Bioscience Reports (2023) 43 BSR20230005

https://doi.org/10.1042/BSR20230005

Research Article

Identification of circRNA-associated ceRNA

networks in peripheral blood mononuclear cells as
potential biomarkers for chronic obstructive
pulmonary disease
Shan Zhong1,2,3 , Chengshui Chen4 , Li Yang4 , Meiling Jin5 , Yiming Zeng6 , Gang-Ming Zou7 , Qingying Zhang2 and
Yun Wang1
1 Collegeof Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong 518055, PR China; 2 Department of Preventive Medicine, Shantou University Medical
College, Shantou, Guangdong 515041, PR China; 3 Institute of Precision Medicine, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518036, PR China; 4 Department of
Respiratory Medicine, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, PR China; 5 Department of Respiratory Medicine, Zhongshan Affiliated
Hospital of Fudan University, Shanghai 200030, PR China; 6 Department of Respiratory Medicine, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian 362000,
PR China; 7 School of Nursing and Dental Health. University of Hawaii at Manoa, 2528 McCarthy Mall, Webster Hall. Honolulu, HI 96822, USA
Correspondence: Yun Wang ([email protected]) and Qingying Zhang ([email protected])

Chronic obstructive pulmonary disease (COPD), which is a common respiratory dis-

order with high morbidity and mortality globally, has a complex pathogenesis that is
not fully understood. Some circular RNAs (circRNAs) have been recognized to serve
as miRNA sponges for regulating target RNA transcripts during the processes of hu-
man diseases. In the present study, we aimed to investigate novel circRNA-associated
biomarkers for COPD, 245 differentially expressed circRNAs were identified, including 111
up-regulated and 134 down-regulated circRNAs. These candidate circRNAs were enriched
in inflammation-associated pathways (such as mTOR, B-cell receptor, and NF-κB sig-
naling pathways) via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes
enrichment analyses. A combination of two circRNAs (up-regulated hsa circ 0067209
and down-regulated hsa circ 0000673) demonstrated good diagnostic value (area un-
der the receiver operating characteristic curve [AUC] = 0.866) for COPD by receiver
operating characteristic curve (ROC) analysis and qRT-PCR validation. Subsequently,
hsa-miR-8082 and hsa-miR-1248 were identified as targets for hsa circ 0067209 and
hsa circ 0000673, respectively, via bioinformatics analysis and a dual-luciferase reporter
assay, and the combination of these two miRNAs displayed better diagnosis potential
for COPD (AUC = 0.967) than each other. Evaluation of COPD-related mRNA profiles re-
vealed that the up-regulated genes ABR and TRPM6 were predicted downstream targets for
hsa circ 0067209/hsa-miR-8082, whereas the down-regulated gene RORC was a predicted
downstream target for hsa circ 0000673/hsa-miR-1248. In summary, hsa circ 0067209 and
hsa circ 0000673 have potential as novel diagnostic biomarkers of COPD. In addition, com-
peting endogenous RNA networks of hsa circ 0067209/hsa-miR-8082/ABR/TRPM6 and
* These authors are both
hsa circ 0000673/hsa-miR-1248/RORC may play critical regulation roles for COPD patho-
corresponding authors. genesis.
Received: 09 January 2023
Revised: 10 August 2023
Accepted: 24 August 2023

Accepted Manuscript online:

31 August 2023 Background
Version of Record published: Chronic obstructive pulmonary disease (COPD) is a heterogeneous respiratory disorder with chronic in-
31 October 2023 flammation and incomplete reversible airflow obstruction, which leads to higher morbidity and mortality

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 1
License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Table 1 Clinical information of participants used in circRNA microarray analysis

Characteristic Control (n=9) COPD (n=9)

Gender (male/female) 9/0 9/0

Age (years) 55.000 +
− 4.583 59.333 +
− 6.834
BMI (kg/m2 ) 24.873 +
− 3.061 22.076 +
− 3.833
Current/ex-smokers 7/9 6/9
Pulmonary functions
FVC (L) 2.939 +
− 0.482 1.781 +
− 0.776
FEV1 (L) 2.741 +
− 0.363 0.927 +
− 0.408
FEV1/FVC% 92.389 +
− 7.088 35.511 +
− 16.894
FEV1% predicted 98.676 +
− 9.788 49.701 +
− 16.503

and poses a heavy social and economic burden globally [1–3]. In 2018, a China Pulmonary Health (CPH) study by
Wang et al. [4] reported that the overall prevalence of spirometry-defined COPD in individuals >40 years of age had
increased by 13.7%, amounting to 99.9 million patients with COPD. A major etiological factor of COPD is long-term
exposure to noxious particles and/or gas [5]. However, the detailed pathophysiological mechanism of COPD is not
well understood.
Non-coding RNA (ncRNA) is classified into two subtypes: linear ncRNA and circular RNA (circRNA). Although
circRNA possesses the primary structural characteristics of linear ncRNA, it contains covalently closed loops without
a 5 cap and a 3 poly (A) tail, resulting in a configuration that is more conserved and stable than linear ncRNAs in the
cytoplasm of eukaryotic cells [6]. In the past decade, increasing evidence has shown that circRNAs play important
roles in the development of various diseases by regulating the expression and function of mRNAs, miRNAs, and
proteins [7–10].
Recently, the regulatory roles of circRNA in gene transcription and alternative splicing have been elucidated, in-
dicating that some circRNAs may serve as miRNA sponges or may combine with RNA-binding proteins to affect
RNA/protein synthesis and degradation [11]. For example, co-expression of cirs-7/CDR1as and miR-7 in nerve tis-
sues in vivo was closely associated with critical regulatory pathways in neurological disorders [12,13]. Moreover,
circRNAs have been recognized as regulators in the processes of cancer. For example, circRNA LARP4 acts as a
sponge for miR-424-5p to regulate LATS1 expression and inhibits cell proliferation in invasive gastric cancer [14]. In
addition, circMTO1 could be directly bound to matched microRNA in hepatocellular carcinoma, thereby behaving
as a prognostic biomarker [15]. Furthermore, aberrant levels of circRNA expression have been reported in a various
respiratory diseases, such as lung cancer [16,17], pulmonary arterial hypertension [18], asthma [19], and idiopathic
pulmonary fibrosis [20]. Zeng and coworkers [21] showed that cigarette smoke extract (CSE) could stimulate primary
human epithelial cells of small airways in a cellular model of COPD, suggesting that some circRNAs may perform
key roles through specific circRNA-mediated competing endogenous RNA (ceRNA) networks in COPD. However,
there were very few circRNA studies on COPD development. Consequently, in the present study, we aimed to screen
out COPD-related biomarkers from peripheral blood mononuclear cells (PBMCs) by a comparison between COPD
patients and healthy controls, and then explore potential roles of circRNA-miRNA networks and associated gene
effectors in the COPD process. The workflow of the present study is shown in Figure 1.

