Systematic analysis of transcriptomic profiles of COPD airway epithelium using next-generation sequencing and bioinformatics

Figures & data

Figure 1 Flowchart of study design.

Notes: In order to investigate the roles of microRNA–mRNA interactions in the microenvironment of COPD, we used normal human bronchial epithelial cells and COPD bronchial epithelial cells for next-generation sequencing (NGS). Then, we analyzed the NGS data of with several bioinformatic tools, including MiRmap, Ingenuity Pathway Analysis (IPA), the Database for Annotation, Visualization, and Integrated Discovery (DAVID), MirDB, TargetScan, and the Gene Expression Omnibus (GEO) database.
Abbreviations: DE, differentially expressed; FC, fold change; RPM, reads per million; FPKM, fragments per kilobase of transcript per million mapped reads; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Table 1 Dysregulated genes with potential microRNA–mRNA interactions in COPD bronchial epithelial cells

44 downregulated genes	36 upregulated genes
CCDC149, ENG, ACSS1, FOXP2, CECR1, FGFR1, C6orf223, SORL1, NOS1, ADM2, RAB3B, SDK1, PCDH7, IL24, QPRT, PAG1, CD40, PAIP2B, CDHR1, LARGE, NCALD, TGFBR3, GJC1, SCN4B, SV2A, IRAK3, CDH26, NT5E, PREX1, BEGAIN, SDK2, TNS1, KIF21B, KCNJ5, PLEKHH2, FZD10, UGT8, BEX4, ADCY9, AKT3, SLC7A8, NRSN2, OLFM2, ABCC9	L1CAM, RTKN2, SYT1, SLC6A11, SUSD5, PCDH1, KRT80, LSP1, KIAA1644, CLDN11, CYGB, ADAMTS15, ADAM19, WDR72, LIF, SLC43A2, FAM84A, MAP1B, ALDH1A3, STC1, HHIP, RIMS3, SLC46A3, DMD, WNT5A, PLEKHA7, GPRC5A, TIMP2, FLI1, GNG2, PEX5L, TFRC, MFI2, TTC9, C4orf26, CDH5

Figure 2 Identification of genes with potential microRNA–mRNA interactions in COPD bronchial epithelial cells.

Notes: Gene-expression heat map (right) of differentially expressed genes revealed 685 genes with fold change >2. The heat map (left) of differentially expressed microRNAs revealed 144 microRNAs with fold change >2 and reads per million (RPM) >1. According to the MiRmap web-based database, we predicted 543 mRNAs as targets of these 144 microRNAs. Venn diagrams of microRNA–mRNA interactions shows that 44 genes were downregulated and 36 genes upregulated in COPD bronchial epithelial (DHBE) cells compared to normal human bronchial epithelial (NHBE) cells. The selection threshold for microRNA-target prediction was MiRmap score ≥99.

