Access the full text.
Sign up today, get DeepDyve free for 14 days.
Circular RNAs (circRNAs) have been reported to be related to the initiation and progression of chronic obstructive pulmonary disease (COPD) by affecting the function of human bronchial epithelial cells (HBECs). Here, we aimed to investigate the function and mechanism of circ_0006872 in regulating COPD process using cigarette smoke extract (CSE)-induced 16HBEC in vitro. The results showed that circ_0006872 was increased in smokers without or with COPD, especially in smokers with COPD. Also, its expression was dose-dependently up-regulated by CSE exposure in 16HBECs. Functionally, circ_0006872 knockdown dramatically attenuated CSE-evoked proliferation arrest, apoptosis, inflammatory response and oxidative stress in 16HBECs. Mechanistically, circ_0006872/miR-485-3p/cyclin-dependent kinase inhibitor 1B (CDKN1B) formed a competitive endogenous RNA (ceRNA) network. CDKN1B was increased and miR-485-3p was decreased in COPD patients and CSE-induced 16HBECs. MiR-485-3p overexpression or CDKN1B knockdown protected 16HBEC against CSE-induced 16HBEC injury mentioned above. Moreover, rescue experiments showed that circ_0006872 regulated CSE-induced 16HBEC injury via miR-485-3p/CDKN1B axis. Circ_0006872 silencing protected against CSE-induced bronchial epithelial cell injury via miR-485-3p/CDKN1B axis, suggesting the potential application of circ_0006872 in preventing cigarette smoke-induced COPD. Keywords Circ_0006872, MiR-485-3p, CDKN1B, COPD, CSE, Smoke Introduction mechanisms are positively implicated in COPD patho- Chronic obstructive pulmonary disease (COPD) is a genesis . Bronchial epithelial cells are the first bar - common chronic inflammatory respiratory disease rier to protect airways against harmful substances, mainly caused by cigarette smoking (CS) [1, 2]. Smok- the integrity of which is responsible in response to CS ing-induced COPD is partly related to the inflammation, . Therefore, in-depth investigations on the molecu - which can also give rise to the elevation of reactive oxy- lar mechanism underlying bronchial epithelial cell dis- gen species resulting in oxidative stress activation [3, 4]. ruption is of great importance for preventing COPD Besides, it has been proposed that increased apoptosis progression. Circular RNAs (circRNAs) are a new kind of non-cod- ing molecules possessing a covalently closed continuous *Correspondence: loop, so they are failed to be degraded by RNase R exonu- Chuanbo Wang firstname.lastname@example.org clease and more stable than linear RNA [7, 8]. CircRNAs Respiratory Department, Tongde hospital of Zhejiang Province, are widely identified in the eukaryotes, and emerging Hangzhou, China 2 reports suggest that they are engaged in modulating Respiratory Department, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, China important biological processes [9, 10]. Recently, some Respiratory Department, The Second Hospital of Anhui Medical reports proposed that dysregulated expression of circR- University, No.678, Furong Road, Hefei 230601, Anhui, China NAs is tightly related to the initiation and progression of © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Wang et al. Applied Biological Chemistry (2023) 66:14 Page 2 of 12 CODP. Zheng et al. showed that silencing of circ-OSBPL2 with serum-free medium into different concentrations could suppress human bronchial epithelial cells (HBECs) for the use in experiments within 1 h. injury in smoke-related COPD by repressing BRD4 through sponging miR-193a-5p . Down-regulation of circ-HACE1 was found to protect HBECs from cigarette Cell culture and treatment smoke extract (CSE)-induced inflammatory response, 16HBECs were obtained from Procell (Wuhan, China) oxidative stress and apoptosis by miR-485-3p/TLR4 and grown in RPMI1640 medium (Gibco, Shanghai, axis . Zhou’s team showed that circFOXO3 knock- China) plus 1% penicillin/streptomycin (Gibco) and down might exert protective effects against pneumonic 10% FBS (Gibco) at 37 ℃ with 5% CO . The prepared inflammation in CS-exposed COPD mice model through 16HBECs at 70–80% confluence were exposed to dif - repressing IKK-β via miR-214-3p . Circ_0006872 is ferent concentrations of CSE for 24 h for experimental generated by the back-splicing of ASCC3 gene on chr6: purpose. 