A Next-Generation Sequencing of Plasma Exosome-Derived microRNAs and Target Gene Analysis with a Microarray Database of Thermally Injured Skins: Identification of Blood-to-Tissue Interactions at Early Burn Stage

Figures & data

Table 1 Demographics for Participants Sampled for NGS and Patients from GSE8056

Research and Sample Type	Control Group	Sex	Age (Years)	Mean Age	Burn Group	Sex	Age (Years)	Mean Age	% TBSA Burn (% Deep Burn)	Mean%TBSA	ABSI Score	Day Postburn
NGS of plasma exosome-derived small RNA	Normal 01	F	24	28.3 ±5.28	Burn 01	F	54	39 ±16.69	60 (32)	79.83 ±19.99	11	5
	Normal 02	F	21		Burn 02	M	28		60 (46)		10	5
	Normal 03	F	29		Burn 03	F	25		65 (20)		11	5
	Normal 04	M	28		Burn 04	M	54		97 (80)		15	4
	Normal 05	M	33		Burn 05	F	54		98 (92)		16	4
	Normal 06	M	35		Burn 06	M	19		99 (95)		13	4
cDNA-microarray analysis of skin	GSM198875-01	F	35	38.4 ±14.37	GSM198869-01	M	8	30.8 ±14.83	6	14.67 ±12.94	No record	5
	GSM198875-02	F	26		GSM198869-02	M	19		8			4
	GSM198875-03	F	43		GSM198869-03	M	30		10			4
	GSM198875-04	F	20		GSM198869-04	F	11		5			5
	GSM198875-05	F	20		GSM198869-05	F	31		15			5
	GSM198876-01	F	32		GSM198870-01	M	26		30			4
	GSM198876-02	F	43		GSM198870-02	M	33		5			5
	GSM198876-03	F	52		GSM198870-03	F	37		4			6
	GSM198876-04	F	56		GSM198870-04	F	38		20			7
	GSM198876-05	F	44		GSM198870-05	M	18		20			7
	GSM198877-01	F	55		GSM198871-01	M	51		50			4
	GSM198877-02	M	43		GSM198871-02	M	25		10			5
	GSM198877-03	F	22		GSM198871-03	M	42		27			5
	GSM198877-04	M	22		GSM198871-04	M	28		5			7
	GSM198877-05	F	63		GSM198871-05	M	65		5			6

Abbreviations: TBSA, total body surface area; ABSI, abbreviate burn severity index; NGS, next-generation sequencing; F, female; M, male.

Figure 1 Characterization of plasma exosomes from burn patients and control group. (A) Visualized exosomal morphology in transmission electron microscopy (TEM). Scale bar = 200 nm. (B) Size distribution of plasma exosomes by nanoparticle tracking analysis (NTA). (C) Western blotting (WB) analyses of the exosome protein marker CD63 and the endoplasmic reticulum protein marker Calnexin.

Table 2 Genome Alignment Ratios of RNA Read Counts Concerning Plasma Exosome-Derived Small RNAs

Reads	Counts	Ratio
All clean reads	16,534,063	100%
Noadapter reads	400,031	2.42%
Short UMIs reads	8252	0.05%
Mapped miRBase mature reads	2,653,251	16.05%
Mapped miRBase hairpin reads	295,149	1.79%
Mapped piRNA reads	107,862	0.65%
Mapped Rfam reads	998,098	6.04%

Table 3 Differentially Expressed microRNAs (DEMs) in Plasma Exosomes at the Early Burn Stage

