RNA Sequencing and Bioinformatics Analysis to Reveal Potential Biomarkers in Patients with Combined Allergic Rhinitis and Asthma Syndrome

Figures & data

Table 1 Primers Sequence in RT-qPCR

Gene	Primer Sequence (5ʹto3ʹ)
GAPDH-F	CTGGGCTACACTGAGCACC
GAPDH-R	AAGTGGTCGTTGAGGGCAATG
ACTB-F	GATCAAGATCATTGCTCCTCCT
ACTB-R	TACTCCTGCTTGCTGATCCA
KRT8-F	AAGACCACCAGCGGCTATG
KRT8-R	ACACCAGCTTCCCATCACG
OVCH1-F	AAGCCGGGTGTATTTGCCAG
OVCH1-R	AACCTGGCCAAGATGCACTG
CLC-F	AGGCTGTGAAGATGGTGCAAGTG
CLC-R	ATGAGCAGGAGTAAGGATTGAAGTGAG
TEC-F	GCAGCAGGACTTGTCACCAG
TEC-R	TTTGCCAAGCCTCACCACTC
HEATR6-DT-F	GGAGGAAGGCTGCCAACTT
HEATR6-DT-R	ACAGGGTGGGTGGCTATTC
LINC00639-F	GTGACATCAAAGCTGTGCCG
LINC00639-R	GGGTTCCCGTGTTCATTCCT
LINC01783-F	GCACCCAATAAGCCCACCT
LINC01783-R	TTCCCAAGGCCTAACGAGC
RP11-178F10.2-F	AGCTCCCATGGAAGAAACGT
RP11-178F10.2-R	CTGAAGCAGCCACCCTCTT
OVCH1-AS1-F	CCGGTCACAGCACAGATCT
OVCH1-AS1-R	AGGTGAGAAGGGCCAAACAC
LINC00452-F	GACCGTGTCTTTCCCGTGT
LINC00452-R	TGCCCATTCCCTTACCTTG
cir-GAPDH-F	CTCCAAAATCAAGTGGGGC
cir-GAPDH-R	TGGTTCACACCCATGACGAA
hsa_circ_0007272-F	GACATTTGGCCCATTGGTTTCA
hsa_circ_0007272-R	TTTTGTCTTTCAGCCAGGGGT
hsa_circ_0008903-F	TGCTGCCAGACTACATGAGG
hsa_circ_0008903-R	GTCGGCCAAAGGTCTTTCAC
hsa_circ_0070934-F	GGTGAAAGGACTGATCAACCAT
hsa_circ_0070934-R	TGTCTTGAGCTTTCCTGCCT

Table 2 The Demographic Data of Recruited Subjects

Group	CARAS			AR			N
Gender	M	F	M	M	F	M	M	F	M
Age (years)	24	18	37	35	30	26	32	35	28
BMI (kg/m^2)	21.4	20.2	22.5	21.2	22.1	24.2	20.4	21.5	24.5
Duration (years)	3	5	12	10	11	5	N/A	N/A	N/A
Keep pets	No	No	No	No	No	No	No	No	No
Family history	No	No	Yes	No	No	No	No	No	No
Smoking history	No	No	No	No	No	No	No	No	No
Drinking history	No	No	No	No	No	No	No	No	No
FEV1	71.58%	92.62%	85%	85%	86.50%	87.60%	85%	86.7%	90.2%
FEV1/FVC	68.30%	102%	86%	98.60%	94.70%	92.60%	95.3%	96.70%	94.20%
Diastolic test	Positive	Positive	Positive	N/A	N/A	N/A	N/A	N/A	N/A
FeNO (ppb)	105	96	47	27	53	29	14	10	7
Eos (*10^9/L)	1.39	0.3	0.31	0.23	0.23	0.04	0.03	0.02	0.03
Eos	13.90%	5.30%	5.40%	4.70%	4.70%	1.40%	1.30%	1.10%	1.20%
IgE	(+)	(+)	(+)	(+)	(+)	(+)	(–)	(–)	(–)
Diagnosis methods^#	1 + 2	1 + 2	1 + 2	1	1	1	N/A	N/A	N/A
Complication	No	No	No	No	No	No	No	No	No

Notes: ^#1: clinical symptoms; 2: pulmonary function.

Abbreviations: CARAS, combined allergic rhinitis and asthma syndrome; AR, allergic rhinitis; N, healthy controls; M, male; F, female; BMI, body mass index; N/A, not applicable; FEV1, forced expiratory volume 1; FVC, forced vital capacity; FeNO, exhaled nitric oxide; Eos, eosinophil; IgE, immunoglobulin E.

Figure 1 DEmRNAs/DElncRNAs/DEcircRNAs in N, AR, and CARAS. (A) Venn diagram of DEmRNAs in N, AR, and CARAS. (B) The heatmap of common DEmRNAs in AR vs N and CARAS vs AR groups. (C) Venn diagram of DElncRNAs in N, AR, and CARAS. (D) The heatmap of common DElncRNAs in AR vs N and CARAS vs AR groups. (E) Venn diagram of DEcircRNAs in N, AR, and CARAS. (F) The heatmap of common DEcircRNAs in AR vs N and CARAS vs AR groups. The color scale represented the expression levels.

Abbreviations: DEmRNA, differentially expressed mRNA; DElncRNA, differentially expressed lncRNA; DEcircRNA, differentially expressed circRNA; N, healthy controls; AR, allergic rhinitis; CARAS, combined allergic rhinitis and asthma syndrome.

Figure 2 KEGG analysis (A), GSEA analysis (B), PPI network (C) and TF regulatory network (D) of common DEmRNAs in AR vs N and CARAS vs AR groups. Red and blue color represent up-regulation and down-regulation, respectively.

Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, Gene Set Enrichment Analysis; PPI, protein–protein interaction; TF, transcription factor; DEmRNA, differentially expressed mRNA; AR, allergic rhinitis; N, healthy controls; CARAS, combined allergic rhinitis and asthma syndrome.

Figure 3 LncRNA-mRNA cis-regulatory (A), trans-regulatory (B) and circRNA-mRNA co-expressed (C) networks. The rectangles, triangles and inverted triangles indicate mRNAs, lncRNAs and circRNAs, respectively. Red and blue color represents the up-regulation and down-regulation, respectively.

Figure 4 Validation in RT-qPCR. RT-qPCR, real time qPCR. Two-sample T-test was used for statistical analysis. **p < 0.01.

Figure 5 Validation in GSE19187 (A) and GSE141623 (B). ANOVA was used for statistical analysis.

Figure 6 The protein expression validation by ELISA. Two-sample T-test was used for statistical analysis. **p < 0.01; ***p < 0.001.

Abbreviation: ELISA, expression analysis by enzyme-linked immunosorbent assay.