Methods
Clinical samples
Peripheral blood samples of all participants in the present study were collected at the First Affiliated Hospital of
Wenzhou Medical University (Wenzhou, China) from December 2017 to December 2019. The study was carried
out in accordance with the World Medical Association Declaration of Helsinki, approved by the Human Medical
Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Approval no. 2016131), and writ-
ten informed consent was provided by each participant. The participants were 40 to 80 years old and were grouped
based on their clinical respiratory symptoms. Participants with a history of respiratory symptoms (e.g., chronic cough,
wheezing, and/or expectoration) and a ratio of forced expiratory volume in 1st second (FEV1) to forced vital capacity
(FVC) less than 70% after inhalation of albuterol were defined as COPD. Exclusion criteria included a history of ma-
lignancy, cardiovascular diseases, Alzheimer’s disease, autoimmune disease, and other respiratory diseases (such as
bronchiectasis, bronchial asthma pulmonary fibrosis, and active tuberculosis, etc.). The basic clinical characteristics
of all subjects are provided in Tables 1-3.

2 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 1. The workflow of the present study

DEcircRNA, differentially expressed circRNA; DEmRNA, differentially expressed mRNA.

Table 2 Clinical information of participants used for circRNA validation

Characteristic Control (n=36) COPD (n=36)

Gender (male/female) 25/11 33/3

Age (years) 56.361 +
− 9.206 66.802 +
− 6.803
BMI (kg/m2 ) 24.123 +
− 3.165 21.406 +
− 3.572
Current/ex-smokers 17/23 10/30
Pulmonary functions
FVC (L) 3.064+
− 0.632 2.330 +
− 0.982
FEV1 (L) 2.582 +
− 0.531 1.458+
− 0.846
FEV1/FVC% 94.589+
− 16.159 53.406 +
− 29.509
FEV1% predicted 85.506 +
− 7.514 59.080 +
− 15.710

Table 3 Clinical information of patients used for miRNA validation

Characteristic Control (n=24) COPD (n=24)

Gender (male/female) 16/6 20/4

Age (years) 55.118 +
− 10.006 63.000 +
− 9.393
BMI (kg/m2 ) 24.384 +
− 2.839 20.331 +
− 2.828
Current/ex-smokers 11/16 10/19
Pulmonary functions
FVC (L) 3.395 +
− 0.658 2.312 +
− 1.024
FEV1 (L) 2.802 +
− 0.542 1.462 +
− 0.821
FEV1/FVC% 93.129 +
− 12.264 51.339 +
− 24.893
FEV1% predicted 85.488 +
− 9.571 55.639 +
− 16.024

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 3
License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Isolation of PBMCs and extraction of RNA

Peripheral blood samples from COPD patients and healthy controls were collected into EDTA-anticoagulated va-
cutainer tubes. PBMCs were isolated from 10 ml of blood sample per case by density gradient centrifugation using
human lymphocyte separation medium (Solarbio Life Sciences, China) and were immediately stored at −80◦ C until
the assay. Total RNA was extracted from the PBMCs of each case with the M5 HiPer Universal Plus RNA Mini Kit
(Mei5 Biotechnology, China), according to the kit instructions. The concentration of total RNAs were measured with
the Nanodrop ND-2000 spectrophotometer.

Microarray analysis
Total RNAs were initially extracted from PBMC samples of nine COPD patients and nine healthy subjects. Three
cases with equal quality of RNAs per group were pooled into one testing sample. Subsequently, three pooled samples
per group were applied to microarray testing. Approximately 3 μg of RNA per sample was digested with 3 U/μg of
RNase R (Epicentre, U.S.A.) for 20 min at 37◦ C to purify the circRNAs. The enriched circRNAs were then reversed
transcribed into cRNA utilizing fluorescent reagents with random primers, and were hybridized onto the Arraystar
Human circRNA Array V2 (8 × 15K, Arraystar, U.S.A.). The circRNA expression profiles were analyzed with the
Arraystar program and the limma package of R software. CircRNAs with |fold change| >2 and adjusted P-value
<0.05 were considered significantly differentially expressed. The disease-related circRNA candidates were screened
out based on the back-splice junction of the special structure of RNA and were confirmed by Sanger sequencing of
amplification products (Sangon Biotech, China).

RNA sequencing analysis

Specific libraries were constructed from three pooled-COPD RNA samples and three pooled-control RNA samples
after removal of rRNAs. Subsequently, paired-end sequencing, generating 150-bp reads, was performed on the Illu-
mina X Ten/Nova™ platform. The differentially expressed genes (DEGs) were obtained from a comparison between
COPD and control groups using the DESeq2 package of R software (Version 1.20.0, http://www.bioconductor.org/
packages/release/bioc/html/DESeq2.html). Genes with cutoff values of |fold change| >2 and adjusted P-value <0.05
were identified as DEGs.

Luciferase activity assay

Wild-type (WT) and mutated (MUT) hsa circ 0067209 sequences were co-transfected with NC-mimic or
hsa-miR-8082 mimic into HEK293T cells with the psiCHECK-2 Luciferase Reporter Vectors (Promega, U.S.A.).
In addition, WT and MUT hsa circ 0000673 sequences were co-transfected with NC-mimic or hsa-miR-1248 into
HEK293T cells using the same technique. Treated cells were lysed after incubating for 48 h, and luciferase activity
was measured by the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s protocol.

Quantitative real-time PCR (qRT-PCR)

Specific paired primers for qRT-PCR were designed and synthesized by Sangon Biotech (Supplementary Table S1).
Universal reverse primers and U6 primers were provided by the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa,
Japan), and used for miRNA expression analysis. cDNAs were synthesized from total RNAs with a cDNA synthesis kit
(TaKaRa) or the Mir-X miRNA First-Strand Synthesis Kit. Subsequently, qRT-PCR amplification was performed via
SYBR Green PCR Premix Ex TaqTM II reagents (TaKaRa) with the QuantStudio 6 FlexI real-time system (Applied
Biosystems, U.S.A.) following the protocol of this product. The levels of targeted genes were determined with the
2(−Ct) method in comparison with endogenous controls (GAPDH or U6).

Gene Expression Omnibus (GEO) datasets for mRNA validation

To validate the expression levels of target mRNAs in the network, two microarray datasets associated with COPD
(GSE57148 and GSE54837) were downloaded from the GEO public data repository (http://www.ncbi.nlm.nih.gov/
geo). The GSE57148 dataset contains 189 samples of lung tissues (including 91 cases of normal individual and 98 cases
of patients with COPD. The GSE54837 dataset contains 226 blood samples from patients with COPD in different
stages (Stage 1: n=90, Stage 2: n=68, Stage 3: n=55, Stage 4: n=13).

Biological function and signal pathway enrichment analyses

An online tool (https://cloud.oebiotech.cn) was used for functional annotation of candidate genes. Gene Ontology
(GO) analysis was performed to enrich biological functions of screened out DEGs, including molecular function,

4 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

cellular components, and biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was
applied to enrich signal pathways of disease-related DEGs.

Protein–protein interaction (PPI) network analysis

STRING version 11.0b (https://cn.string-db.org/), Cytoscape version 3.5 (https://cytoscape.org/), and the MCODE
app were applied for establishing the PPI networks of mRNA. Parameter sets were Network Scoring (Include Loops
= false, Degree Cutoff = 2) and Cluster Finding (Node Score Cutoff = 0.2, Haircut = true, Fluff = false, K-Core =
2, Max. Depth from Seed = 100).