DEMs	Log2FC	P-value	FDR	Regulation	Targets Number in Focus Genes
hsa-miR-6873-3p	3.6939	2.79E-24	7.41E-21	Up	79
hsa-miR-6751-3p	2.8164	2.98E-14	3.95E-11	Up	50
hsa-miR-1281	2.6124	2.33E-11	1.65E-08	Up	51
hsa-miR-6741-3p	2.6198	2.49E-11	1.65E-08	Up	51
hsa-miR-7111-3p	2.4319	4.00E-10	2.08E-07	Up	68
hsa-miR-6776-3p	3.0672	4.69E-10	2.08E-07	Up	61
hsa-miR-6809-3p	2.4835	6.62E-09	2.51E-06	Up	79
hsa-miR-6830-3p	2.1379	8.48E-09	2.82E-06	Up	87
hsa-miR-6754-3p	2.5296	1.03E-08	3.03E-06	Up	62
hsa-miR-6880-3p	2.2448	1.55E-08	4.12E-06	Up	51
hsa-miR-718	2.9613	2.05E-08	4.94E-06	Up	50
hsa-miR-6739-5p	2.5980	2.27E-08	5.01E-06	Up	69
hsa-miR-4646-3p	2.2586	3.31E-08	6.76E-06	Up	65
hsa-miR-6894-3p	2.1834	8.96E-08	1.70E-05	Up	68
hsa-miR-6739-3p	2.6488	1.53E-07	2.71E-05	Up	74
hsa-miR-10398-5p	2.1290	1.67E-07	2.77E-05	Up	27
hsa-miR-4667-5p	2.1068	2.27E-07	3.55E-05	Up	67
hsa-miR-1224-3p	1.9389	5.33E-07	7.87E-05	Up	45
hsa-miR-4434	2.7177	6.80E-07	9.18E-05	Up	72
hsa-miR-150-5p	−1.7822	7.03E-07	9.18E-05	Down	87
hsa-miR-6823-3p	1.8625	7.26E-07	9.18E-05	Up	48
hsa-miR-6759-3p	2.1785	9.26E-07	1.12E-04	Up	57
hsa-miR-12116	1.6649	1.96E-06	2.26E-04	Up	42
hsa-miR-6884-3p	1.7718	2.56E-06	2.83E-04	Up	47
hsa-miR-4716-5p	1.8601	5.63E-06	5.98E-04	Up	61
hsa-miR-6883-3p	1.7636	6.57E-06	6.71E-04	Up	59
hsa-miR-7847-3p	1.4644	7.91E-06	7.78E-04	Up	61
hsa-miR-4516	1.7274	9.01E-06	8.54E-04	Up	72
hsa-miR-6797-3p	1.9338	1.18E-05	1.08E-03	Up	51
hsa-miR-335-3p	2.1197	1.38E-05	1.23E-03	Up	67
hsa-miR-6510-5p	1.7775	1.61E-05	1.38E-03	Up	57
hsa-miR-7107-3p	1.8947	1.76E-05	1.46E-03	Up	66
hsa-miR-550b-3p	2.8765	2.03E-05	1.63E-03	Up	72
hsa-miR-4667-3p	1.5593	3.42E-05	2.67E-03	Up	56
hsa-miR-657	1.5340	4.03E-05	3.05E-03	Up	46
hsa-miR-6886-3p	1.6442	4.21E-05	3.10E-03	Up	51
hsa-miR-6833-3p	1.5770	5.57E-05	4.00E-03	Up	71
hsa-miR-4646-5p	1.7058	5.73E-05	4.01E-03	Up	56
hsa-miR-10401-3p	2.2197	7.39E-05	5.03E-03	Up	26
hsa-miR-6756-3p	1.5415	7.73E-05	5.13E-03	Up	56
hsa-miR-365b-5p	−5.0048	9.60E-05	6.20E-03	Down	60
hsa-miR-6760-3p	1.4502	9.80E-05	6.20E-03	Up	54
