Study On The Mechanism Of Circular RNA Circ0061052 Regulating MiR-515-5p In Airway Remodeling Induced By Smoking

Chronic obstructive pulmonary disease（COPD）is one of the main causes of morbidity and death in the global population,and its pathological characteristics are mainly airway remodeling and emphysema.Cigarette smoke（CS）contains more than 7,000 chemicals that are closely related to the development of many diseases and considered to be one of the major causes of COPD.Airway remodeling is an important physiological and pathological change in COPD.Airway remodeling leads to airway obstruction to a large extent and leads to airflow limitation in COPD patients.Human bronchial epithelial（HBE）cells are the first anatomical barrier exposed to toxic and harmful substances and CS,and they are also the most vulnerable to injury.HBE cells can cause airway wall thickening through epithelial-mesenchymal transition（EMT）.Airway remodeling,but the mechanism of its occurrence and development is still unclear.Non-coding RNA（ncRNA）is a type of functional RNA that does not encode protein.Circular RNA（circRNA）is a ring structure connected by typical 5’to 3’-phosphodiester bonds.A new class of functional molecules,it does not contain 5’or 3’free ends,and is more stable in the cytoplasm.circRNA contains many miRNA binding sites and plays a role in inhibiting the translation of target mRNA through sponge action.Therefore,as a new type of regulatory molecule,circRNA has become a new therapeutic and diagnostic target.It is necessary to learn more about these molecules in health.Regulation and function in tissues and diseases.Therefore,to explore the role and regulation mechanism of circRNA combined with miRNA to play competitive endogenous RNA（ceRNA）in the occurrence of EMT,airway remodeling and COPD in HBE cells caused by CS,in order to understand the role of these molecules in airway remodeling and COPD caused by smoking the role of the process,to find its new therapeutic and diagnostic targets to provide a scientific basis.ObjectiveTo investigate the mechanism of circRNA₀061052（circ0061052）regulating FoxC1/Snail-mediated EMT of HBE cells through miR-515-5p in smoking-induced airway remodeling and COPD.MethodsHBE cells were exposed to CSE at a concentration of 0,1%,2%or 4%for 48 hours to construct CSE in vitro cell exposure models;HBE cells were treated with 50 nM circ0061052 siRNA or Con siRNA for 24 hours,and then exposed to 4%CSE for 48 h;HBE cells were treated with 50 nM miR-515-5p mimic or Con mimic for 24 h,and then exposed to 4%CSE for 48 h;50 nM circ0061052 siRNA and 50 nM miR-515-5p inhibitor were co-transfected with HBE cells for 24 h,and then exposed to 4%CSE for 48 h;HBE cells were fixed with 4%paraformaldehyde for 10 minutes and permeabilized with 0.5%Triton X-100 at 4℃ cells were labeled with probes for circ0061052 location detection;6-8 weeks old male Balb/c mice were randomly divided into 4 groups and exposed to 0,100,200,300 mg/m3 TPM CS respectively,lung function was measured,and tissues were collected The samples were subjected to Western blot,qRT-PCR,Masson staining,and immunohistochemical analysis.CCK-8 experiment to evaluate the effect of different concentrations of CSE on the viability of HBE cells;Western blot to detect the expression of EMT marker proteins and FoxC1/Snail signaling pathway related proteins in HBE cells;qRT-PCR to detect circ0061052,miR-515-5p in HBE cells and mouse lung tissue expression level;immunofluorescence detection of E-cadherin and Vimentin expression levels in HBE cells;FISH experiment and nuclear-plasma separation technology to detect the location and distribution of circ0061052 in the nucleus and cytoplasm;bioinformatics website CircInteractome predicts circ0061052 and the binding site of miR-515-5p;the luciferase reporter gene verifies the binding of circ0061052 to miR-515-5p and miR-515-5p to FoxC1;the whole body plethysmography method measures AHR（Penh value）to evaluate the mouse Lung function;Mason staining to evaluate the accumulation of collagen around the mouse airways,and immunohistochemistry to evaluate the expression of α-SMA in lung tissue.Results1.The effects of CSE on EMT and circ0061052 levels of HBE cellsHBE cells were exposed to CSE at a concentration of 0、1%、2%、4%、8%、10%、20%or 30%for 48 hours to determine the effect on cell viability.It was found that when the CSE concentration was greater than or equal to 8%,the cell vitality decreased;With the increase of CSE exposure level,the expression levels of epithelial marker E-cadherin decreased,the expression levels of mesenchymal markers N-cadherin,Vimentin and α-SMA increased,the expression levels of circ0061052 increased,and the cells developed EMT;immunofluorescence results confirmed CSE treatment causes EMT in HBE cells.It shows that CSE exposure can cause EMT in HBE cells and increase the level of circ0061052.2.The ring characterization of circ0061052 in HBE cellsThe results showed that circ0061052 is derived from the host gene oxysterol binding protein 2（OSBPL2）;the results of FISH and nuclear-plasma separation experiments show that circ0061052 is mainly located in the cytoplasm of HBE;after treatment with Rnase R,it is found that circ0061052 is positive for Rnase R is resistant to digestion.It shows that circ0061052 has a ring structure and mainly exists in the cytoplasm of HBE cells.3.The effects of CSE on miR-515-5p level and FoxC1/Snail signaling pathway in HBE cellsThe results showed that circ0061052 can target miR-515-5p.The levels of miR-515-5p