Construction of circRNA-miRNA-mRNA networks

The Circular RNA Interactome online tool (https://circinteractome.irp.nia.nih.gov/), miRadna (http://www.
microrna.org/), and TargetScan (http://www.targetscan.org/) were used to predict targeted miRNAs of candidate cir-
cRNAs in the present study. The binding sites of circRNA-miRNA were visualized via a platform from the online web-
site (https://cloud.oebiotech.cn/). The interactions of miRNA-mRNA were predicted via miRDB (http://mirdb.org/)
and TargetScan software. Next, special circRNA-miRNA and miRNA-mRNA pairs were combined to construct
circRNA-miRNA-mRNA networks that were visualized by Cytoscape version 3.5 (https://cytoscape.org/).

Statistical analysis
GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, U.S.A.) and SPSS V21.0 (NY: IBM Corp, Armonk,
U.S.A.) were used as statistical tools. Student’s t-test and Mann–Whitney U-test were applied to measure the dif-
ferences of gene expression levels between COPD and control groups after normalizing data. Receiver operating
characteristic (ROC) curve analysis was performed to evaluate the power of candidate genes. The correlation of gene
expression and clinical characteristics in the cohort study was calculated using Spearman rank correlation. P<0.05
was considered statistically significant.

Results
Screening out COPD-related circRNAs by circRNA microarray
To screen for COPD-related circRNAs, the circRNA expression levels in PBMCs from COPD patients and healthy
controls (three pooled cases per group) were detected with a human circRNA microarray. Comparison and bioin-
formatics analyses were subsequently conducted to select disease-related differentially expressed circRNAs from the
COPD group versus the control group. Hierarchical clustering results (Figure 2A) showed that there were various
differentially expressed circRNAs between the COPD patients and healthy controls. Volcano plots based on the spe-
cific threshold of |fold change| >2 and P<0.05 were used to identify differentially expressed circRNAs between the
COPD patients and controls (Figure 2B). A total of 245 differentially expressed circRNAs were found in COPD pa-
tients, compared with healthy controls, including 111 up-regulated and 134 down-regulated circRNAs. The top 20
circRNAs are listed in Table 4, according to the values of expressive fold change. Analysis of the chromosome local-
ization and classification of the circRNA candidates in COPD (Figure 2C) indicated that the differentially expressed
circRNAs were widely distributed in all chromosomes but were primarily derived from chromosomes 1, 12, 16, and
19. Moreover, approximately three-quarters of the identified candidates were rooted in exons (Figure 2D).

GO and KEGG enrichment analysis of COPD-related circRNAs

GO analysis results are illustrated in Figure 3A. In molecular function, the COPD-related circRNAs were chiefly
incorporated into RNA binding and chromatin DNA binding. In cellular component, the circRNAs were primarily
included in the cytosol, focal adhesion, and nucleosome. In biological process, the candidate circRNAs were princi-
pally involved in the regulation of cell adhesion and nucleosome assembly. Concurrently, KEGG enrichment analysis
of the COPD-related candidate circRNAs indicated that they were primarily enriched in genetic information process-
ing consisting of ribosome and RNA transport, and in signal pathways such as the mTOR, B-cell receptor, and NF-κB
signaling pathways (Figure 3B).

Validation of candidate circRNAs in PBMCs of patients with COPD

Through expression abundance and specificity target sequencing (Supplementary Figure S1), ten differentially ex-
pressed circRNAs were selected for validation by qRT-PCR, including five up-regulated circRNAs (hsa circ 0010906,
hsa circ 0009362, hsa circ 0067209, hsa circ 0040823, and hsa circ 0023523) and five down-regulated cir-
cRNAs (hsa circ 0001535, hsa circ 0005699, hsa circ 0025460, hsa circ 0000673, and hsa circ 0008274). The

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 5
License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 2. Identification of differentially expressed circRNAs in COPD by microarray analysis

(A) Heatmaps of differentially expressed circRNAs between COPD patients and healthy controls. ‘Red’ indicates higher relative
expression, and ‘green’ indicates lower relative expression. (B) Volcano map of differentially expressed circRNAs from COPD and
control groups. The red and blue points in the plot represent the up-regulated and down-regulated circRNAs, respectively. The
circRNAs with fold change >2 and P<0.05 were considered to be differential genes, and ten circRNAs investigated in the present
study are annotated in the volcano plot. (C) The distribution of differentially expressed circRNAs located in the chromosomes. (D)
Types of differentially expressed circRNAs.

6 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 3. Enrichment analyses of differentially expressed circRNAs in COPD

Differentially expressed circRNAs were subjected to (A) GO analysis and (B) KEGG pathway analysis.

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 7
License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Table 4 Top 20 differentially expressed circRNAs related to COPD

circRNA ID Circbase ID Fold change P-value Gene symbol circRNA type

Upregulated circRNAs
hsa circRNA 089763 hsa circ 0089763 5.465 0.016 JA760600 Exonic
hsa circRNA 406587 - 4.890 0.008 TRIO Intronic
hsa circRNA 001678 hsa circ 0000517 4.399 0.018 RPPH1 Sense overlapping
hsa circRNA 101903 hsa circ 0040823 4.319 0.016 BANP Exonic
hsa circRNA 023523 hsa circ 0023523 4.298 0.017 UCP2 Exonic
hsa circRNA 009054 hsa circ 0009054 4.262 0.004 MCC Exonic
hsa circRNA 051238 hsa circ 0051238 4.149 0.002 ATP5SL Exonic
hsa circRNA 001065 hsa circ 0001065 3.912 0.005 GYPC Antisense
hsa circRNA 060102 hsa circ 0060102 3.784 0.049 ERGIC3 Exonic
hsa circRNA 040730 hsa circ 0040730 3.770 0.017 GSE1 Exonic
Downregulated circRNAs
hsa circRNA 101287 hsa circ 0008274 13.516 0.001 UGGT2 Exonic
hsa circRNA 406083 - 12.981 0.022 TASP1 Intronic
hsa circRNA 025460 hsa circ 0025460 9.997 0.001 YBX3 Exonic
hsa circRNA 101707 hsa circ 0000673 8.991 0.002 RSL1D1 Exonic
hsa circRNA 404837 - 8.093 0.003 NUP98 Intronic
hsa circRNA 101744 hsa circ 0005699 7.999 0.003 C16orf62 Exonic
hsa circRNA 001655 hsa circ 0001655 7.947 0.018 - Intergenic
hsa circRNA 050649 hsa circ 0050649 7.317 0.020 HSPB6 Exonic
hsa circRNA 100983 hsa circ 0024766 7.311 0.005 STT3A Exonic
hsa circRNA 033628 hsa circ 0033628 7.269 0.025 CRIP1 Exonic