hsa-miR-6867-3p	1.5707	1.04E-04	6.43E-03	Up	68
hsa-miR-550a-3p	2.1836	1.21E-04	7.32E-03	Up	68
hsa-miR-10400-5p	3.7818	1.45E-04	8.36E-03	Up	40
hsa-miR-4522	2.0049	1.45E-04	8.36E-03	Up	66
hsa-miR-4786-5p	−2.4633	2.01E-04	1.11E-02	Down	56
hsa-miR-5006-5p	1.7414	2.01E-04	1.11E-02	Up	61
hsa-miR-4684-3p	−3.7701	2.31E-04	1.25E-02	Down	75
hsa-miR-1539	−3.4463	2.37E-04	1.26E-02	Down	60
hsa-miR-6891-3p	1.5370	2.46E-04	1.27E-02	Up	57
hsa-miR-375-3p	−2.9319	2.49E-04	1.27E-02	Down	63
hsa-miR-4649-3p	2.5969	2.95E-04	1.45E-02	Up	62
hsa-miR-125a-5p	−1.4474	2.96E-04	1.45E-02	Down	64
hsa-miR-8052	−4.4882	3.01E-04	1.45E-02	Down	58
hsa-miR-7114-3p	1.7273	3.25E-04	1.54E-02	Up	44
hsa-miR-6795-3p	1.5891	3.47E-04	1.62E-02	Up	44
hsa-miR-7110-3p	1.3498	3.90E-04	1.78E-02	Up	76
hsa-miR-365a-5p	−3.0986	3.95E-04	1.78E-02	Down	55
hsa-miR-4685-3p	1.6637	4.35E-04	1.92E-02	Up	58
hsa-miR-4728-5p	1.7391	4.41E-04	1.92E-02	Up	63
hsa-miR-4739	1.2684	5.12E-04	2.18E-02	Up	65
hsa-miR-6747-3p	1.3237	5.16E-04	2.18E-02	Up	74
hsa-miR-3059-5p	1.4961	5.28E-04	2.19E-02	Up	62
hsa-miR-5193	1.1997	5.68E-04	2.32E-02	Up	65
hsa-miR-6882-3p	1.4466	6.14E-04	2.47E-02	Up	59
hsa-miR-10398-3p	3.1236	6.31E-04	2.50E-02	Up	37
hsa-miR-122b-3p	2.0180	7.49E-04	2.92E-02	Up	63
hsa-miR-1184	2.5324	7.84E-04	3.02E-02	Up	49
hsa-miR-6848-3p	−1.9548	8.17E-04	3.06E-02	Down	66
hsa-miR-6513-5p	1.4819	8.19E-04	3.06E-02	Up	81
hsa-miR-6826-3p	1.2170	8.49E-04	3.13E-02	Up	56
hsa-miR-6726-5p	−3.1241	9.51E-04	3.41E-02	Down	50
hsa-miR-4488	1.5293	9.58E-04	3.41E-02	Up	50
hsa-miR-4754	2.9626	9.63E-04	3.41E-02	Up	54
hsa-miR-6817-3p	3.3331	9.81E-04	3.43E-02	Up	76
hsa-miR-4644	1.1645	1.03E-03	3.56E-02	Up	59
hsa-miR-520g-5p	−1.9903	1.12E-03	3.82E-02	Down	60
hsa-miR-663b	1.6953	1.17E-03	3.94E-02	Up	48
hsa-miR-6878-3p	1.3082	1.25E-03	4.17E-02	Up	74
hsa-miR-6727-3p	1.3258	1.35E-03	4.43E-02	Up	60
hsa-miR-6816-5p	−3.4400	1.45E-03	4.66E-02	Down	52
hsa-miR-4783-5p	1.4944	1.45E-03	4.66E-02	Up	37
hsa-miR-4478	1.3517	1.56E-03	4.93E-02	Up	73
hsa-miR-631	−2.6145	1.59E-03	4.97E-02	Down	45

