LincRNA00612 Inhibits Apoptosis And Inflammation In LPS-Induced BEAS-2B Cells Via Enhancing Interaction Between P-STAT3 And A2M Promoter

Purposes: Chronic obstructive pulmonary disease(COPD)is a progressive,degenerative disease involving the respiratory system,characterized by chronic airway inflammation.Long non-coding RNAs(LncRNAs)have been reported as key regulators of chronic obstructive pulmonary disease(COPD),which may be potential targets for the clinical treatment of COPD,so it is of great significance to explore the mechanism of LncRNA.This study aimed to investigate the value of LncRNA00612(LINC00612)as a diagnostic marker for COPD and its role and regulatory mechanism in lipopolysaccharide(LPS)-induced apoptosis and inflammation in BEAS-2B cells.Methods: Clinical samples of peripheral venous blood from healthy control group(n=34)and COPD group(n=32)were collected from Changzhou Second People’s Hospital(January 2022 to July 2022),and the clinical data of patients were retrospectively analyzed and sorted.Treat BEAS-2B cells with LPS to construct a cellular model of COPD.In vitro transfection of plasmids/small interfering RNA of LINC00612 and α-2-macroglobulin(A2M)was used to detect the expression levels of LINC00612 and A2 M using real-time quantitative polymerase chain reaction(RT-qPCR).Western blot detects the expression of apoptosis-associated proteins,caspase3 proteins,transcriptional activator 3(STAT3)proteins,and A2 M proteins.Cell Counting Kit-8(CCK-8)detects cell viability.Flow cytometry to detect apoptosis.Inflammatory factors are detected by enzyme-linked immunosorbent assay(ELISA).The binding sites between LINC00612,STAT3 and A2 M promoters were predicted by catRAPID and JASPAR,and RNA antisense purification(RAP)was performed to verify the binding of LINC00612 and STAT3,and chromatin immunoprecipitation(CHIP)to verify the binding of STAT3 to A2 M promoter fragments.All data were analyzed by the SPSS26.0 program and expressed as the means ± standard error of the mean(SEM)from three independent replicates.One-way ANOVA and t-test were used to assess differences between groups.The Pearson correlation analysis method was used to analyze the correlation between LINC00612 and A2 M expression in patients with COPD.The value of LINC00612 in the diagnosis of COPD was investigated using a subject-operating characteristic(ROC)curve.When the P value < 0.05,the difference is considered to be statistically significant.Results: Compared with the control group,the expression of LINC00612 and A2 M in peripheral venous blood in COPD patients was significantly downregulated(P < 0.0001).The ROC curve suggests that LINC00612 has the ability to distinguish COPD from healthy controls,and the area under the curve(AUC)is 0.9292(95% CI,0.8703 0.9882;P <0.0001).In addition,Pearson’s correlation analysis showed that LINC00612 was positively correlated with A2 M in peripheral venous blood of COPD patients(r=0.6788,P <0.0001).In LPS-induced BEAS-2B cells,LINC00612(P <0.001)and A2M(P < 0.01)expression were significantly down-regulated.Overexpression of LINC00612 attenuated the effect of LPS-induced BEAS-2B cells on apoptosis and inflammation.After knocking down LINC00612,the expression levels of mRNA(P <0.01)and protein(P <0.05)of A2 M decreased,while after overexpression of LINC00612,the levels of mRNA(P <0.01)and protein(P <0.05)of A2 M increased,knocking down A2 M reversed the effect of overexpression of LINC00612(P <0.05).Overexpression of LINC00612 promotes BEAS-2B cells against LPS-mediated apoptosis and inflammation,while knockdown A2 M attenuates this effect.RAP experiments showed that LINC00612 interacted directly with p-STAT3(Tyr705)(P <0.05),and significant enrichment of A2 M mRNA in LINC00612 pull-down samples was observed.After knocking down STAT3,the expression of mRNA and protein of A2 M decreased(P <0.05),and knocking down LINC00612 disrupted the binding of p-STAT3 to the A2 M promoter.Conclusion: LINC00612 is a potential biomarker for COPD,which improves the apoptosis and inflammatory response of LPS-induced BEAS-2B cells by recruiting STAT3 to bind to A2 M.This finding will provide a experimental basis for the treatment of COPD.