101,073,067–101,086,697. A previous study manifested that circ_0006872 level was higher in COPD patients, and might contribute to CSE-triggered HBECs dam- Cell transfection age through miR-145-5p/NF-κB pathway . However, Small interfering RNA (siRNA) targeting circ_0006872 or large-scale identifications of the mechanism underlying CDKN1B (si-circ_0006872 or si-CDKN1B) and the non- circ_0006872 in CSE-induced HBEC dysfunction were target siRNA (si-NC), miR-485-3p mimic (miR-485-3p), not yet reported. inhibitor (anti-miR-485-3p) and negative control oligos Hence, this study used CSE-treated 16HBECs, a human (miR-NC or anti-miR-NC), pcDNA3.1 CDKN1B overex- bronchial epithelial cell line, to mimic CS-induced pression plasmids (CDKN1B) and scrambled pcDNA3.1 COPD in vitro, then the functions of circ_0006872 on plasmids (pcDNA) was constructed by Sangon (Shang- CSE-evoked bronchial epithelial cell injury were inves- hai, China). After 48 h of transfection, 16HBECs were tigated. It has been proposed that circRNAs can act incubated with 2% CSE for 24 h to conduct subsequent as the sponges for microRNA (miRNA/miR) to par- functional experiments. ticipate in the process of gene translation, namely com- petitive endogenous RNA (ceRNA) hypothesis [15, 16]. Furthermore, the underlying miRNA/mRNA axis of Reverse transcription, RNase R digestion and qRT-PCR circ_0006872 on CSE-induced 16HBEC injury was also Total RNA was extracted by the use of TRIzol reagent explored to state the potential regulatory network of (Takara, Dalian, China). Approximately 3 µg of total circ_0006872 in COPD. RNAs were mixed with RNase R (3 U/µg) or Mock for 30 min incubation at 37 ℃. The PrimeScript RT Reagent Kit (Takara) was applied for the generation of first-strand Materials and methods cDNA, then qRT-PCR analysis with the TB Green Pre- Subjects mix Ex Taq II (Takara) was carried out. U6 or β-actin was Blood samples (5 mL) were collected from non-smokers utilized as an internal control. The sequences of primers without COPD (n = 35), and smokers without (n = 35) were listed in Table 1. or with COPD (n = 35). All subjects were recruited at Tongde hospital of Zhejiang Province. The diagnosis of COPD patients was based on the guidelines of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) Table 1 Primers sequences used for qRT-PCR . The exclusion criteria included interstitial lung Name Primers for qRT-PCR (5’-3’) diseases, asthma, neuromuscular disease, and/or heart failure. circ_0006872 ForwardTTG ATC GCC TTC CTG GCT AC ReverseGGC TGA AGT TGT ACA GGG CT miR-485-3p ForwardGCC GAG GTC ATA CAC GGC TCTC Cigarette smoke extract (CSE) preparation ReverseCAG TGC GTG TCG TGG AGT The mainstream smoke from two cigarettes (Jinsheng CDKN1B ForwardTCG GGG TCT GTG TCT TTT GG Tobacco Corporate Ltd., Nanchang, China) containing ReverseAGA CAC TCG CAC GTT TGA CA 0.1 g nicotine was bubbled through 20 mL of cell growth GAPDH ForwardGAC AGT CAG CCG CAT CTT CT medium. After removing insoluble particles by filtration ReverseGCG CCC AAT ACG ACC AAA TC with a 0.22 μm filter membrane (Merck Millipore, Shang - U6 ForwardCTC GCT TCG GCA GCACA hai, China), the resultant CSE solution was collected and ReverseAAC GCT TCA CGA ATT TGC GT regarded as 100% CSE solution, and then was diluted W ang et al. Applied Biological Chemistry (2023) 66:14 Page 3 of 12 Cell counting kit-8 (CCK-8) assay a commercial Superoxide Dismutase (SOD) assay kit. Single 16HBEC suspensions (1 × 10 cells/mL) were Finally, the OD value at 450 nm was assayed. cultivated in a 96-well plate (0.1 mL per well) overnight, then per well was added with 10 µL CCK-8 solution Dual-luciferase reporter assay for 2 h incubation. At last, OD values at 450 nm were The fragments of circ_0006872 or CDKN1B 3’UTR com - read using a microplate reader (Bio-Rad, Hercules, CA, prising the binding sites of miR-485-3p or the mutant USA) to represent cell viability ability. version without miR-485-3p binding sites were amplified and cloned into psiCHECK -2 vector (Promega, Madi- son, WI, USA) to construct wild-type (WT) or mutated EdU assay (MUT) luciferase reporter vector, named as WT/MUT- 16HBECs were seeded into a 24-well plate all night and circ_0006872 or WT/MUT-CDKN1B 3ʹUTR. Then 50 ng incubated with 50 µM EdU labeling solution (RiboBio) above recombinant plasmids and 50 nM miR-485-3p in growth medium for 3 h. Then cells were stained with mimic or the control (miR-NC) were co-transfected into Click-It reaction mixture for 30 min, followed by DAPI 16HBECs, and firefly activities were detected following staining. Finally, a fluorescence microscope (Leica, 48 h of transfection. Wetzlar, Germany) was utilized to observe and test EdU positive cells. RNA pull-down assay Biotin-labeled miR-485-3p probe (bio-miR-485-3p) Flow cytometry and nonsense control probe (bio-miR-NC) were syn- 16HBECs were harvested and resuspended in buffer thesized by Geneseed Biotech (Shanghai, China) and solution (500 µL) to the density of 1 × 10 cells/mL. then incubated with 16HBECs for 2 h. After lysing, the Then cells (1 × 105) were then stained with Annexin lysates of 16HBECs were incubated with M-280 Strepta- V-FITC and propidium iodide (KenGen Biotech, Nan- vidin magnetic beads for 1 h. Finally, the abundance of jing, China) for 20 min under darkness. Cell apoptosis circ_0006872 or CDKN1B was analyzed. was detected by flow cytometer within 1 h. Statistical analysis Western blotting The data from three repetitions were exhibited as RIPA lysis buffer (KenGen Biotech) harboring 1% pro - mean ± standard deviation (SD). Student’s t test, Mann- teinase inhibitor was applied for proteins extraction. Whitney or Analysis of Variance (ANOVA) followed by Then 40 µg of proteins were separated by 8% SDS- Tukey’s post-test was used for the group comparisons. PAGE and electrophoretically transferred onto cellulose Pearson’s correlation coefficient assay was used for corre - nitrate membranes. After 1 h block by 5% skim milk lation analysis between two variables. *P < 0.05, **P < 0.01, powder at 37 ℃, membranes were incubated with the ***P < 0.001 or ****P < 0.0001 suggested statistically specific primary antibodies against CDKN1B (ab32034, significant. 1:1000), Bcl-2 (ab692, 1:1000), Bax (ab32503, 1:1000), and GAPDH (ab181602, 1:10000) (Abcam, Cambridge, Results MA, USA) at 4 ℃overnight, followed by probing with CSE treatment induces 16HBECs imjury secondary antibody (D110058, 1:4000, Sangon Biotech, To investigate the effects of CS on bronchial epithelial Shanghai, China) for 2 h at 37 ℃. Protein bands were cells, 16HBECs were exposed to different concentrations quantified using an ECL reagent (Beyotime). of CSE (1%, 2%, or 4%) for 24 h. CSE treatment induced arrest of 16HBEC viability and DNA synthesis activity ELISA in a dose-dependent manner (Fig. 1A, B). On the con- The culture supernatant of indicated 16HBECs were trary, CSE treatment dose-dependently evoked apopto- collected, and then levels of interleukin (IL)-6 and sis in 16HBECs (Fig. 1C), accompanied with the increase tumor necrosis factor-α (TNF-α) were measured as of Bax and decrease of Bcl-2 in 16HBECs (Fig. 1D, E). per the instructions of the corresponding ELISA kits Besides, with the increasing concentrations of CSE, the (Abcam). release of IL-6 and TNF-α was significantly enhanced (Fig. 1F). Moreover, CSE exposure had obstructive effects on SOD content, an endogenous antioxidant enzyme, Measurement of superoxide dismutase (SOD) since SOD level declined upon the exposure of increasing 16HBECs were lysed using RIPA lysis buffer (Ken - doses of CSE in (Fig. 1G). u Th s, these data suggested that Gen Biotech) and SOD content was determined using CSE could lead to 16HBECs injury. Wang et al. Applied Biological Chemistry (2023) 66:14 Page 4 of 12 Fig. 1 CSE treatment induces 16HBECs injury. A–G 16HBECs were exposed to different concentrations of CSE (1%, 2%, or 4%) for 24 h. Analyses of (A, B) 16HBEC proliferation, and C apoptosis. D, E Levels of Bax and Bcl-2 in 16HBECs. F Detection of IL-6 and TNF-α contents in 16HBECs. G Measurement of SOD content in 16HBECs using a commercial kit. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Knockdown of circ_0006872 attenuates CSE-induced CSE were attenuated by circ_0006872 down-regulation 16HBEC injury in 16HBECs (Fig. 2I). Besides that, circ_0006872 dele- As shown in Fig. 2A, the level of circ_0006872 was tion rescued CSE-evoked decrease of SOD content in higher in smokers with or without COPD than those in 16HBECs (Fig. 2J). non-smokers, especially in COPD group. In addition, circ_0006872 expression was dose-dependently elevated by CSE treatment in 16HBECs (Fig. 2B). Additionally, the Circ_0006872 acts as a sponge for miR-485-3p experiment showed that RNase R could rapidly degrade Circinteractome database predicted that circ_0006872 linear GAPDH rather than circ_0006872 in 16HBECs, possesses the binding site of miR-485-3p (Fig. 3A). indicating the round structure of circ_0006872 (Fig. 2C). The miR-485-3p expression was significantly elevated Thereafter, the impacts of circ_0006872 on CSE-induced after miR-485-3p mimic introduction in 16HBECs bronchial epithelial injury were elucidated. Following (Fig. 3B). Then we found that miR-485-3p mimics could the transfection of circ_0006872 siRNA in 16HBECs, overtly reduce the luciferase activity of WT group but cells were exposed to 2% CSE for 24 h, then we observed not the mutant one in 16HBECs (Fig. 3C). Moreover, that si-circ_0006872 introduction reduced CSE-induced circ_0006872 was found to be significantly enriched in elevation of circ_0006872 in 16HBECs (Fig. 2D). Func- bio-miR-485-3p group in 16HBECs (Fig. 3D). Thereafter, tionally, it was proved that circ_0006872 silencing it was proved that miR-485-3p expression was decreased reversed CSE-induced proliferation arrest (Fig. 2E, F) in smokers, especially in smokers with COPD (Fig. 3E), and apoptosis (Fig. 2G, H) in 16HBECs. Furthermore, which was negatively correlated with circ_0006872 both the up-regulations of IL-6 and TNF-α mediated by expression in COPD patients (Fig. 3F). Besides that, W ang et al. Applied Biological Chemistry (2023) 66:14 Page 5 of 12 Fig. 2 Knockdown of circ_0006872 attenuates CSE-induced 16HBEC injury. A The expression level of circ_0006872 was detected using qRT-PCR in non-smokers (n = 35), smokers (n = 35), and smokers with COPD (n = 35). B Levels of circ_0006872 in 16HBECs exposed to different concentrations of CSE. C Stability analysis by RNase R treatment. D–J 16HBECs were transfected with si-circ_0006872 or si-NC and then exposed with 2% CSE for 24 h. D Transfection efficiency. E, F 16HBEC proliferation analysis. G 16HBEC apoptosis detection. H Levels of Bax and Bcl-2 in 16HBECs. I Levels of IL-6 and TNF-α in 16HBECs. J Measurement of SOD content in 16HBECs using a commercial kit. **P < 0.01, ***P < 0.001, ****P < 0.0001 we also showed that CSE treatment dose-dependently as well as elevation of SOD content (Fig. 4H) in 16HBECs decreased miR-485-3p in 16HBECs (Fig. 3G). exposed with 2% CSE. Knockdown of circ_0006872 attenuates CSE-induced CDKN1B is targeted by miR-485-3p, and circ_0006872/ 16HBEC injury via miR-485-3p miR-485-3p/CDKN1B constitutes a feedback loop To investigate whether circ_0006872 exerted its func- in 16HBECs tions by miR-485-3p, 16HBECs were co-transfected Next, starbase software showed that CDKN1B contains with si-circ_0006872 and/or anti-miR-485-3p, followed conserved target site of miR-485-3p (Fig. 5A). Then it by 2% CSE exposure for 24 h. As expected, miR-485-3p was showed the luciferase activity of wild-type CDKN1B inhibitor reduced circ_0006872 knockdown-induced 3′UTR reporter vector was significantly reduced by elevation of miR-485-3p in 16HBECs under CSE treat- miR-485-3p overexpression (Fig. 5B). Besides that, RNA ment (Fig. 4A). Functionally, miR-485-3p lack attenu- pull-down analysis indicated a specific enrichment of ated circ_0006872 down-regulation-evoked promotion CDKN1B in the biotin-labeled miR-485-3p probe group of cell proliferation (Fig. 4B–D), arrest of cell apoptosis (Fig. 5C). CDKN1B mRNA was found to be increased (Fig. 4E, F), reduction of IL-6 and TNF-α levels (Fig. 4G), in smokers, especially in smokers with COPD (Fig. 5D), Wang et al. Applied Biological Chemistry (2023) 66:14 Page 6 of 12 Fig. 3 Circ_0006872 acts as a sponge for miR-485-3p. A The binding site of miR-485-3p on circ_0006872 predicted by circinteractome database was listed. B The transfection efficiency detection. C, D The interaction between miR-485-3p and circ_0006872 was confirmed using dual-luciferase reporter assay and RNA pull-down assay. E The expression level of miR-485-3p was detected using qRT-PCR in non-smokers (n = 35), smokers (n = 35), and smokers with COPD (n = 35). F The correlation between miR-485-3p and circ_0006872 expression level in smokers with COPD (n = 35) was analyzed by Pearson’s