qRT-PCR results indicated that the expression levels of hsa circ 0010906, hsa circ 0067209, hsa circ 0040823, and
hsa circ 0000673 had similar trends to those of the microarray analysis (Figure 4A). The expression levels of these
four circRNAs were reconfirmed with new collected 72 samples for extentional testing. The results showed that
hsa circ 0067209 was significantly up-regulated and hsa circ 0000673 was markedly down-regulated in COPD, com-
pared with the controls (Figure 4B). The circRNA hsa circ 0067209 was derived from the eukaryotic elongation fac-
tor selenocysteine-tRNA specific (EEFSEC) gene, which had a spliced sequence length of 470 bp and consisted of the
head-to-tail splicing of exons 2, 3, and 4 (Supplementary Figure S2A). The circRNA hsa circ 0000673 was derived
from exon regions 4 and 5 within the ribosomal L1 domain-containing protein 1 (RSL1D1) gene locus, and the spliced
mature sequence was 251 bp in length (Supplementary Figure S2B). The circular characteristics of hsa circ 0067209
and hsa circ 0000673 were verified after RNase R digestion, and both circRNAs exhibited more resistance to RNase
R digestion compared with that of matched linear mRNAs (Supplementary Figure S2C).

Diagnostic value evaluation for hsa circ 0067209 and hsa circ 0000673
in COPD
As a sensitive and reliable indicator, the lung function decrease (FEV1/FVC%≤70%) is the most important diagnosis
index of COPD [1]. In the present study, the clinical characteristics of all subjects suggested that abnormal expression
of hsa circ 0000673 and hsa circ 0067209 were significantly related to FEV1/FVC% (Figure 5A,B). Data from ROC
curve analysis showed that the area under the ROC curve (AUC) values of hsa circ 0067209 and hsa circ 0000673
were 0.710 (95% confidence interval [CI]: 0.591–0.829) and 0.708 (95% CI: 0.587–0.828), respectively. Moreover, the
AUC value of the two circRNAs in combination was 0.866 (95% CI: 0.782–0.950) (Figure 5C), suggesting that the
combination provided better diagnostic value than the circRNAs individually.

Verification of targeted miRNAs for hsa circ 0067209 or hsa circ 0000673
CircRNAs are recognized to have negative regulatory capability for miRNA expression, based on the hypothe-
sis of ceRNA [11–13]. In the present study, the interaction of targeted miRNAs with either hsa circ 0067209 or
hsa circ 0000673 was predicted via miRadna and TargetScan softwares, and 13 miRNAs could bind to matched nu-
cleic acid sequences in hsa circ 0067209 and hsa circ 0000673 (Figure 6A,B). Subsequently, the expression trends of
these candidate miRNAs were measured in PBMC samples from 48 subjects by qRT-PCR assay. By comparing with
healthy controls, the results confirmed a marked decrease in expression of hsa-miR-8082 in COPD, which showed

8 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 4. Expression validation of candidate circRNAs between COPD patients and controls
(A) Preliminary comparison of microarray and qRT-PCR data for the relative expression levels of selected circRNAs. (B) Expressions
of hsa circ 0010906, hsa circ 0067209, hsa circ 0040823, and hsa circ 0000673 were measured with 72 additional samples by
qRT-PCR. Control: n=36, COPD: n=36. Relative expression levels were presented as 2(−Ct) , GAPDH was used as an internal
reference; **P<0.01, *P<0.05.

an inverse correlation to the expression of hsa circ 0067209 (Figure 6C,E). In contrast, hsa-miR-1248 expression was
dramatically increased in COPD and was negatively associated with hsa circ 0000673 expression (Figure 6D,F).
The predicted interactions of circRNAs and miRNAs in the present study, as shown in Figure 7A,B, in-
cluded three binding sites for the interaction of hsa circ 0067209/hsa-miR-8082, and one binding site for
hsa circ 0000673/hsa-miR-1248. Concurrently, results from the dual-luciferase reporter assay indicated that
hsa-miR-8082 and hsa-miR-1248 mimics could significantly decrease the luciferase activity of hsa circ 0067209
and hsa circ 0000673, individually, in the WT group, but not in the MUT group (Figure 7C,D). This suggested
that there were direct links between hsa-miR-8082 and hsa circ 0067209, as well as between hsa-miR-1248 and
hsa circ 0000673. In addition, Spearman correlation analysis showed a negative correlation of the expression of
hsa-miR-1248 and FEV1/FVC% (Figure 8A). In contrast, hsa-miR-8082 expression was positively correlated with

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 9
License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 5. Correlation analysis of clinical characteristics and ROC curve analysis of hsa circ 0000673 and hsa circ 0067209
(A,B) Spearman correlation analysis for the link between the FEV1/FVC% of lung function and the expression of hsa circ 0000673
or hsa circ 0067209. (C) ROC curve analysis for hsa circ 0000673, hsa circ 0067209, and the combination of both circRNAs.

FEV1/FVC% (Figure 8B). The AUC values of hsa-miR-8082 and hsa-miR-1248 were 0.846 (95% CI: 0.736–0.957)
and 0.825 (95% CI: 0.705–0.944), respectively, whereas the AUC value of the combination of the two miRNAs was
0.967 (95% CI: 0.924–1.000) (Figure 8C). This suggested that a much better diagnostic value could be provided via
the combination of two miRNAs compared with a single-targeted miRNA.

Construction of circRNA-miRNA-mRNA networks involved in regulation of

the COPD process
Since most circRNAs regulate the expression of downstream genes positively through competitive binding with
matched miRNAs, the expression profiles of targeted mRNAs were evaluated in three pooled RNA samples per group
in PBMCs of COPD patients and healthy controls by RNA sequencing to identify functional circRNA-miRNA-mRNA
networks in the present study. Compared with healthy controls, there were 80 DEGs in patients with COPD, based
on a threshold (|fold change| >2 and adjusted P<0.05), including 44 up-regulated and 36 down-regulated genes
(Supplementary Figure S3A–C). The expression changes of all candidate genes are listed in Supplementary Table S2.
All 80 COPD-related DEGs were used to construct PPI networks. Consequently, four clusters were generated (see
Supplementary Figure S4A). Cluster 1 included seven genes (MMP8, OLFM4, BPI, CEACAM8, CXCL1, CAMP,
and DEFA3) that are primarily associated with immune response, neutrophil degranulation, and regulation of
macrophage activation and are enriched in the NOD-like receptor signaling pathways (Supplementary Figure S4B
and S4C). Subsequently, expression profiles of COPD-related DEGs and predicted target genes of hsa-miR-8082
and hsa-miR-1248 were employed to further reveal the molecular functions of circRNA-miRNA-mRNA networks.
Overall, seven downstream genes (TRPM6, ABR, MME, MMP8, MT-ND4L, LTF, and KCNJ15) corresponded to

10 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 6. Prediction and expression validation of sponging miRNAs of hsa circ 0067209 and hsa circ 0000673
(A,B) The interactive network of circRNA-miRNA was constructed for hsa circ 0067209 and hsa circ 0000673. (C,D) Expression
levels of predicted candidate miRNAs for hsa circ 0067209 or hsa circ 0000673 by qRT-PCR. Control: n=24, COPD: n=24. Relative
expression was presented as 2(−Ct) , and U6 was used as an internal reference. (E) Correlation analysis of hsa circ 0067209 and
hsa-miR-8082. (F) Correlation analysis hsa circ 0000673 and hsa-miR-1248; **P<0.01, *P<0.05.

