L
Group		High density lipoprotein	Total cholesterol	Triglyceride	Blood platelet count	Cardiactroponin (cTn)	Creatine kinase isoenzyme(CK-MB)	Electrocardiograph	Percutaneous coronary intervention (PCI) treatment	Time for stenosis after PCI	Coronary angiography results (percentage of restenosis in the original lesion)
Normal controls (NC) group		1.4 mmol/L	3.8 mmol/L	2.2 mmol/L	228	0.01 ug/L	8 U/L	Normal
		1.5 mmol/L	4.5 mmol/L	1.5 mmol/L	247	0.02 ug/L	14 U/L	Normal
		1.8 mmol/L	5.6 mmol/L	1.9 mmol/L	254	0.01 ug/L	11 U/L	Normal
		1.2 mmol/L	4.1 mmol/L	2.1 mmol/L	221	0.03 ug/L	12 U/L	Normal
Acute coronary syndrome (ACS) group		1.2 mmol/L	6.8 mmol/L	2.6 mmol/L	225	0.4 ug/L	22 U/L	ST-T changed
		1.3 mmol/L	6.5 mmol/L	3.7 mmol/L	126	2.5 ug/L	46 U/L	ST-T changed
		1.1 mmol/L	7.1 mmol/L	3.1 mmol/L	256	0.3 ug/L	12 U/L	ST-T changed
		1.5 mmol/L	6.6 mmol/L	2.7 mmol/L	187	0.4 ug/L	25 U/L	ST-T changed
		1.6 mmol/L	7.2 mmol/L	2.1 mmol/L	207	0.2 ug/L	20 U/L	ST-T changed
Percutaneous coronary intervention_restenosis (PCI_Re) group		1.1 mmol/L	6.2 mmol/L	2.2 mmol/L	210	2.8 ug/L	66 U/L	ST-T changed	2 anterior descending brace	3 months	85%
		1.1 mmol/L	6.3 mmol/L	2.4 mmol/L	214	0.09 ug/L	17 U/L	ST-T changed	1 diagonal brace, 2 right crown braces	6 months	96%
		1.1 mmol/L	6.5 mmol/L	4.3 mmol/L	258	0.05 ug/L	9 U/L	ST-T changed	1 anterior descending branch open stent, 1 right crown middle stent	10 months	80%
		1.4mmol/L	6.6mmol/L	2.1mmol/L	187	0.06ug/L	10U/L	ST-T changed	2 anterior descending stents, 1 right crown stent	12 months	90%
		1.5mmol/L	7.2mmol/L	2.9mmol/L	245	0.1ug/L	18U/L	ST-T changed	2 circumflex supports	1 month	95%

Table 3. Primer sequence in the RT-qPCR

Primer name	Primer sequence (5ʹ to 3ʹ)
GAPDH-F (internal reference)	5-CTGGGCTACACTGAGCACC-3
GAPDH-R (internal reference)	5-AAGTGGTCGTTGAGGGCAATG-3
ACTB-F (internal reference)	5-GATCAAGATCATTGCTCCTCCT-3
ACTB-R (internal reference)	5-TACTCCTGCTTGCTGATCCA-3
RNF213-F	5-GCTGCTGTGAAAAACGAGAAG-3
RNF213-R	5-TCCCATTTTGACTCCCCAAATTC-3
LAMP3-F	5-AGCAAGCACCTCACCAAACTT-3
LAMP3-R	5-TGTAGTCGCTGGGGTAGTTGT-3
IFI44-F	5-GGTGGGCACTAATACAACTGG-3
IFI44-R	5-CACACAGAATAAACGGCAGGTA-3
IFIT1-F	5-TCAGCACTTCGATGGGACG-3
IFIT1-R	5-ACACTGCAGCCTCGAACTC-3
IFIT3-F	5-AAAAGCCCAACAACCCAGAAT-3
IFIT3-R	5-CGTATTGGTTATCAGGACTCAGC-3
IFIT5-F	5-CCGGAAAGCTCTTCGTCTGG-3
IFIT5-R	5-TGCGAAGGGGTGATCTGTCT-3
XAF1-F	5-GCTCCACGAGTCCTACTGTG-3
XAF1-R	5-GTTCACTGCGACAGACATCTC-3
NRIR-F	5-TCTGTCGCCAGGCTGGAGTG-3
NRIR-R	5-GGCTGAGGCAGGATAATCGCTTG-3
PROK2-F	5-CTGCCATCCACTGACTCGT-3
PROK2-R	5-GTCCGTAAACAGGCCAAGC-3
LINC00570-F	5-GGGGATCAACGAACAGGCT-3
LINC00570-R	5-ACTCAGTCTCCAGCACTCCT-3

Figure 10. RT-PCR validation of PROK2, LINC00570, RNF213, LAMP3, IFI44, IFIT1, IFIT3, IFIT5, XAF1 and NRIR in blood samples.

* represent P < 0.05, ** represent P < 0.01, P < 0.05 was considered significant. NC, normal control; ACS, acute coronary syndrome; PCI_Re, percutaneous coronary intervention_ restenosis.