correlation coefficient assay (r=-0.8966, P < 0.001). G Levels of miR-485-3p in16HBECs exposed to different concentrations of CSE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and was negatively correlated with miR-485-3p expres- elevation of CDKN1B expression level. Then transfected sion in COPD patients (Fig. 5E). Similarly, its protein 16HBECs were treated with 2% CSE for 24 h. In CCK-8 expression was also elevated in smokers with COPD and EdU assays, CDKN1B knockdown suppressed CSE- (Fig. 5F). In addition, CSE dose-dependently elevated induced inhibition of 16HBEC proliferation (Fig. 6B–D). CDKN1B expression in 16HBECs (Fig. 5G). Importantly, Flow cytometric analysis suggested that the apoptosis of circ_0006872 down-regulation was accompanied by the CSE-induced 16HBECs was decreased after CDKN1B decrease of CDKN1B, which was subsequently rescued silencing (Fig. 6E). And western blotting showed the by miR-485-3p inhibitor (Fig. 5H). In all, circ_0006872 decrease of Bax protein level and increase of Bcl-2 level could indirectly regulated CDKN1B through sponging in 16HBECs after CDKN1B down-regulation in the pres- miR-485-3p in 16HBECs. ence of CSE (Fig. 6F). Additionally, CDKN1B depletion in ELISA analysis gave rise to IL-6 and TNF-α release CDKN1B silencing reverses CSE-induced 16HBEC injury enhancement in CSE-induced 16HBECs (Fig. 6G). In Next, we evaluated the functions of CDKN1B on CSE- final SOD measurement, a remarkable elevation of SOD induced bronchial epithelial injury. CDKN1B siRNAs level was unveiled in response to CDKN1B siRNA in were established to knock down CDKN1B expression in CSE-treated 16HBECs (Fig. 6H). These data suggested 16HBECs. As exhibited in Fig. 6A, the introduction of si- that knockdown of CDKN1B protected CSE-induced CDKN1B in 16HBECs significantly reduced CSE-evoked 16HBEC injury. W ang et al. Applied Biological Chemistry (2023) 66:14 Page 7 of 12 Fig. 4 Knockdown of circ_0006872 attenuates CSE-induced 16HBEC injury via miR-485-3p. A–H 16HBECs were co-transfected with si-circ_0006872 and/or anti-miR-485-3p, followed by 2% CSE exposure for 24 h. A Transfection efficiency. B–D 16HBEC proliferation analysis. E 16HBEC apoptosis detection. F Levels of Bax and Bcl-2 in 16HBECs. G Levels of IL-6 and TNF-α in 16HBECs. H Measurement of SOD content in 16HBECs using a commercial kit. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Mir-485-3p protects 16HBEC from CSE-induced injury CDKN1B axis on CSE-induced bronchial epithelial via CDKN1B injury, we conducted rescue experiments by transfect- In order to investigate the action of miR-485-3p/ ing miR-485-3p mimics and/or CDKN1B overexpression Wang et al. Applied Biological Chemistry (2023) 66:14 Page 8 of 12 Fig. 5 CDKN1B is a target of miR-485-3p, and circ_0006872/miR-485-3p/CDKN1B constitutes a feedback loop in 16HBECs. A The conserved target site of miR-485-3p on CDKN1B predicted by starbase software was listed. B, C The interaction between miR-485-3p and CDKN1B was confirmed using dual-luciferase reporter assay and RNA pull-down assay. D Levels of CDKN1B mRNA in non-smokers (n = 35), smokers (n = 35), and smokers with COPD (n = 35). E The correlation between miR-485-3p and CDKN1B mRNA expression level in smokers with COPD (n = 35) was assessed by Pearson’s correlation coefficient assay (r=-0.8606, p < 0.001). F CDKN1B protein level in non-smokers (n = 35), smokers (n = 35), and smokers with COPD (n = 35). G CDKN1B expression in 16HBECs exposed to different concentrations of CSE. H The impacts of circ_0006872/miR-485-3p axis on CDKN1B expression in 16HBECs under CSE treatment. **P < 0.01, ***P < 0.001, ****P < 0.0001 plasmids into 16HBECs. After treating with 2% CSE (Fig. 7E, F). Both the increases of IL-6 and TNF-α medi- for 24 h, the transfection efficiency was validated by ated by CSE in 16HBECs were reduced by miR-485-3p CDKN1B expression in 16HBECs (Fig. 7A). Function- mimic, and subsequently promoted in response to ally, 16HBEC proliferative ability was potentiated upon CDKN1B plasmids (Fig. 7G). In addition, the SOD con- miR-485-3p overexpression in the presence of CSE, while tent was increased with miR-485-3p mimic introduc- CDKN1B overexpression could reverse this condition tion in CSE-induced 16HBECs, which was abolished by (Fig. 7B–D). Furthermore, miR-485-3p