Figure 2 DEMs in plasma exosomes and DEGs in skin. (A) 85 DEMs identified from NGS of plasma exosomal small RNAs, including 14 downregulated miRNAs in green and 71 upregulated miRNAs in red, are exhibited in the volcano plot. (B) Heatmap of all DEMs with the intensity plot showing a relatively low expression in green and a relatively high expression in red. (C) 1861 DEGs identified from microarray data of skin gene expressions (GSE8056), including 1030 downregulated genes in green and 831 upregulated genes in red, are exhibited in the volcano plot. (D) Heatmap of top 100 DEGs with greatest significance, with the intensity plot showing a relatively low expression in green and a relatively high expression in red.

Abbreviations: DEMs, differentially expressed miRNAs; DEGs, differentially expressed genes; NGS, next-generation sequencing.

Figure 3 GO terms and KEGG pathways enriched in DEMs’ target genes, DEGs and focus genes. (A) Top 10 GO terms enriched in target genes of plasma exosome-derived DEMs. (B) Top 10 KEGG pathways of DEMs’ target genes. (C) Top 10 GO terms enriched in DEGs of skins (from GSE8056). (D) Top 10 KEGG pathways of DEGs. (E) Top 10 GO terms enriched in the focus genes. (F) All 7 KEGG pathways of the focus genes.

Abbreviations: DEMs, differentially expressed miRNAs; DEGs, differentially expressed genes.

Figure 4 Venn analysis of DEMs’ target genes and DEGs. Up/down DEGs, differentially expressed genes significantly upregulated/downregulated in thermally injured skins from GSE8056. Up/down DEM targets, target genes of differentially expressed miRNAs significantly upregulated/downregulated in plasma exosomes from burn patients.

Table 4 Top 9 Hub Genes in the PPI Network of Focus Genes

Gene Symbol	Gene Name	GeneCards Summary of Protein Encoded	Degree Score	Log2FC in GSE8056	Regulation	P-value	FDR	Downregulated Upstream DEMs	Upregulated Upstream DEMs
CDK1	Cyclin dependent kinase 1	Ser/Thr protein kinase essential for G1/S and G2/M phase transitions	51	1.481599	Up	6.31E-04	1.99E-03		hsa-miR-4754
CCNB1	Cyclin B1	Cyclin member necessary to the G2/M transition	41	2.535214	Up	6.42E-06	6.90E-05		hsa-miR-6884-3p, hsa-miR-718
CCNA2	Cyclin A2	Cyclin member promoting G1/S and G2/M transition	39	2.514916	Up	1.45E-05	1.22E-04		hsa-miR-550b-3p, hsa-miR-550a-3p, hsa-miR-6795-3p
BUB1B	BUB1 Mitotic Checkpoint Serine/Threonine Kinase B	Checkpoint kinase delaying anaphase and ensuring proper chromosome segregation	35	2.196643	Up	3.54E-06	4.65E-05		hsa-miR-6754-3p
PLK1	Polo like kinase 1	Serine/threonine-protein kinase prominent throughout M phase	31	2.341512	Up	7.04E-05	3.84E-04		hsa-miR-1224-3p, hsa-miR-4754
KIF11	Kinesin family member 11	Motor protein required for establishing a bipolar spindle	29	2.446886	Up	9.00E-04	2.61E-03	hsa-miR-6848-3p	hsa-miR-4739
AURKA	Urora kinase A	Mitotic serine/threonine kinase regulating microtubules at the spindle pole during chromosome segregation	27	1.387036	Up	9.31E-06	8.90E-05	hsa-miR-4684-3p	hsa-miR-4739, hsa-miR-6739-5p, hsa-miR-6751-3p
NUSAP1	Nucleolar and spindle-associated protein 1	Regulator to bundle and stabilize microtubules	27	2.091017	Up	1.41E-04	6.41E-04	hsa-miR-4786-5p, hsa-miR-365a-5p	hsa-miR-4754
CDCA8	Cell division cycle associated 8	Component of the chromosomal passenger complex essential for mitosis and cell division	27	1.059758	Up	7.16E-04	2.19E-03		hsa-miR-4739, hsa-miR-6878-3p

Abbreviations: PPI, protein–protein interaction; DEMs, differentially expressed miRNAs.

Figure 5 Networks of potential blood-to-tissue interactions. (A) The protein–protein interaction network of focus genes. Size of the node is scaled by | log2FC (fold change) | of gene expressions; color of the node and its border represents the expression regulation of mRNA and predicted upstream miRNA, respectively. (B) An integrated miRNA-mRNA and PPI network of the key miRNAs and their target genes (including 9 hub genes) in the focus genes. Edge line between genes (proteins) is solid; edge line between miRNA (round rectangle) and mRNA (round) is in dots. Red: upregulated. Green: downregulated.