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 11
License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 7. Hsa-miR-8082 and hsa-miR-1248 were direct targets for hsa circ 0067209 and hsa circ 0000673, respectively
(A,B) Potential binding sites were predicted for hsa circ 0067209/hsa-miR-8082 and hsa circ 0000673/hsa-miR-1248. (C,D) The di-
rected interaction of hsa circ 0067209/hsa-miR-8082 or hsa circ 0000673/hsa-miR-1248 was identified by dual-luciferase reporter
assay; *P<0.05, **P<0.01.

hsa circ 0067209/hsa-miR-8082, and eight genes (ADAMTS1, NEFL, RGS16, MYOM2, EFNB2, MDGA1, RORC,
and CD248) were the downstream targets of hsa circ 0000673/hsa-miR-1248 as interaction networks in the COPD
process (Figure 9A,B). Expression profiles of all predicted target genes for hsa-miR-8082 and hsa-miR-1248 were ver-
ified by comparing the GSE57148 dataset associated with COPD. This analysis showed that COPD-related TRPM6
and ABR were significantly up-regulated and RORC was significantly down-regulated when these gene expressions
were compared with those in healthy controls (Figure 9C,D). In addition, these findings were consistent with the
negative correlation of miRNAs to corresponding mRNAs.

12 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 8. Correlation analysis for clinical characteristics and ROC curve analysis of hsa-miR-1248 and hsa-miR-8082 for
COPD
(A,B) Spearman correlation analysis for the link between FEV1/FVC% and hsa-miR-1248 or hsa-miR-8082 expression. (C) ROC
curve analysis of hsa-miR-1248 or hsa-miR-8082, and a combination of that two.

Ultimately, expression levels of ABR, TRPM6, and RORC were detected in the blood of patients with COPD in
the clinical stages of disease (data derived from GEO dataset GSE54837), and based on the severity of disease, mRNA
expression levels of ABR and TRPM6 were increased and that of RORC was decreased (Figure 9E). The interactive
network maps of hsa circ 0067209/hsa-miR-8082/ABR/TRPM6 and hsa circ 0000673/hsa-miR-1248/RORC in the
present study were constructed by Cytoscape software (Figure 9F). Hsa circ 0067209 and hsa circ 0000673 may serve
as key diagnostic biomarkers for the early diagnosis of COPD and be important regulators of COPD pathogenesis via
ceRNA network.

Discussion
Owing to the high prevalence, morbidity, and mortality, COPD is a serious global threat to human life and health
[3,22]. In recent years, numerous studies have focused on screening and identifying novel diagnostic and therapeu-
tic biomarkers of COPD [23–25]. As a special and widespread type of endogenous non-coding RNA, circRNAs are
becoming more prominent because they exhibit critical roles in regulating many disease processes. However, there
are limited studies on the expression and function of circRNAs in patients with COPD. In the present study, 245
differentially expressed circRNAs, including 111 up-regulated and 134 down-regulated circRNAs, were screened out
as COPD-related candidates using microarray analysis. The differentially expressed circRNAs selected for transcrip-
tomics analysis were primarily derived from exons, which was consistent with their distribution in other diseases
[26,27]. Validation experiments showed that hsa circ 0067209 was significantly increased in the PBMC samples of
patients with COPD. The targeted host gene of hsa circ 0067209 is EEFCEC, which is a specialized elongation factor

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 13
License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Figure 9. Construction of COPD-related circRNA-miRNA-mRNA networks

(A) Up-regulated mRNAs predicted to bind to hsa-miR-8082. (B) Down-regulated mRNAs predicted to bind to hsa-miR-1248. (C,D)
Expression levels of targeted genes of hsa-miR-8082 and hsa-miR-1248 in lung tissues of COPD patients and negative controls
(COPD: n=98, Control: n=91). Recalculated datasets were downloaded from the NCBI GEO dataset GSE57148, and the result was
presented with probe intensity. (E) Expression levels of ABR, TRPM6, and RORC in the blood of patients with COPD were evaluated
(Stage 1: n=90, Stage 2: n=68, Stage 3: n=55, Stage 4: n=13), according to NCBI GEO dataset GSE54837. (F) Construction
of the hsa circ 0067209/hsa-miR-8082/ABR/TRPM6 network and the hsa circ 0000673/ hsa-miR-1248/RORC network; *P<0.05,
**P<0.01.

14 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

that is crucial in selenoprotein synthesis and may play an important role in maintaining oxidant/antioxidant bal-
ance and regulating inflammatory responses [28,29]. Therefore, it is of interest to better understand the roles of
hsa circ 0067209 in the pathogenesis of COPD. A circRNA that was significantly decreased in COPD patients in
the present study was hsa circ 0000673, which comprised the head-to-tail splicing of RSL1D1 exons 4 and 5. A pre-
vious study reported that overexpressed hsa circ 0000673 could act as an oncogene with promising diagnostic value
in cholangiocarcinoma [30]. Furthermore, hsa circ 0000673 could serve as a sponge for miR-767-3p by promoting
cell proliferation and invasion in the progression of liver cancer [31]. Collectively, these studies have demonstrated
that the expression of hsa circ 0000673 varies across different diseases.
Based on many reports [14,15,32–34], the effectors of exon circRNAs can serve as miRNA sponges to pro-
tect targeted mRNAs from miRNA-mediated degradation. CircRNA-miRNA-mRNA networks have crucial roles
in regulating the post-transcription of genes in numerous physiological and pathophysiological processes. To fur-
ther understand the effects of hsa circ 0067209 and hsa circ 0000673 in COPD pathogenesis, based on special
axises of circRNA-miRNA and/or miRNA-mRNA were used to construct the ceRNA cross-talk. The interactions of
hsa circ 0067209/hsa-miR-8082 or hsa circ 0000673/hsa-miR-1248 were subsequently identified by validating the
prediction and the regulation of miRNA response elements. Few reports have been published on hsa-miR-8082 re-
search; one clinical trial indicated that hsa-miR-8082 was significantly increased in the prodromal phase of Hunting-
ton’s disease [35]. In the present study, hsa-miR-8082 was found to be significantly decreased in PBMCs of patients
with COPD, compared with normal controls. Seven DEGs (TRPM6, ABR, MME, MMP8, MT-ND4L, LTF, and
KCNJ15) were identified as candidate target genes through integration analysis to identify the mRNA expression
profiles of COPD. The expression levels of TRPM6 and ABR could be increased due to the severity of COPD, which
was consistent with the expectations of the present study. TRPM6, a potential transient receptor channel, was pre-
viously reported to play a prominent role in regulating vertebrate embryonic development, hypomagnesemia, and
metabolic disorders, and may be a promising drug target [36,37]. ABR, an activator of RhoGEF and GTPase, is as-
sociated with mitosis in human embryonic stem cells, and acts as an apoptotic promoter in dissociated cells [38,39].
Several studies have found that apoptosis of lung structural cells is a factor in the pathogenesis of COPD [40–42]. Re-
cent studies have reported that the dysregulation of hsa-miR-1248 is associated with certain cancers [43–45], diabetes
mellitus [46], Sjögren’s syndrome [47], and aging [48], which depends on the roles of inflammatory responses greatly.
In the present study, eight mRNAs (ADAMTS1, NEFL, RGS16, MYOM2, EFNB2, MDGA1, RORC, and CD248)
were predicted to be target genes of hsa-miR-1248, with one of them-RORC-exhibiting decreased expression with
the severity of disease. RORC is a key transcription factor for the differentiation of Th17, which can control the ex-
pression of several inflammatory genes, and plays key roles in the pathogenesis of COPD [49,50]. Collectively, the
results of the present study and previous reports suggest that the hsa circ 0000673/hsa-miR-1248/RORC axis has a
potent effect on the progression of COPD by regulating the inflammatory response.
Clinical data have suggested that a decline in lung function could be accompanied by increased risks of both
morbidity and mortality in patients with COPD [51–53]. Therefore, the relationship of lung function and diagnos-
tic value of hsa circ 0067209/hsa-miR-8082 and/or hsa circ 0000673/hsa-miR-1248 in COPD were analyzed in the
present study. Both hsa circ 0067209 and hsa circ 0000673, as well as their miRNAs targets, were notably corre-
lated with FEV1/FVC%. ROC analysis indicated a better AUC value from the combination of hsa circ 0067209 and
hsa circ 0000673, as well as hsa-miR-8082 and hsa-miR-1248; the AUC values were 0.866 (95% CI: 0.782–0.950)
and 0.967 (95% CI: 0.924–1.000), respectively. This finding suggests that the expression levels of the two non-coding
RNAs have potential for the clinical application of COPD diagnosis and therapeutics.
Although there are a few novel discoveries revealed by the present study, some limitations remain in our work
(such as not big enough sample size, and less verifying tests of biological functions for that two circRNA-associated
ceRNA networks). Therefore, we will collect more related-samples of clinical patients and healthy controls, conduct
additional follow-up studies to elucidate candidate biomarker profiles, their signal pathways and mechanisms for
regulating the COPD process in vivo and in vitro.