mimic reversed CDKN1B overexpression (Fig. 7H). Collectively, miR- CSE evoked apoptosis in 16HBECs, and this phenom- 485-3p suppressed CSE-induced16HBEC injury via enon was counteracted via CDKN1B up-regulation CDKN1B. W ang et al. Applied Biological Chemistry (2023) 66:14 Page 9 of 12 Fig. 6 CDKN1B silencing reverses CSE-induced 16HBEC injury. A–H 16HBECs were transfected with si-CDKN1B or si-NC, followed by treatment with 2% CSE for 24 h. (A) Transfection efficiency. B–D 16HBEC proliferation analysis. E 16HBEC apoptosis detection. F Levels of Bax and Bcl-2 in 16HBECs. G Levels of IL-6 and TNF-α in 16HBECs. H Measurement of SOD content in 16HBECs using a commercial kit. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Discussion and ubiquitous abundance in eukaryotes . In our As the fourth leading cause of death through the world, study, we found an increased circ_0006872 in smok- over 40% of COPD deaths are attributed to smoking [18, ers without or with COPD, especially in smokers with 19]. Long-term exposure to CS is the leading cause of COPD. Furthermore, its expression was also up-regu- COPD through inducing the activation of epithelial cells lated by CSE exposure in 16HBECs in a dose-dependent and macrophages to cause apoptosis and inflammation manner. Furthermore, deletion of circ_0006872 abolished and the subsequent release of mediators in response to CSE-triggered proliferation arrest, inflammation, apopto - oxidative stress . Studies have exhibited that circRNA sis, and oxidative stress in 16HBECs. Thus, we speculated dysregulation is related to CSE-induced HBECs damage that knockdown of circ_0006872 might exert protective [11, 12, 21]. CircRNAs hold great promise as potential effects against CS-evoked bronchial epithelial cell injury biomarkers in COPD since their highly stable structure in the process of COPD. Wang et al. Applied Biological Chemistry (2023) 66:14 Page 10 of 12 Fig. 7 miR-485-3p protects 16HBEC from CSE-induced injury via CDKN1B. A–H 16HBECs were co-transfected with miR-485-3p mimics and/or CDKN1B overexpression and then treated with 2% CSE for 24 h. A Transfection efficiency. B–D 16HBEC proliferation analysis. E 16HBEC apoptosis detection. F Levels of Bax and Bcl-2 in 16HBECs. G Levels of IL-6 and TNF-α in 16HBECs. H Measurement of SOD content in 16HBECs using a commercial kit. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 According to the ceRNA hypothesis [15, 16], circRNAs 16HBECs. Previous studies have showed the implication of are able to act as miRNA sponges to extricate the degra- miRNAs in multiple processes of COPD, modulating path- dation of the downstream mRNA mediated by miRNAs. ways related to apoptosis, inflammation and stress response u Th s, the underlying miRNA/mRNA axis of circ_0006872 [23–25]. miR-485-3p had been unveiled to be down-regu- in 16HBECs was explored. This work confirmed the lated in patients with COPD . Besides that, miR-485-3p circ_0006872/miR-485-3p/CDKN1B feedback loop in could attenuate CSE-triggered 16HBEC dysfunction via W ang et al. Applied Biological Chemistry (2023) 66:14 Page 11 of 12 2. Labaki WW, Rosenberg SR (2020) Chronic obstructive pulmonary disease. circ-HACE/miR-485-3p/TLR4 axis . Consistent with Ann Intern Med. https:// doi. org/ 10. 7326/ AITC2 02008 040 previous findings, we also confirmed the protective action 3. Zuo L, Prather ER, Stetskiv M, Garrison DE, Meade JR, Peace TI, Zhou T of miR-485-3p on CSE-induced 16HBECs. Moreover, miR- (2019) Inflammaging and oxidative stress in human diseases: from molec- ular mechanisms to novel treatments. International J Mol Sci 20:4472 485-3p could abolish the functions of circ_0006872 knock- 4. Wiegman CH, Li F, Ryffel B, Togbe D, Chung KF (2020) Oxidative stress in down on 16HBEC injury caused by CSE. CDKN1B belongs ozone-induced chronic lung inflammation and emphysema: a facet of to the Kip/Cip family of CDK inhibitors, and suppresses the chronic obstructive pulmonary disease. Front Immunol 11:1957 5. Tan WSD, Shen HM, Wong WSF (2019) Dysregulated autophagy in COPD: function of multiple cyclin-CDK complexes, thus showing a pathogenic process to be deciphered. Pharmacol Res 144:1–7 anti-proliferative activity by impairing cell cycle progres- 6. Gohy ST, Hupin C, Pilette C, Ladjemi MZ (2016) Chronic