For COPD, regulation of miR-515-5p by hsa_circ_0061052, acting via the FoxC1/Snail pathway, is involved in cigarette smoke-induced airway remodeling CURRENT STATUS: POSTED

Background Regulatory networks involving non-coding RNA (ncRNA) are involved in various lung diseases. The function of circular RNA (circRNA), a recently recognized type of ncRNA, in chronic obstructive pulmonary disease (COPD), has not been elucidated. Methods Western blots and cellular immunofluorescence are used to check molecular biological changes and molecular interactions in cells, bioinformatics tools and luciferase reporter genes are used to assess molecular binding relationships, immunohistochemical examination of mouse lung tissue-related proteins A mouse model of COPD was used to validate related concubine mechanisms. In the present study, we aimed to determine whether hsa_circ_0061052 participates in the EMT of HBE cells and to elucidate its biological mechanism. Experiments with cultured cells and animals showed that exposure to cigarette smoke extract (CSE) or cigarette smoke (CS) induced the EMT and led to lung dysfunction and airway remodeling. For HBE cells and the lung tissues of CS-exposed mice, the expression of hsa_circ_0061052 was elevated. To verify the function of this circRNA, knocking it out in HBE cells reversed the CSE-induced EMT. We analyzed the regulatory relationship between hsa_circ_0061052 and miR-515-5p using bioinformatics, a luciferase reporter gene, and qRT-PCR. We found that hsa_circ_0061052 was mainly distributed in the cytoplasm and acted as a sponge for miR-515-5p. Assays with a luciferase reporter gene showed that miR-515-5p binds to the 3'UTR region of FoxC1 mRNA to inhibit its transcription. For HBE cells, overexpression of miR-515-5p reversed the CSE-induced EMT. In addition, hsa_circ_0061052 regulated the expression of FoxC1/Snail by competitively binding to miR-515-5p. When an hsa_circ_0061052 siRNA and an miR-515-5p inhibitor were co-transfected into HBE cells, the effect of hsa_circ_0061052 maintained RPMI-1640 medium 10% fetal bovine serum Life Technologies/Gibco, 100 µg/mL streptomycin, and 100 U/mL penicillin (Life Technologies/Gibco, Gaithersburg, MD) under 5% CO 2 at 37°C. Cells were passaged at a ratio 1:3 every 2 days. After reaching 70-80% confluence, the cells were washed with phosphate-buffered saline, grown in RPMI-1640 medium supplemented with 10% FBS, and exposed to 0, 1, 2, or 4 % CSE for 48 hours. acts on the 3'-UTR region of FoxC1 mRNA. The results of luciferase reporter experiments showed that FoxC1 mRNA is a target of miR-515-5p and that, in HBE cells, CSE increased the levels of FoxC1 and Snail. Results of experiments with mice were consistent with these findings. After transfection of cells with an miR-515-5p mimic, the ectopic expression of miR-515-5p attenuated the CSE-induced EMT process and reduced the expression levels of FoxC1 and Snail, indicating that miR-515-5p and FoxC1/Snail participate in the EMT of HBE cells. CircRNAs, long non-coding RNAs, and various other RNA transcripts, including those of pseudogenes, can function as ceRNAs and participate in the development of human diseases The present results indicate that hsa_circ_0061052 is a ceRNA


RNA preparation and reverse-transcriptase polymerase chain reaction (RT-PCR)
The nuclear and cytoplasmic fractions of cells were extracted by use of the PARIS Kit Protein and RNA Isolation System (Thermo Fisher Scientific). Total RNA (1 μg) was treated with 10 U of RNase R (Epicentre Technologies Corp., Madison, WI) in 1× RNase R reaction buffer in a total volume of 10 µl.
The mixture was incubated at 37 °C for 1 hour. Total RNA (1 μg) was transcribed into cDNA by HiScript II Q RT Supermix (Vazyme Biotech, Nanjing, China). The polymerase chain reaction reactions (PCR) were evaluated by checking the PCR products on 2% w/v agarose gels. The primers are listed in Table   1. Table 1 Primer sequences used

Shanghai, China) and transfected into HBE cells. Luciferase activity was determined with Dual
Luciferase Reporter Gene Assay Kits (Beyotime) according to the manufacturer's protocol. The Renilla luciferase activity was normalized by firefly luciferase.

