The Role And Mechanism Of LncRNA NONMMUT083950 As CeRNA In Smoke Inhalation Injury

Objective:Smoke inhalation injury is one of the important factors affecting the prognosis of burns,but the molecular mechanism of its occurrence and development is not very clear.It has been confirmed that long non-coding RNA(lncRNAs)plays an important role in various diseases such as tumor and sepsis-induced acute lung injury.In the first part of this study,microarray detection and literature review suggested that lncRNA NONMMUT083950(3950 for short)may act as competing endogenous RNA(ce RNA)to mediate inflammatory and apoptotic responses in smoke inhalation injury;then,the second part intends to study the role and molecular mechanism of lncRNA 3950 in smoke inhalation injury in vitro,so as to lay a theoretical and experimental foundation for later clinical application.Methods:Step 1.Based on the previous experimental research on smoke inhalation injury,a mouse smoke inhalation injury model was established by using a self-made smoke inhalation injury instrument.The general morphology of lung tissue was observed,the pathological changes of lung tissue were detected by hematoxylin eosin staining,and the expression levels of inflammatory factors and apoptosis related proteins in lung tissue were detected by Western blot.Step 2.Microarray technology was used to detect the lung tissue of smoke inhalation injury mice and control mice,and the aberrantly expressed lncRNAs that were significantly positively correlated with aberrantly expressed m RNAs were selected as the target lncRNAs for ce RNA analysis.The database mi RBase22 was used to predict the target of mi RNA-lncRNA and mi RNA-m RNA,and the ternary regulatory network of lncRNA-mi RNA-m RNA was established by using mi RNA as a bridge,and the ce RNA network map was drawn.Step 3.To verify the microarray results,quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the expression of lncRNA3950,mi R-149-3p and CXCL14 in mice lung tissue,and Western blot was used to detect the expression level of CXCL14 protein in mice lung tissue.Step 4.A self-made smoke inhalation injury instrument was used to establish a smoke-induced lung epithelial cells(MLE-12 cells and TC-1 cells)injury model,and the scratch assay was used to detect the cell migration and repair ability,the Cell Counting Kit-8(CCK-8)was used to detect the cell viability,the lactate dehydrogenase(LDH)colorimetric assay kit was used to detect the LDH content in the cell culture supernatant,and the enzyme linked immunosorbent assay(ELISA)was used to detect the expression of inflammatory factors in the cell culture supernatant,and Western blot was used to detect the expression levels of apoptosis response-related proteins.Step 5.After smoke-induced,q RT-PCR was used to detect the expression of lncRNA3950,mi RNA-149-3p and CXCL14 in cells,and Western blot was used to detect the expression of CXCL14 protein in cells.Step 6.Fluorescence in situ hybridization(FISH)was used to detect the subcellular localization of lncRNA 3950 in MLE-12 cells and TC-1 cells,and dual-luciferase was used to detect the interaction between lncRNA 3950 and mi R-149-3p,the interaction between mi R-149-3p interacts with CXCL14.Step 7.On the basis of lung epithelial cell injury caused by smoke,lncRNA3950,mi R-149-3p and CXCL14 were silenced or overexpressed by cell transfection.The expression of inflammatory factors in cell culture supernatant was detected by ELISA,the expression of apoptosis related proteins and CXCL14 were detected by Western blot,the changes of apoptosis were detected by Flow Cytometry,and the expression of lncRNA3950,mirna-149-3p and CXCL14 were detected by q RT-PCR.Results:1.The envelope of lung tissue in smoke inhalation injury mice was tense and swollen obviously,scattered bleeding points or even local flake bleeding could be seen on the surface,and the edge became blunt;the results of lung histopathological examination showed that inflammatory cell infiltration and bleeding in varying degrees could be seen in the alveolar cavity,alveolar cavity collapse or cystic expansion,partial alveolar septum thickening and local hyaline membrane formation could be seen in some visual fields;when compared to the control group,in the Injury group,the expression levels of IL-1β,TNF-α,Bax,caspase-7 and caspase-3 in the lung tissue of mice increased,while Bcl-2 decreased.All these differences were statistically significant.2.Compared with the control group,in the Injury group,577 lncRNAs were differentially expressed in the lung tissue of mice,of which 322 were down-regulated and 255 were up-regulated;there were 517 differentially expressed m RNAs,of which106 were down-regulated and 411 were up-regulated;the ce RNA network was constructed;the expression of lncRNA3950 and CXCL14 were up-regulated,and the expression of mi R-149-3p was down regulated in the injury group;all these differences were statistically significant.3.Compared with the control group,in the Injury group,the cell migration and repair ability decreased;the contents of LDH and inflammatory factors TNF-α and IL-1β in the cell culture supernatant increased;the cell viability decreased;the expression level of apoptosis-related proteins,including Bax,caspase-7 and caspase-3was increased,while Bcl-2 was decreased;the expression of lncRNA3950 and CXCL14 was up-regulated,and the expression of mi R-149-3p was down-regulated;all these differences were statistically significant.4.On the basis of smoke-induced lung epithelial cell injury,silencing lncRNA3950,compared with the Injury+si-NC group,in the Injury+si-3950 group,the cell viability was improved;the contents of TNF-α and IL-1β in the cell culture supernatant were reduced;the cell apoptosis rate was decreased;the expression levels of Bax,caspase-7 and caspase-3 in the cells were decreased,while Bcl-2 was increased;the expressions of mi R-149-3p was increased and CXCL14 was decreased;all these differences were statistically significant.5.The results of FISH detection showed that: lncRNA 3950 was mainly located in the cytoplasm in MLE-12 cells and TC-1 cells;the results of dual-luciferase experiments showed that: lncRNA 3950 directly binds and adsorbs mi R-149-3p,and mi R-149-3p directly binds and adsorbs CXCL14.6.On the basis of smoke-induced lung epithelial cell injury,mi R-149-3p was overexpressed,compared with the Injury+mi R-NC group,in the Injury+mi R-149-3p group,the contents of TNF-α and IL-1β in the culture supernatant were decreased;the expression levels of Bax,caspase-7 and caspase-3 in cells were decreased,while Bcl-2 was increased;and the expression of CXCL14 was decreased.CXCL14 was silenced,compared with Injury+si-NC group,in the si-CXCL14 group,the contents of TNF-α and IL-1β in the culture supernatant of cells were decreased;the expression levels of Bax,caspase-7 and caspase-3 in the cells were decreased,and Bcl-2 was increased;the expression of lncRNA3950 was decreased;All these differences were statistically significant.7.On the basis of smoke-induced lung epithelial cell injury,silencing of mi R-149-3p or overexpression of CXCL14 could reverse the effect of silencing lncRNA3950 on reducing the expression of inflammatory factors and apoptosisrelated proteins.Conclusion:1.The expression profiles of lncRNAs and m RNAs in lung tissue of smoke inhalation injury mice were different from those of the control group.Among them,577 lncRNAs and 517 m RNAs were aberrantly expressed;2.Lnc RNA3950 mediates inflammation and apoptosis by acting as ce RNA to sponge mi R-149-3p and upregulate CXCL14 expression in smoke inhalation injury;3.Silencing lncRNA 3950 can effectively reduce the inflammatory response,reduce cell apoptosis,and improve cell viability in post-injury cells,which lays a theoretical and experimental foundation for the later clinical targeted therapy of smoke inhalation injury.