inflammatory air - sion . In COPD, Yang et al. showed that CDKN1B was way diseases: the central role of the epithelium revisited. Clin Exp Allergy 46:529–542 decreased in COPD patients, and could abate the action 7. Chen LL, Yang L (2015) Regulation of circRNA biogenesis. RNA Biol of miR-221-3p on the inhibition of CSE-evoked apoptotic 12:381–388 and inflammatory damages in 16HBECs . In our study, 8. Geng Y, Jiang J, Wu C (2018) Function and clinical significance of circRNAs in solid tumors. J Hematol Oncol 11:98 the increased expression of CDKN1B in smokers without 9. Han B, Chao J, Yao H (2018) Circular RNA and its mechanisms in disease: or with COPD, and CSE-treated 16HBECs was observed, from the bench to the clinic. Pharmacol Ther 187:31–44 moreover, we proved that CDKN1B down-regulation 10. Kristensen LS, Andersen MS, Stagsted LVW, Ebbesen KK, Hansen TB, Kjems J (2019) The biogenesis, biology and characterization of circular RNAs. Nat could prevent CSE-induced16HBEC injury. In addition, Rev Genet 20:675–691 miR-485-3p exerted its protective functions by targeting 11. Zheng C, Zhang Y, Zhao Y, Duan Y, Mu Q, Wang X (2021) Circ-OSBPL2 con- CDKN1B. tributes to smoke-related chronic obstructive pulmonary disease by tar- geting miR-193a-5p/BRD4 axis. Int J Chronic Obstr Pulm Dis 16:919–931 In all, this work demonstrated that circ_0006872 silenc- 12. Zhou F, Cao C, Chai H, Hong J, Zhu M (2021) Circ-HACE1 aggravates ciga- ing could protect against CSE-induced inflammatory, rette smoke Extract-Induced Injury in Human bronchial epithelial cells apoptotic, and oxidative injury via miR-485-3p/CDKN1B via regulating toll-like receptor 4 by sponging miR-485-3p. Int J Chronic Obstr Pulm Dis 16:1535–1547 axis, which may provide a novel insight into COPD pre- 13. Zhou L, Wu B, Yang J, Wang B, Pan J, Xu D, Du C (2021) Knockdown of circ- vention in subjects with smoking addiction. FOXO3 ameliorates cigarette smoke-induced lung injury in mice. Respir Res 22:294 Acknowledgements 14. Xue M, Peng N, Zhu X, Zhang H (2021) Hsa_circ_0006872 promotes Not applicable. cigarette smoke-induced apoptosis, inflammation and oxidative stress in HPMECs and BEAS-2B cells through the miR-145-5p/NF-κB axis. Biochem Author contributions Biophys Res Commun. 534:553–560 Conceptualization and Methodology: ZL and LZ; Formal analysis and Data 15. Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, Damgaard curation: JT and CW; Validation and Investigation: JW and ZL; Writing - original CK, Kjems J (2013) Natural RNA circles function as efficient microRNA draft preparation and Writing - review and editing: JW, ZL and LZ; All authors sponges. Nature 495:384–388 read and approved the final manuscript. 16. Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP (2011) A ceRNA hypoth- esis: the rosetta stone of a hidden RNA language? Cell 146:353–358 Funding 17. Vestbo J, Hurd SS, Agustí AG, Jones PW, Vogelmeier C, Anzueto A, Barnes No funding was received. PJ, Fabbri LM, Martinez FJ, Nishimura M, Stockley RA, Sin DD (2013) Rodriguez-Roisin, global strategy for the diagnosis, management, and Availability of data and materials prevention of chronic obstructive pulmonary disease: GOLD executive The analyzed data sets generated during the present study are availablefrom summary. Am J Respir Crit Care Med 187:347–365 the corresponding author on reasonable request. 18. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, Ahn SY, Alvarado M, Anderson HR, Anderson LM, Andrews KG, Atkinson C, Baddour LM, Barker-Collo S, Bartels DH, Bell Declarations ML, Benjamin EJ, Bennett D, Bhalla K, Bikbov B, Bin Abdulhak A, Birbeck G, Blyth F, Bolliger I, Boufous S, Bucello C, Burch M, Burney P, Carapetis J, Ethics approval and consent to participate Chen H, Chou D, Chugh SS, Coffeng LE, Colan SD, Colquhoun S, Colson The present study was approved by the ethical review committee of Tongde- KE, Condon J, Connor MD, Cooper LT, Corriere M, Cortinovis M, de Vaccaro hospital of Zhejiang Province. Written informed consent was obtained from KC, Couser W, Cowie BC, Criqui MH, Cross M, Dabhadkar KC, Dahodwala allenrolled patients. N, De Leo D, Degenhardt L, Delossantos A, Denenberg J, Des Jarlais DC, Dharmaratne SD, Dorsey ER, Driscoll T, Duber H, Ebel B, Erwin PJ, Consent for publication Espindola P, Ezzati M, Feigin V, Flaxman AD, Forouzanfar MH, Fowkes FG, Patients agree to participate in this work. Franklin R, Fransen M, Freeman MK, Gabriel SE, Gakidou E, Gaspari F, Gil- lum RF, Gonzalez-Medina D, Halasa YA, Haring D, Harrison JE, Havmoeller Competing interests R, Hay RJ, Hoen B, Hotez PJ, Hoy D, Jacobsen KH, James SL, Jasrasaria R, The authors declare that they have no competing interests. Jayaraman S, Johns N, Karthikeyan G, Kassebaum N, Keren A, Khoo JP, Knowlton LM, Kobusingye O, Koranteng A, Krishnamurthi R, Lipnick M, Lipshultz SE, Ohno SL, Mabweijano J, MacIntyre MF, Mallinger L, March L, Received: 9 June 2022 Accepted: 5 February 2023 Marks GB, Marks R, Matsumori A, Matzopoulos R, Mayosi BM, McAnulty JH, McDermott MM, McGrath J, Mensah GA, Merriman TR, Michaud C, Miller M, Miller TR, Mock C, Mocumbi AO, Mokdad AA, Moran A, Mulhol- land K, Nair MN, Naldi L, Narayan KM, Nasseri K, Norman P, O’Donnell M, Omer SB, Ortblad K, Osborne R, Ozgediz D, Pahari B, Pandian JD, Rivero References AP, Padilla RP, Perez-Ruiz F, Perico N, Phillips D, Pierce K, Pope CA, Porrini E, 1. Rabe KF, Watz H (2017) Chronic obstructive pulmonary disease. Lancet Pourmalek F, Raju M, Ranganathan D, Rehm JT, Rein DB, Remuzzi G, Rivara 389:1931–1940 FP, Roberts T, De León FR, Rosenfeld LC, Rushton L, Sacco RL, Salomon Wang et al. Applied Biological Chemistry (2023) 66:14 Page 12 of 12 JA, Sampson U, Sanman E, Schwebel DC, Segui-Gomez M, Shepard DS, Singh D, Singleton J, Sliwa K, Smith E, Steer A, Taylor JA, Thomas B, Tleyjeh IM, Towbin JA, Truelsen T, Undurraga EA, Venketasubramanian N, Vijayakumar L, Vos T, Wagner GR, Wang M, Wang W, Watt K, Weinstock MA, Weintraub R, Wilkinson JD, Woolf AD, Wulf S, Yeh PH, Yip P, Zabetian A, Zheng ZJ, Lopez AD, Murray CJ, AlMazroa MA, Memish ZA (2012) Global andregional mortality from 235 causes of death for 20 age groups in 1990 and 2010:a systematic analysis for the global burden of disease study 2010. Lancet 380:2095–2128 19. Adeloye D, Chua S, Lee C, Basquill C, Papana A, Theodoratou E, Nair H, Gasevic D, Sridhar D, Campbell H, Chan KY, Sheikh A, Rudan I (2015) Global and regional estimates of COPD prevalence: systematic review and meta-analysis. J global health 5:020415 20. Salvi SS, Barnes PJ (2009) Chronic obstructive pulmonary disease in non- smokers. Lancet (London England) 374:733–743 21. Wang Z, Zuo Y, Gao Z (2021) CircANKRD11 Knockdown protects HPMECs from cigarette smoke extract-induced injury by regulating miR-145-5p/ BRD4 axis. Int J Chronic Obstr Pulm Dis 16:887–899 22. Patop IL, Wüst S, Kadener S (2019) Past, present, and future of circRNAs. EMBO J 38:e100836 23. Cañas JA, Rodrigo-Muñoz JM, Sastre B, Gil-Martinez M, Redondo N (2020) Del Pozo, MicroRNAs as potential regulators of immune response networks in asthma and chronic obstructive pulmonary disease. Front Immunol 11:608666 24. Tan BWQ, Sim WL, Cheong JK, Kuan WS, Tran T, Lim HF (2020) MicroRNAs in chronic airway diseases: clinical correlation and translational applica- tions. Pharmacol Res 160:105045 25. Baker JR, Vuppusetty C, Colley T, Papaioannou AI, Fenwick P, Donnelly L, Ito K, Barnes PJ (2016) Oxidative stress dependent microRNA-34a activation via PI3Kα reduces the expression of sirtuin-1 and sirtuin-6 in epithelial cells. Sci Rep 6:35871 26. Musri MM, Coll-Bonfill N, Maron BA, Peinado VI, Wang RS, Altirriba J, Blanco I, Oldham WM, Tura-Ceide O, García-Lucio J, de la Cruz-Thea B, Meister G, Loscalzo J, Barberà JA (2018) MicroRNA dysregulation in pul- monary arteries from chronic obstructive pulmonary disease. Relation- ships with vascular remodeling. Am J Respir Cell Mol Biol 59:490–499 27. Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, Koff A (1994) p27Kip1, a cyclin-cdk inhibitor, links transforming growth factor- beta and contact inhibition to cell cycle arrest. Genes Dev 8:9–22 28. Yang H, Zhang L, Wang Q (2021) MicroRNA-221-3p alleviates cell apop- tosis and inflammatory response by targeting cyclin dependent kinase inhibitor 1B in chronic obstructive pulmonary disease. Bioengineered 12:5705–5715 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
Applied Biological Chemistry – Springer Journals
Published: Feb 22, 2023
Keywords: Circ_0006872; MiR-485-3p; CDKN1B; COPD; CSE; Smoke
Access the full text.
Sign up today, get DeepDyve free for 14 days.