Conclusions
The present study identifys two novel COPD-related circRNAs, and constructed their own circRNA-associated
ceRNA networks (hsa circ 0067209/hsa-miR-8082/ABR/ and hsa circ 0000673/hsa-miR-1248/RORC). New find-
ings demonstrate that the expression levels of the two novel circRNAs and their targets might have potential biomarker
values for precision diagnosis and therapeutic intervention in patients with COPD.

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons 15
Attribution License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Competing Interests
The authors declare that there are no competing interests associated with the manuscript.

Funding
This study was supported by the Shenzhen Basic Research Program of Science and Technology Innovation Commission [grant
number JCYJ20190808122413582] and National Key R&D Program of China [grant number 2016YFC1304000].

CRediT Author Contribution

Shan Zhong: Conceptualization, Data curation, Software, Validation, Investigation, Visualization, Methodology, Writing—original
draft, Project administration, Writing—review & editing. Chengshui Chen: Resources, Data curation. Li Yang: Resources, In-
vestigation. Meiling Jin: Resources, Funding acquisition. Yiming Zeng: Resources, Funding acquisition. Gang-Ming Zou:
Writing—review & editing. Qingying Zhang: Supervision, Project administration, Writing—review & editing. Yun Wang: Conceptu-
alization, Supervision, Funding acquisition, Project administration, Writing—review & editing.

Ethics Approval
Human Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Approval no. 2016131).

Acknowledgements
The authors are grateful to the Instrumental Analysis Center of Shenzhen University for providing research instruments.

Abbreviations
CI , confidence interval; circRNA , circular RNA; COPD, chronic obstructive pulmonary disease; DEG, differentially expressed
gene; PPI, protein–protein interaction; qRT-PCR, quantitative real-time PCR; ROC, receiver operating characteristic curve.

References
1 (2020) Global Strategy for Diagnosis, Management and Prevention of COPD. The Global Initiative for Chronic Obstructive Lung Diseases (GOLD). report.
Available from: https://goldcopd.org/gold-reports/Access: 01.01.2020]
2 GBD 2015 Chronic Respiratory Disease Collaborators (2017) Global, regional, and national deaths, prevalence, disability-adjusted life years, and years
lived with disability for chronic obstructive pulmonary disease and asthma, 1990-2015: a systematic analysis for the global burden of disease study
2015. Lancet Respir. Med. 5, 691–706, https://doi.org/10.1016/S2213-2600(17)30293-X
3 Ur Rehman, A., Ahmad Hassali, M.A., Muhammad, S.A., Shah, S., Abbas, S., Hyder Ali, I.A.B. et al. (2020) The economic burden of chronic obstructive
pulmonary disease (COPD) in the USA, Europe, and Asia: results from a systematic review of the literature. Expert. Rev. Pharmacoecon. Outcomes Res.
20, 661–672, https://doi.org/10.1080/14737167.2020.1678385
4 Wang, C., Xu, J., Yang, L., Xu, Y., Zhang, X., Bai, C. et al. (2018) Prevalence and risk factors of chronic obstructive pulmonary disease in China (the
China Pulmonary Health [CPH] study): a national cross-sectional study. Lancet 391, 1706–1717, https://doi.org/10.1016/S0140-6736(18)30841-9
5 Salvi, S. (2014) Tobacco smoking and environmental risk factors for chronic obstructive pulmonary disease. Clin. Chest Med. 35, 17–27,
https://doi.org/10.1016/j.ccm.2013.09.011
6 Kristensen, L.S., Andersen, M.S., Stagsted, L.V.W., Ebbesen, K.K., Hansen, T.B. and Kjems, J. (2019) The biogenesis, biology and characterization of
circular RNAs. Nat. Rev. Genet. 20, 675–691, https://doi.org/10.1038/s41576-019-0158-7
7 Panni, S., Lovering, R.C., Porras, P. and Orchard, S. (2020) Non-coding RNA regulatory networks. Biochim. Biophys. Acta Gene Regul. Mech. 1863,
194417–194427, https://doi.org/10.1016/j.bbagrm.2019.194417
8 Zhong, S., Chen, C., Liu, N., Yang, L., Hu, Z., Duan, P. et al. (2019) Overexpression of hsa-miR-664a-3p is associated with cigarette smoke-induced
chronic obstructive pulmonary disease via targeting FHL1. Int. J. Chron. Obstruct. Pulmon. Dis. 14, 2319–2329,
https://doi.org/10.2147/COPD.S224763
9 Zheng, M., Hong, W., Gao, M., Yi, E., Zhang, J., Hao, B. et al. (2019) Long noncoding RNA COPDA1 promotes airway smooth muscle cell proliferation in
chronic obstructive pulmonary disease. Am. J. Respir. Cell Mol. Biol. 61, 584–596, https://doi.org/10.1165/rcmb.2018-0269OC
10 Ma, H., Lu, L., Xia, H., Xiang, Q., Sun, J., Xue, J. et al. (2020) Circ0061052 regulation of FoxC1/Snail pathway via miR-515-5p is involved in the
epithelial-mesenchymal transition of epithelial cells during cigarette smoke-induced airway remodeling. Sci. Total Environ. 746, 141181–141192,
https://doi.org/10.1016/j.scitotenv.2020.141181
11 Li, X., Yang, L. and Chen, L.L. (2018) The biogenesis, functions, and challenges of circular RNAs. Mol. Cell 71, 428–442,
https://doi.org/10.1016/j.molcel.2018.06.034
12 Hansen, T.B., Jensen, T.I., Clausen, B.H., Bramsen, J.B., Finsen, B., Damgaard, C.K. et al. (2013) Natural RNA circles function as efficient microRNA
sponges. Nature 495, 384–388, https://doi.org/10.1038/nature11993