Mice exposed to CS
Male BALB/c mice at 6-8 weeks of age were purchased from Animal Core Facility of Nanjing Medical University and housed in Jiangsu Province Medicine, Pesticide and Veterinary Drug Safety Evaluation and Research Center. Animals were treated humanely and with regard for alleviation of suffering according to a protocol approved by the Nanjing Medical University Animal Care and Use Committee.
To observe the effects of CS on airway obstruction of lungs, 16 male BALB/c mice (age 6-8 weeks) were divided into four groups: normal control and low-, medium-, and high-CS exposure groups. The low-, medium-, and high-CS groups were exposed in a whole-body exposure system (Beijing increasing exposure, as follows: mice were placed in the chamber and exposed for 20 min on the first day, 30 min on the second day, and 60 min on the third day until the end. Age-matched mice kept in a similar environment without exposure to CS served as controls. Experiments were accomplished with n = 4 randomized animals per group.

Lung function measurement
For mice, airway hyper-responsiveness (AHR) was measured as the change in airway function by use of whole-body plethysmography (Buxco Electronics Ltd., USA), as previously reported [26]. Individual mice were placed unrestricted in a chamber connected to a pressure transducer to measure pressure changes inside the chamber. After acclimation, methacholine (0, 12.5, 25, or 50 mg/mL) were nebulized for 2 min, and enhanced pause (Penh) was recorded during the response period using FinePoint software (Buxco Electronics Ltd., USA). Penh, a dimensionless unit, correlates with pulmonary resistance. Values were averaged and expressed as absolute Penh values.

Masson trichrome staining
The lungs of mice were fixed with 4% paraformaldehyde and embedded in paraffin. For histological analysis and detection of collagen deposition, successive 5-µm lung sections were placed on slides and subjected to staining with trichrome (Masson) kits (Sigma-Aldrich, Germany), according to the manufacturer's instructions. Collagen content was determined by the ratio of collagen surface area (blue) to total surface area (red). Image J software was used to evaluate collagen deposition.

Statistical analyses
All experiments were performed in triplicate. Derived values are presented as means ± SD.
Comparison of means among multiple groups was accomplished by one-way analysis of variance (ANOVA), and a multiple-range least significant difference (LSD) was used for inter-group comparisons. P values < 0.05 were considered statistically significant. All statistical analyses were performed with SPSS 18.0.

CSE exposure induces the EMT and increases the levels of hsa_circ_0061052 in HBE cells
In organs, the EMT occurs during repair of chronic damage and in response to inflammation, such as fibrosis and COPD [28,29]. HBE cells were exposed to 0, 1, 2, or 4% CSE for 48 hours. With the increased CSE exposure, levels of the epithelial marker protein, E-cadherin, decreased; there were elevated levels of the mesenchymal markers, N-cadherin, vimentin, and α-SMA ( Fig. 1a and b).
Immunofluorescence microscopy of E-cadherin and vimentin confirmed the location of EMT-related markers. The cells formed epithelial-like cell junctions and showed high levels of mesenchymal cell markers (Fig. 1c). After CSE treatment, hsa_circ_0061052 increased in a dose-response relationship ( Fig. 1d and e). Thus these results show that, for HBE cells, CSE exposure induces the EMT and increases the levels of hsa_circ_0061052.

Characterization of hsa_circ_0061052 in HBE cells
Hsa_circ_0061052, derived from a host gene, oxysterol-binding protein 2 (OSBPL2), has a closed loop structure and a length of 253 nucleotides (Fig. 2a). Fluorescence in situ hybridization (FISH) revealed that hsa_circ_0061052 is located mainly in the cytoplasm of HBE cells (Fig. 2b). RT-PCR and qRT-PCR analysis of RNA in the nucleus and cytoplasm showed that hsa_circ_0061052 was concentrated in the cytoplasm ( Fig. 2c and d). To verify the circular characteristics of hsa_circ_0061052, total RNA was processed with or without 10 U Rnase R for 1 hour. Hsa_circ_0061052 was resistant to exonucleases, but linear OSBPL2 mRNA was digested by exonucleases ( Fig. 2e and f). These results show that hsa_circ_0061052 has a ring structure and is mainly concentrated in the cytoplasm.