decreased with the increase of CSE exposure dose,and showed a dose-response relationship;the luciferase reporter gene confirms that circ0061052 targets miR-515-5p;the pathway protein FoxC1/Snail increased with the increase of CSE exposure dose,and showed a dose-response relationship;the luciferase reporter gene confirms that miR-515-5p acts on the downstream target gene FoxC1 and affects the FoxC1 protein expression.It shows that circ0061052 regulates the expression of FoxCl/Snail through miR-515-5p,and thus plays a role.4.The effects of circ0061052 on the CSE-induced EMT and FoxC1/Snail signaling pathway in HBE cellsThe results showed that in HBE cells,circ0061052 siRNA transfection significantly reduced the levels of circ0061052;compared with the CSE exposure group,the expression levels of E-cadherin in the circ0061052 siRNA group increased,and the levels of N-cadherin,Vimentin and α-SMA decreased.The levels of FoxCl/Snail protein decreased,and the effect of CSE was inhibited;immunofluorescence results confirmed that the EMT effect caused by CSE was reversed after transfection with circ0061052 siRNA.It indicates that circ0061052 is involved in regulating the expression of EMT and FoxCl/Snail induced by CSE in HBE cells.5.The effects of miR-515-5p on the CSE-induced EMT and FoxCl/Snail signaling pathway in HBE cellsThe results showed that in HBE cells,miR-515-5p mimic transfection significantly increased the levels of miR-515-5p;compared with the CSE exposure group,the levels of E-cadherin in the miR-515-5p mimic group increased,the levels of cadherin,Vimentin and α-SMA decreased,and the levels of pathway protein FoxC1/Snail decreased,and the effects of CSE was inhibited;immunofluorescence results confirmed that after transfection with miR-515-5p mimic,the EMT caused by CSE was reversed effect.It shows that miR-515-5p is involved in regulating the expression of EMT and FoxC1/Snail induced by CSE in HBE cells.6.Circ0061052,via miR-515-5p regulation of FoxC1 and Snail,is involved in the CSE-induced EMTThe results showed that in HBE cells,compared with the CSE exposure group,the expression levels of E-cadherin increased in the circ0061052 siRNA group,the levels of N-cadherin,Vimentin and α-SMA decreased,and the levels of pathway proteins FoxCl/Snail decreased,the effect of CSE is inhibited;based on knocking down the level of circ0061052,transfection of miR-515-5p inhibitor can further down-regulate the levels of miR-515-5p.The results showed that the EMT inhibition caused by reducing the levels of circ0061052 and the FoxCl/Snail inhibitory pathway was reversed by the downregulation of miR-515-5p.It indicates that miR-515-5p is involved in regulating the expression of CSE-induced EMT and FoxCl/Snail in HBE cells.It shows that circ0061052 regulates the FoxCl/Snail signaling pathway through miR-515-5p and plays a role in the EMT process of HBE cells exposed to CSE.7.The effects of circ0061052 in airway remodeling induced by chronic exposure to CS in miceThe results of animal experiments showed that with the increase in the dose of CS exposure in mice,the levels of circ0061052 in lung tissue increased,and the levels of miR-515-5p decreased;as the dose of CS increased,the Penh value showed an upward trend.The increase in airway resistance in mice has a greater impact on the lung function of mice;Western blot results showed that CS exposure leads to a decrease in the expression of E-cadherin in lung tissues,an increase in the levels of N-cadherin,Vimentin and α-SMA,and levels of the pathway protein FoxCl/Snail increased;Mason staining results showed that with the increase of CS exposure,the airway wall of mice became thicker and collagen was deposited.The immunohistochemical results showed that the levels of α-SMA increased.These results indicated that CS exposure causes lung dysfunction and small airway remodeling in mice,and CS exposure can increase the levels of circ0061052,decrease the levels of miR-515-5p,and activate FoxCl/Snail pathway to participate in the airway remodeling process.8.The effects of smoking on circ0061052,miR-515-5p and EMT in human lung tissueFurther validation was carried out at the population level,and baseline data and lung tissue samples were collected for analysis.The results showed that,compared with the control group,the levels of circ0061052 in lung tissues of smoking group and smoking COPD group was increased,and the levels of miR-515-5p was decreased,and the change was more significant in smoking COPD group,and there was a negative correlation between the levels of circ0061052 and miR-515-5p.These results indicated that the levels of circ0061052 was increased and the levels of miR-515-5p was decreased by smoking,and the levels of circ0061052 was negatively correlated with the levels of miR-515-5p.The levels of EMT key protein E-cadherin in lung tissue was decreased,while Vimentin was increased,and the change was more significant in smoking COPD group.These results indicate that smoking can increase the levels of circ0061052 in lung tissue,decrease the levels of miR-515-5p,and change the EMT index in lung tissue.Conclusion1.Cigarette smoke exposure can induce airway remodeling and the occurrence and development of COPD.2.circ0061052 plays an important role in the development of airway remodeling and COPD induced by cigarette smoke exposure.3.circ0061052 regulates the FoxCl/Snail signaling pathway through miR-515-5p to mediate the EMT of HBE cells and participate in the development of airway remodeling and COPD caused by cigarette smoke.