16 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY).
Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

13 Memczak, S., Jens, M., Elefsinioti, A., Torti, F., Krueger, J., Rybak, A. et al. (2013) Circular RNAs are a large class of animal RNAs with regulatory
potency. Nature 495, 333–338, https://doi.org/10.1038/nature11928
14 Zhang, J., Liu, H., Hou, L., Wang, G., Zhang, R., Huang, Y. et al. (2017) Circular RNA LARP4 inhibits cell proliferation and invasion of gastric cancer by
sponging miR-424-5p and regulating LATS1 expression. Mol. Cancer 6, 151–166, https://doi.org/10.1186/s12943-017-0719-3
15 Han, D., Li, J., Wang, H., Su, X., Hou, J., Gu, Y. et al. (2017) Circular RNA circMTO1 acts as the sponge of microRNA-9 to suppress hepatocellular
carcinoma progression. Hepatology 66, 1151–1164, https://doi.org/10.1002/hep.29270
16 Qiu, B.Q., Zhang, P.F., Xiong, D., Xu, J.J., Long, X., Zhu, S.Q. et al. (2019) CircRNA fibroblast growth factor receptor 3 promotes tumor progression in
non-small cell lung cancer by regulating Galectin-1-AKT/ERK1/2 signaling. J. Cell. Physiol. 234, 11256–11264, https://doi.org/10.1002/jcp.27783
17 Cheng, Z., Yu, C., Cui, S., Wang, H., Jin, H., Wang, C. et al. (2019) circTP63 functions as a ceRNA to promote lung squamous cell carcinoma
progression by upregulating FOXM1. Nat. Commun. 10, 3200–3213, https://doi.org/10.1038/s41467-019-11162-4
18 Zhou, S., Jiang, H., Li, M., Wu, P., Sun, L., Liu, Y. et al. (2019) Circular RNA hsa circ 0016070 is associated with pulmonary arterial hypertension by
promoting PASMC proliferation. Mol. Ther. Nucleic Acids 18, 275–284, https://doi.org/10.1016/j.omtn.2019.08.026
19 Huang, Z., Cao, Y., Zhou, M., Qi, X., Fu, B., Mou, Y. et al. (2019) Hsa circ 0005519 increases IL-13/IL-6 by regulating hsa-let-7a-5p in CD4+ T cells to
affect asthma. Clin. Exp. Allergy 49, 1116–1127, https://doi.org/10.1111/cea.13445
20 Li, R., Wang, Y., Song, X., Sun, W., Zhang, J., Liu, Y. et al. (2018) Potential regulatory role of circular RNA in idiopathic pulmonary fibrosis. Int. J. Mol.
Med. 42, 3256–3268, https://doi.org/10.3892/ijmm.2018.3892
21 Zeng, N., Wang, T., Chen, M., Yuan, Z., Qin, J., Wu, Y. et al. (2019) Cigarette smoke extract alters genome-wide profiles of circular RNAs and mRNAs in
primary human small airway epithelial cells. J. Cell. Mol. Med. 23, 5532–5541, https://doi.org/10.1111/jcmm.14436
22 Singh, D., Agusti, A., Anzueto, A., Barnes, P.J., Bourbeau, J., Celli, B.R. et al. (2019) Global strategy for the diagnosis, management, and prevention of
chronic obstructive lung disease: the GOLD science committee report 2019. Eur. Respir. J. 53, 1900164–1900186,
https://doi.org/10.1183/13993003.00164-2019
23 Hollander, Z., DeMarco, M.L., Sadatsafavi, M., McManus, B.M., Ng, R.T. and Sin, D.D. (2017) Biomarker development in COPD: moving from P values to
products to impact patient care. Chest 151, 455–467, https://doi.org/10.1016/j.chest.2016.09.012
24 Fermont, J.M., Masconi, K.L., Jensen, M.T., Ferrari, R., Di Lorenzo, V.A.P., Marott, J.M. et al. (2019) Biomarkers and clinical outcomes in COPD: a
systematic review and meta-analysis. Thorax 74, 439–446, https://doi.org/10.1136/thoraxjnl-2018-211855
25 Shi, T. and Feng, L. (2022) Blood biomarkers associated with acute type II respiratory failure in COPD: A meta-analysis. Clin. Respir. J. 16, 75–83,
https://doi.org/10.1111/crj.13464
26 Shao, Y., Li, J., Lu, R., Li, T., Yang, Y., Xiao, B. et al. (2017) Global circular RNA expression profile of human gastric cancer and its clinical significance.
Cancer Med. 6, 1173–1180, https://doi.org/10.1002/cam4.1055
27 Zhao, W., Su, J., Wang, N., Zhao, N. and Su, S. (2021) Expression profiling and bioinformatics analysis of CircRNA in mice brain infected with rabies
virus. Int. J. Mol. Sci. 22, 6537–6553, https://doi.org/10.3390/ijms22126537
28 Simonović, M. and Puppala, A.K. (2018) On elongation factor eEFSec, its role and mechanism during selenium incorporation into nascent
selenoproteins. Biochim. Biophys. Acta Gen. Subj. 1862, 2463–2472, https://doi.org/10.1016/j.bbagen.2018.03.018
29 Labunskyy, V.M., Hatfield, D.L. and Gladyshev, V.N. (2014) Selenoproteins: molecular pathways and physiological roles. Physiol. Rev. 94, 739–777,
https://doi.org/10.1152/physrev.00039.2013
30 Zhao, X., Zhang, X., Zhang, Z., Liu, Z., Zhu, J., Lyu, S. et al. (2020) Comprehensive circular RNA expression profiling constructs a ceRNA network and
identifies hsa circ 0000673 as a novel oncogene in distal cholangiocarcinoma. Aging (Albany NY) 12, 23251–23274,
https://doi.org/10.18632/aging.104099
31 Jiang, W., Wen, D., Gong, L., Wang, Y., Liu, Z. and Yin, F. (2018) Circular RNA hsa circ 0000673 promotes hepatocellular carcinoma malignance by
decreasing miR-767-3p targeting SET. Biochem. Biophys. Res. Commun. 500, 211–216, https://doi.org/10.1016/j.bbrc.2018.04.041
32 Chen, Q., Liu, T., Bao, Y., Zhao, T., Wang, J., Wang, H. et al. (2020) CircRNA cRAPGEF5 inhibits the growth and metastasis of renal cell carcinoma via
the miR-27a-3p/TXNIP pathway. Cancer Lett. 469, 68–77, https://doi.org/10.1016/j.canlet.2019.10.017
33 Zhou, C., Liu, H.S., Wang, F.W., Hu, T., Liang, Z.X., Lan, N. et al. (2020) circCAMSAP1 promotes tumor growth in colorectal cancer via the
miR-328-5p/E2F1 axis. Mol. Ther. 28, 914–928, https://doi.org/10.1016/j.ymthe.2019.12.008
34 Kong, Z., Wan, X., Lu, Y., Zhang, Y., Huang, Y., Xu, Y. et al. (2020) Circular RNA circFOXO3 promotes prostate cancer progression through sponging
miR-29a-3p. J. Cell. Mol. Med. 24, 799–813, https://doi.org/10.1111/jcmm.14791
35 Reed, E.R., Latourelle, J.C., Bockholt, J.H., Bregu, J., Smock, J., Paulsen, J.S. et al. (2018) MicroRNAs in CSF as prodromal biomarkers for Huntington
disease in the PREDICT-HD study. Neurology 90, e264–e272, https://doi.org/10.1212/WNL.0000000000004844
36 Chubanov, V. and Gudermann, T. (2014) TRPM6. Handb. Exp. Pharmacol. 222, 503–520, https://doi.org/10.1007/978-3-642-54215-2˙20
37 Runnels, L.W. and Komiya, Y. (2020) TRPM6 and TRPM7: Novel players in cell intercalation during vertebrate embryonic development. Dev. Dyn. 249,
912–923, https://doi.org/10.1002/dvdy.182
38 Ohgushi, M., Minaguchi, M., Eiraku, M. and Sasai, Y. (2017) A RHO small GTPase regulator ABR secures mitotic fidelity in human embryonic stem cells.
Stem Cell Rep. 9, 58–66, https://doi.org/10.1016/j.stemcr.2017.05.003
39 Vaughan, E.M., Miller, A.L., Yu, H.Y. and Bement, W.M. (2011) Control of local Rho GTPase crosstalk by Abr. Curr. Biol. 21, 270–277,
https://doi.org/10.1016/j.cub.2011.01.014
40 Demedts, I.K., Demoor, T., Bracke, K.R., Joos, G.F. and Brusselle, G.G. (2006) Role of apoptosis in the pathogenesis of COPD and pulmonary
emphysema. Respir. Res. 7, 53–62, https://doi.org/10.1186/1465-9921-7-53
41 Song, Q., Chen, P. and Liu, X.M. (2021) The role of cigarette smoke-induced pulmonary vascular endothelial cell apoptosis in COPD. Respir. Res. 22,
39–53, https://doi.org/10.1186/s12931-021-01630-1