Exposure of HBE cells to CSE decreases their levels of miR-515-5p and increases their levels of FoxC1/Snail, which is a target of miR-515-5p
CircRNA can act as a miRNA sponge and form a circRNA-miRNA regulatory network [30,31]. Using the CircInteractome (http://circinteractome.nia.nih.gov/) database, we predicted the miRNAs that may be regulated by hsa_circ_0061052, and selected possible binding sites of hsa_circ_0061052 with miRNAs ( Fig. 3a). The top five miRNAs (miR-515-5p, miR-1182, miR-1304, miR-136, and miR-571) were selected for analysis. QRT-PCR was performed after HBE cells were treated with 0, 1, 2, or 4% CSE for 48 hours. With increasing concentrations, CSE reduced the levels of miR-515-5p, but levels of miR-1182, miR-1304, miR-136, and miR-571 levels were not decreased (Fig. 3b). To verify the results, a luciferase reporter gene was constructed based on the complementary pairing binding site of hsa_circ_0061052 and miR-515-5p (Fig. 3c) and co-transfected into HBE cells with an miR-515-5p mimic or control (con) mimic. The relative luciferase activity was reduced in cells co-transfected with psiCHECK2-hsa_circ_0061052-wt and the miR-515-5p mimic (Fig. 3d). These results indicate that hsa_circ_0061052 can act as a miRNA sponge for miR-515-5p. . HBE cells were exposed to 0, 1, 2, or 4% CSE for 48 hours. With the increase of CSE exposure, the levels of FoxC1 and Snail increased ( Fig. 3e and f). We used the bioinformatics tools miRanda (http: // www.microrna.org/) and TargetScan (http://www.targrtscan.org/) to predict that FoxC1 is a target of miR-515-5p. Consistent with the binding site of miR-515-5p in the 3'UTR region of FoxC1 mRNA (Fig. 3g), a luciferase reporter gene was constructed and co-transfected with an miR-515-5p mimic or control. Co-transfection of the miR-515-5p mimic reduced the relative luciferase activity with the wt-FoxC1 3'UTR vector (Fig. 3h). These results show that, in HBE cells, FoxC1 is a target of miR-515-5p and that CSE increases the levels of FoxC1 and Snail.

In HBE cells, hsa_circ_0061052 is involved in the CSE-induced EMT and regulates the expression of FoxC1 and Snail
In determine if hsa_circ_0061052 is involved in the EMT, three siRNAs targeting hsa_circ_0061052 were designed to suppress its expression; their efficiency was tested (Fig. 4a). All three siRNAs downregulated hsa_circ_0061052 in HBE cells. Since hsa_circ_0061052 siRNA #1 was most effective, for further research, it was transfected into HBE cells.
Knockdown of hsa_circ_0061052 in HBE cells reduced the CSE-induced decrease in the levels of E-cadherin and the increase in levels of N-cadherin, vimentin, and α-SMA ( Fig. 4b and c).
Immunofluorescence microscopy showed that, after transfection of CSE-HBE cells with hsa_circ_0061052 siRNA, the level of E-cadherin increased, and the level of vimentin decreased (Fig.   4d). To determine if hsa_circ_0061052 was involved in the CSE-induced EMT through miR-515-5p/FoxC1 signaling, we determined whether hsa_circ_0061052 regulated the expression of FoxC1/Snail. In CSE-HBE cells transfected with hsa_circ_0061052 siRNA, the expressions of FoxC1 and Snail were lower compared with the control group ( Fig. 4e and f). Thus, in the process of CSEinduced EMT in HBE cells, hsa_circ_0061052 regulates the expression of FoxC1 and Snail.

In HBE cells, miR-515-5p is involved in the CSE-induced EMT and regulates the expression of FoxC1 and Snail
We used bioinformatics tools to predict that FoxC1 is a target of miR-515-5p. To determine whether miR-515-5p reverses the CSE-induced EMT process, we transfected a miR-515-5p mimic into HBE cells (Fig. 5a). Western blots showed that ectopic expression of miR-515-5p attenuated the CSE-induced reduction of the levels of E-cadherin and the increased levels of N-cadherin, vimentin, and α-SMA ( Fig.   5b and c). Immunofluorescence microscopy revealed that, after transfection of CSE-HBE cells with the miR-515-5p mimic, the levels of E-cadherin increased, and the levels of vimentin decreased (Fig. 5d).
Compared with the CSE-HBE group, the levels of FoxC1 and Snail were lower in CSE-HBE cells transfected with miR-515-5p ( Fig. 5e and f). These results show that, for HBE cells, miR-515-5p participates in the CSE-induced EMT and regulates the expression of FoxC1 and Snail.

