The Regulatory Effect And Molecular Mechanism Of LncRNA NEAT1/miR-204-5p/SIX1 Axis On Airway Remodeling In Asthma

Background: Asthma is a chronic airway inflammation and airway hyperresponsiveness of the immune allergic disease,clinical often at night or in the early morning recurrent or exacerbated wheezing,cough,shortness of breath or chest tightness as the performance.Respiratory structural changes,the so-called airway remodeling,are important pathological outcomes.In the long-term inflammatory process,in addition to environmental factors and immune factors,gene regulation is also particularly important.Current studies have shown that non-coding RNA(ncRNA)plays a role in many chronic inflammatory processes,but there are few reports on the role of ncRNA in asthma airway remodeling.Studying the role of ncRNA in asthma airway remodeling may have high value in preventing and treating asthma airway remodeling.This study explored the interaction of long non-coding RNA(lncRNA)NEAT1,micro RNA(mi RNA)mi R-204-5p and Sine oculis homeobox homolog 1(SIX1)and their regulation of downstream signals.These genes may play a regulatory role in the process of airway remodeling and may provide valuable reference information for the prevention and treatment of asthma airway remodeling.Methods: To find and identify lncRNAs related to human transforming growth factor-β1(TGF-β1),epithelial-mesenchymal transition(EMT)and binding sites with mi R-204-5p from the bioinformatics database.Human bronchial epithelial cells(16HBE)were cultured in vitro and treated with TGF-β1 to induce EMT.RT-q PCR was used to detect the expression levels of lncRNA,mi R-204-5p and SIX1 in 16 HBE with EMT changes.The antisense oligonucleotide of NEAT1(ASO-NEAT1)was transfected into 16 HBE to interfere with the expression of NEAT1.Then the expression levels of NEAT1,mi R-204-5p and SIX1 were detected by RT-q PCR,and the expression of E-cadherin,N-cadherin,keratin,vimentin,fibronectin and phosphorylated SMAD3(p-SMAD3)was detected by Western Blot(WB).After co-transfection of ASO-NEAT1 and mi R-204-5p overexpression plasmids,the expression levels of NEAT1,mi R-204-5p and SIX1 were detected by RT-q PCR,and the expression levels of E-cadherin,N-cadherin,keratin,vimentin,fibronectin and p-SMAD3 were detected by WB.After constructing the wild-type and mutant vector plasmids of NEAT1,the dual luciferase reporter gene analysis experiment was used to verify whether NEAT1 and mi R-204-5p bind to each other.Results: From the bioinformatics database,the related lncRNAs were found,namely TBILA,NKILA,ATB,HOTAIR and NEAT1.After TGF-β1 intervention in 16 HBE,the inverted microscope observed that the cell morphology was long fusiform,the connection between cells was not close,the gap increased,and the cells showed EMT changes.After TGF-β1 intervention,RT-q PCR showed that the expression of lncRNA TBILA,NKILA and HOTAIR was up-regulated,while the expression of ATB and NEAT1 was downregulated,and the expression of mi R-204-5p was down-regulated and the expression of SIX1 was up-regulated.WB results showed that the expression of E-cadherin and keratin was down-regulated,and the expression of N-cadherin,vimentin,fibronectin and p-SMAD3 was up-regulated.After 16 HBE transfected with ASO-NEAT1,the expression of NEAT1 and mi R-204-5p decreased,and the expression of SIX1 increased.WB showed that the expression of E-cadherin and keratin was down-regulated,and the expression of N-cadherin,vimentin,fibronectin and p-SMAD3 was up-regulated.After co-transfection of ASONEAT1 and overexpression plasmids of mi R-204-5p in 16 HBE,RT-q PCR suggested that NEAT1 expression was still down-regulated,but mi R-204-5p expression was up-regulated,while SIX1 expression was down-regulated,WB results showed that the expression of Ecadherin and keratin was up-regulated,and the expression of N-cadherin,vimentin,fibronectin and p-SMAD3 was down-regulated.The results of dual luciferase gene report analysis showed that the fluorescence value of firefly in the co-transfection group of NEAT1wild-type sequence plasmid and mi R-204-5p overexpression plasmid decreased relatively,which proved that NEAT1 had a binding effect with mi R-204-5p.Conclusion: In vitro cultured 16 HBE,TGF-β1 promotes the EMT process through the SMAD3 signaling pathway.LncRNA NEAT1 interacts with mi R-204-5p to regulate the expression of SIX1 and regulates the EMT process through the SMAD3 signaling pathway.NEAT1 may be a biomarker target for the prevention and treatment of airway remodeling in asthma.