Table 2 Downregulated genes with microRNA–mRNA interactions and their functions

MicroRNA	mRNA	Function (IPA)	MiRmap score	MirDB (target score)	TargetScan (total context score)
hsa-miR1285-3p	PLEKHH2	Tumorigenesis of malignant tumor	99.6181	66	−0.17
hsa-miR195-5p	FGFR1	Proliferation of connective tissue cells, depressive disorder, angiogenesis, proliferation of lipoblasts, migration of cells, major depression	99.8560	72	−0.55
hsa-miR3173-3p	CDH26	Eosinophilia	99.1171	50	−0.87
	SDK1	Tumorigenesis of malignant tumors	99.9039	62	−0.58
hsa-miR3613-3p	GJC1	Cell–cell contact, muscle contraction	99.8337	89	−0.63
hsa-miR378g	ENG	Autophagy of vascular endothelial cells, proliferation of connective tissue cells, detachment of vascular endothelial cells, cellular homeostasis, angiogenesis, autophagy of cells, migration of cells, organism death	99.8158	80	−0.25
	ADM2	Respiration of adipocytes, angiogenesis, quantity of M2 macrophages, quantity of leukocytes	99.9646	67	−0.93
hsa-miR424-5p	AKT3	Autophagy of vascular endothelial cells, proliferation of connective tissue cells, autophagy of cells, migration of cells, cellular homeostasis	99.0670	99	−0.67
	FGFR1	Proliferation of connective tissue cells, depressive disorder, angiogenesis, proliferation of lipoblasts, migration of cells, major depression	99.8506	72	−0.55
hsa-miR4435	TNS1	Migration of cells	99.8208	74	−0.16
hsa-miR449a	SDK2	Tumorigenesis of malignant tumor	99.8641	83	−0.41
hsa-miR4532	KIF21B	Long-QT syndrome	99.5702	94	−0.46
hsa-miR4745-5p	SLC7A8	Digestive organ tumor	99.6903	54	−0.25
hsa-miR50015p	TGFBR3	Angiogenesis	99.7104	82	−0.29
	KIF21B	Long-QT syndrome	99.9772	95	−0.34
	SDK1	Tumorigenesis of malignant tumor	99.7629	78	−0.17
hsa-miR5190	FOXP2	Tumorigenesis of carcinoma	99.1872	93	−0.20
hsa-miR570-3p	FOXP2	Tumorigenesis of carcinoma	99.5231	95	−0.21
hsa-miR641	PCDH7	Neurological signs	99.4883	94	−0.47
hsa-miR6505-5p	NCALD	Digestive organ tumor	99.2089	96	−0.56
hsa-miR6511a-5p	TNS1	Migration of cells	99.9283	91	−0.13
	OLFM2	Secretion of protein	99.9239	100	−1.27
	NT5E	Cell–cell contact, activation of leukocytes, activation of T lymphocytes, cellular homeostasis	99.5498	95	−0.35
hsa-miR6724-5p	NCALD	Digestive organ tumor	99.7041	91	−0.75

Note: Predicted by Ingenuity Pathway Analysis (IPA) and meta-analysis with MiRmap, MirDB, and TargetScan.

Figure 3 Functional analysis of dysregulated genes identified in COPD epithelial cells by Ingenuity Pathway Analysis (IPA).

Notes: The 80 identified dysregulated genes with potential microRNA–mRNA interactions were analyzed by IPA. (A) Pathways related to 44 downregulated genes; (B) pathways related to 36 upregulated genes.

Abbreviations: TR, thyroid hormone receptor; RXR, retinoid X receptor; PTEN, phosphatase and tensin homolog; FAK, focal adhesion kinase; NFAT, nuclear factor of activated T-cell.

Figure 4 Possible mechanisms of dysregulated genes identified in COPD epithelial cells analyzed by Database for Annotation, Visualization, and Integrated Discovery (DAVID).

Notes: Functional annotation of the 44 downregulated genes was determined by gene ontology using DAVID. These 44 genes are involved in the functioning of membrane, transmembrane helix, transmembrane, nucleotide bonding, cell adhesion, calcium, disulfide bonds, and signals.

Figure 5 Gene Expression Omnibus (GEO) database analysis of 18 downregulated genes with potential microRNA–mRNA interactions in COPD small airway.

Notes: Gene expression of the 18 downregulated genes with potential microRNA–mRNA interactions was analyzed using GSE4498 microarray data from the GEO database. The results showed that expression of NT5E, SDK1, TNS1, and PCDH7 was significantly downregulated in patients with COPD compared to normal controls. *P<0.05; **P<0.01; ***P<0.001.
Abbreviation: NS, not significant.

Figure 6 Gene Expression Omnibus database analysis of the four genes downregulated in COPD small airway, large airway, and alveolar macrophages.

Notes: The four downregulated gene with potential microRNA–mRNA interactions validated in the GSE4498 database (COPD small-airway bronchial epithelial cells) were further analyzed in the GSE5056 database (large airway) and GSE2125 database (alveolar macrophages). NT5E was the only significantly downregulated mRNA in all databases. *P<0.05; **P<0.01; ***P<0.001.
Abbreviation: NS, not significant.

Figure 7 MicroRNA–mRNA interactions in the microenvironment of COPD.

Notes: The miR6511a-5p–NT5E interaction plays an important role in COPD, and may be associated with cell–cell contact, activation of leukocytes, activation of T lymphocytes, and cellular homeostasis. miR3173-3p–SDK1, miR4435–TNS1, and miR641–PCDH7 interactions might also be associated with COPD pathogenesis in small-airway bronchial epithelial cells.