In HBE cells, hsa_circ_0061052, via miR-515-5p regulation of FoxC1 and Snail, is involved in the CSE-induced EMT
Since hsa_circ_0061052 works in combination with miR-515-5p, rescue experiments were performed to determine if miR-515-5p is involved in the function of hsa_circ_0061052 in the CSE-induced EMT.
Hsa_circ_0061052 siRNA or hsa_circ_0061052 siRNA and an miR-515-5p inhibitor were co-transfected into HBE cells. The qRT-PCR results showed that hsa_circ_0061052 siRNA did not affect the expression of miR-515-5p (Fig. 6a). However, the miR-515-5p inhibitor reversed the increase in E-cadherin caused by knockdown of hsa_circ_0061052 and the decreases in N-cadherin, vimentin, and α-SMA ( Fig. 6b and c). Immunofluorescence microscopy showed that co-transfection of hsa_circ_0061052 siRNA and the miR-515-5p inhibitor reversed the effect of hsa_circ_0061052 siRNA in reducing the EMT (Fig. 6d). Further, knockdown of hsa_circ_0061052 reduced the levels of FoxC1 and Snail; this decrease was reversed by inhibition of miR-515-5p (Fig. 6e And f). Thus, these results show that, for HBE cells, hsa_circ_0061052 regulates FoxC1 and Snail by acting as a sponge for miR-515-5p and that it participates in the CSE-induced EMT. respectively. After exposure of mice to CS, the levels of has_circ_0061052 increased in a dosedependent manner; the levels of miR-515-5p decreased in a dose-dependent manner ( Fig. 7a and b).

Exposure of mice to CS increases
For each group of mice dosed with 0, 12.5, 25, or 50 μg/mL methacholine, AHR (Penh value) was measured by whole body plethysmography as changes in mouse airway function [32]. As the concentration of methacholine increased, mice in each group showed enhanced AHR, and as the CS concentration increased, the Penh value showed an upward trend, indicating that the small airway resistance of the mice increased (Fig. 7c). With increasing exposure to CS, the levels of E-cadherin decreased, and those of N-cadherin, vimentin, and α-SMA increased ( Fig. 7d and e). Masson trichrome staining showed that, as CS exposure increased, the small airways of mice thickened, and collagen was deposited. IHC assessment of mouse lung tissue showed an increase in collagen content and in the expression of α-SMA (Fig. 7f, g and h). These results show that, for mice, CS causes lung dysfunction and small airway remodeling. We conclude that hsa_circ_0061052 is involved in the effect of CS by inducing the EMT in airway epithelial cells through regulation of miR-515-5p, causing the airway remodeling associated with COPD (Fig. 8). In the present study, we used bioinformatics tools to predict that miR-515-5p acts on the 3'-UTR

Conclusion
In conclusion, our findings show that, by regulating miR-515-5p through a FoxC1/Snail regulatory axis, hsa_circ_0061052 is involved in the airway remodeling of COPD caused by CS and indicate that hsa_circ_0000515 is a target for therapy of COPD. Declarations the article and its additional files.

Ethics approval and consent to participate
No ethics approval was required for this study that did not involve patients or patient data.

Consent for publication
All authors consent to publication.   Exposure of HBE cells to CSE decreases their levels of miR-515-5p and increases their levels of FoxC1/Snail, which is a target of miR-515-5p. Band density was quantified by Image J  In HBE cells, miR-515-5p is involved in the CSE-induced EMT and regulates the expression of FoxC1 and Snail. Band density was quantified by Image J software. GAPDH, measured in Figure 6 In HBE cells, hsa_circ_0061052, via miR-515-5p regulation of FoxC1 and Snail, is involved in the CSE-induced EMT. Band density was quantified by Image J software. GAPDH, measured in parallel, served as a control. HBE cells were co-transfected with hsa_circ_0061052 siRNA or with hsa_circ_0061052 siRNA + the miR-515-5p inhibitor for 24 hours, then exposed to 2% CSE for 48 hours. a The relative levels of hsa_circ_0061052 and miR-515-5p were  Exposure of mice to CS increases the levels of hsa_circ_0061052 and decreases the levels of miR-515-5p in lung tissue, induces the EMT in lung tissue, and promotes airway obstruction.
Male BALB/c mice at 6-8 weeks of age were exposed to 0, 100, 200, or 300 mg/m3 TPM CS for 16 weeks. n=4 randomized animals per group. The levels of hsa_circ_0061052 in lung tissue a and the levels of miR-515-5p in lung tissue b were determined by quantitative RT-PCR. AHR is represented as Penh in normal control mice and CS-exposed mice in response to 0, 12.5, 25, or 50 μg/mL of methacholine. c Penh values were measured by use of wholebody plethysmography. Densities of bands were quantified by Image J software. GAPDH, measured in parallel, served as a control. d Western blots were performed, and e relative protein levels of EMT-related markers (E-cadherin, N-cadherin, vimentin, and α-SMA), FoxC1, and Snail in lung tissue of mice were determined. f Representative images of lung sections after Masson trichrome staining and α-SMA IHC, bars = 200 μm. g Quantification of collagen content by Masson trichrome staining. h The levels of α-SMA as determined by IHC analyses.
All data are presented as means ± SD for experiments conducted in triplicate.