© 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons 17
Attribution License 4.0 (CC BY).
 Bioscience Reports (2023) 43 BSR20230005
https://doi.org/10.1042/BSR20230005

42 Lee, H., Park, J.R., Kim, E.J., Kim, W.J., Hong, S.H., Park, S.M. et al. (2016) Cigarette smoke-mediated oxidative stress induces apoptosis via the
MAPKs/STAT1 pathway in mouse lung fibroblasts. Toxicol. Lett. 240, 140–148, https://doi.org/10.1016/j.toxlet.2015.10.030
43 Yang, T., Li, M., Li, H., Shi, P., Liu, J. and Chen, M. (2020) Downregulation of circEPSTI1 represses the proliferation and invasion of non-small cell lung
cancer by inhibiting TRIM24 via miR-1248 upregulation. Biochem. Biophys. Res. Commun. 530, 348–354, https://doi.org/10.1016/j.bbrc.2020.06.106
44 Tanic, M., Yanowski, K., Gómez-López, G., Rodriguez-Pinilla, M.S., Marquez-Rodas, I., Osorio, A. et al. (2015) MicroRNA expression signatures for the
prediction of BRCA1/2 mutation-associated hereditary breast cancer in paraffin-embedded formalin-fixed breast tumors. Int. J. Cancer 136, 593–602,
https://doi.org/10.1002/ijc.29021
45 Zhang, L., Chen, J., Cheng, T., Yang, H., Pan, C. and Li, H. (2020) Identification of Differentially Expressed Genes and miRNAs Associated with
Esophageal Squamous Cell Carcinoma by Integrated Analysis of Microarray Data. Biomed. Res. Int. 2020, 1980921–1980936,
https://doi.org/10.1155/2020/1980921
46 Xiao, S., Zhang, D., Liu, Z., Jin, W., Huang, G., Wei, Z. et al. (2020) Diabetes-induced glucolipotoxicity impairs wound healing ability of adipose-derived
stem cells-through the miR-1248/CITED2/HIF-1α pathway. Aging (Albany NY) 12, 6947–6965, https://doi.org/10.18632/aging.103053
47 Jang, S.I., Tandon, M., Teos, L., Zheng, C., Warner, B.M. and Alevizos, I. (2019) Dual function of miR-1248 links interferon induction and calcium
signaling defects in Sjögren’s syndrome. EBioMed. 48, 526–538, https://doi.org/10.1016/j.ebiom.2019.09.010
48 Noren Hooten, N., Fitzpatrick, M., Wood, 3rd, W.H., De, S., Ejiogu, N., Zhang, Y. et al. (2013) Age-related changes in microRNA levels in serum. Aging
(Albany NY) 5, 725–740, https://doi.org/10.18632/aging.100603
49 Ivanov, I.I., McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., Lafaille, J.J. et al. (2006) The orphan nuclear receptor RORgammat directs the
differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133, https://doi.org/10.1016/j.cell.2006.07.035
50 Zhang, J.C., Chen, G., Chen, L., Meng, Z.J., Xiong, X.Z., Liu, H.J. et al. (2016) TGF-β/BAMBI pathway dysfunction contributes to peripheral Th17/Treg
imbalance in chronic obstructive pulmonary disease. Sci. Rep. 6, 31911–31921, https://doi.org/10.1038/srep31911
51 Drummond, M.B., Hansel, N.N., Connett, J.E., Scanlon, P.D., Tashkin, D.P. and Wise, R.A. (2012) Spirometric predictors of lung function decline and
mortality in early chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 185, 1301–1306,
https://doi.org/10.1164/rccm.201202-0223OC
52 Baughman, P., Marott, J.L., Lange, P., Martin, C.J., Shankar, A., Petsonk, E.L. et al. (2012) Combined effect of lung function level and decline increases
morbidity and mortality risks. Eur. J. Epidemiol. 27, 933–943, https://doi.org/10.1007/s10654-012-9750-2
53 Jin, J., Liu, X. and Sun, Y. (2014) The prevalence of increased serum IgE and Aspergillus sensitization in patients with COPD and their association with
symptoms and lung function. Respir. Res. 15, 130–141, https://doi.org/10.1186/s12931-014-0130-1

18 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).