LncRNAs NR-026690 and ENST0000447867 are upregulated in CD4⁺ T cells in patients with acute exacerbation of COPD

Figures & data

Table 1 Characteristics of subjects providing peripheral blood

Characteristics	Microarray analysis			Validation
	AECOPD (n=5)	Stable COPD (n=5)	Control (n=5)	AECOPD (n=51)	Stable COPD (n=51)	Control (n=30)
Age (years)	66.75±2.250	67.93±2.453	66.45±2.39	66.08±1.072	68.09±0.9601	67.04±0.932
Sex (M/F)	5/0	5/0	5/0	50/1	50/1	29/1
Smoking (packs/year)	38.72±3.723	37.28±2.670	35.24±2.12	36.00±2.382	36.76±1.872	34.52±1.934
Smoking (total packs)	360.6±63.72	323.9±58.18	377.2±51.76	390.2±53.42	387.5±37.64	342.5±49.64
FEV1	NA	1.83±0.12a	2.81±0.1	NA	1.93±0.09b	2.75±0.1
FEV1/predicted	NA	54.12±3.29a	92.84±2.53	NA	52.08±3.02b	92.74±3.74
FEV1/FVC	NA	48.67±2.01a	78.60±1.42	NA	49.72±1.53b	79.23±1.45

Notes:

a P<0.05 compared stable COPD with control in microarray analysis group;

b P<0.05 compared stable COPD with control in validation group.

Abbreviations: NA, not available; AECOPD, acute exacerbation of COPD.

Table 2 Primers of the candidate differentially expressed lncRNAs

Primer name	Sequence
ENST0000447867-forward	GGACTCTTAGCAGAGTGACTGCC
ENST0000447867-reverse	GAAGAGAACGAATTTGCGGAG
NR-026690-forward	AGAGAGGCCAAGGGTACTCG
NR-026690-reverse	CAGGGAGAATCTGGATCGGG
ENST0000584109-forward	ACAGCCTGGGAGCAAATTCA
ENST0000584109-reverse	TTTCAGTCACTTGCATGTCTTCT
ENST0000437350-forward	TGGTTTAGACACAACGGTGGA
ENST0000437350-reverse	TTCAAGCTCTTCTCCCCATGA
IL-17A-forward	AGATTACTACAACCGATCCACCT
IL-17A-reverse	GGGGACAGAGTTCATGTGGTA
IL-10-forward	GACTTTAAGGGTTACCTGGGTTG
IL-10-reverse	TCACATGCGCCTTGATGTCTG
IL-6-forward	ACTCACCTCTTCAGAACGAATTG
IL-6-reverse	CCATCTTTGGAAGGTTCAGGTTG
IL-4-forward	ATGGGTCTCACCTCCCAACT
IL-4-reverse	GATGTCTGTTACGGTCAACTCG
TNF-α-forward	GGAGGGGTCTTCCAGCTGGAGA
TNF-α-reverse	CAATGATCCCAAAGTAGACCTGC
β-actin-forward	AGAAAATCTGGCACCACACCT
β-actin-reverse	GATAGCACAGCCTGGATAGCA

Abbreviations: LncRNA, long noncoding RNA; TNF-α, tumor necrosis factor-alpha.

Figure 1 Differentially expressed lncRNAs in CD4⁺ T cells of the AECOPD group, stable COPD group, and control group.

Notes: (A–C) After normalization, the scatter plot showed difference in the expression of lncRNAs between different groups. Red, upregulated; green: downregulated; gray; not differentially expressed. (D–F) Hierarchical cluster analysis was performed to show the differentially expressed lncRNAs between different groups. Red represents higher expression level and green represents lower expression level.
Abbreviations: LncRNA, long noncoding RNA; AECOPD, acute exacerbation of COPD.

Figure 2 (A) Venn graph showed the overlap of differentially expressed lncRNAs between different groups. Yellow: upregulated lncRNAs in AECOPD vs control; purple: upregulated lncRNAs in AECOPD vs stable COPD; green: upregulated lncRNAs in stable COPD vs control; red: downregulated lncRNAs in stable COPD vs control. (B) Validation of the selected lncRNAs in CD4⁺ T cells in the AECOPD group compared with the stable COPD group and control by qRT-PCR. The relative expression levels of NR-026690 and ENST0000447867 are shown. **P<0.01, ***P<0.001. (C) ROC curve of lncRNAs. The diagnostic values of NR-026690 and ENST0000447867 are shown.

Abbreviations: LncRNA, long noncoding RNA; AECOPD, acute exacerbation of COPD; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; ROC, receiver operating characteristic.

Figure 3 (A) LncRNA-mRNA co-expression network. Green: lncRNA; red: mRNA; solid line: positive correlation; dotted line: negative correlation. (B) Venn graph showed the overlap of co-expressed mRNAs of NR-026690 and ENST0000447867.

Abbreviation: LncRNA, long noncoding RNA.

Figure 4 GO (A) and KEGG pathway analyses (B) of the overlapping part of co-expressed mRNAs of NR-026690 and ENST0000447867. The top ten items are listed. Note: The omitted text in A can be searched by the reader according to the GO number on the website http://wego.genomics.org.cn/tools.

Abbreviations: GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5 (A) Venn graph shows the overlap of co-expressed mRNAs and target mRNAs predicted by ceRNA analysis. Purple: target mRNAs of NR-026690 predicted by ceRNA analysis; yellow: co-expressed mRNAs of NR-026690; green: target mRNAs of ENST0000447867 predicted by ceRNA analysis; red: co-expressed mRNAs of ENST0000447867. (B) lncRNA-microRNA-mRNA network. Blue: lncRNA; green: microRNA; red: mRNA; blue line: relationship between lncRNA and microRNA; green line: relationship between microRNA and mRNA.

Abbreviations: LncRNA, long noncoding RNA; ceRNA, competing endogenous RNA.

Table 3 Spearman’s rank correlation test of cytokines and qRT-PCR-confirmed lncRNAs in CD4⁺ T cells from the AECOPD patients

Cytokines	NR-026690		ENST0000447867
	P-value	Spearman’s r	P-value	Spearman’s r
IL-17A	0.6349	−0.06811	0.7556	0.04561
IL-10	0.1600	−0.1997	0.3292	−0.1424
IL-6	0.7367	0.04875	0.5538	0.08761
IL-4	0.4842	−0.1012	0.3587	0.1354
TNF-α	0.9425	0.01047	0.1976	0.1893

Abbreviations: LncRNAs, long noncoding RNAs; TNF-α, tumor necrosis factor-α; AECOPD, acute exacerbation of COPD; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction.

Figure 6 (A) The transcript level of RAPGEF3 in CD4⁺ T cells of the different groups was determined by qRT-PCR. The transcript level was expressed as ln transformed and relative to the mean of the control group. *P<0.05, **P<0.01. (B) Correlation between NR-026690 and RAPGEF3 transcript levels in CD4⁺ T cells of AECOPD patients (n=51). (C) Correlation between ENST0000447867 and RAPGEF3 transcript levels in CD4⁺ T cells of AECOPD patients (n=51).

Abbreviations: LncRNA, long noncoding RNA; AECOPD, acute exacerbation of COPD; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; NC, negative control.

Figure S1 (A–E) The transcript level of cytokines in CD4⁺ T cells of the different groups was determined by qRT-PCR. *P<0.05, **P<0.01, ***P<0.005. (F–H) After normalization, the volcano plots showed the difference in the expression of lncRNAs between different groups.

Abbreviations: lncRNAs, long noncoding RNAs; AECOPD, acute exacerbation of COPD; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction.