The Function And Mechanism Of Long Non-coding RNA Fantom3_F830212l20 In Cigarette Smoke-induced Oxidative Stress In COPD

Background Chronic Obstructive Pulmonary Disease（COPD）is a disease,which has caused high mortality rates and serious disease burdens worldwide.Therefore,COPD has become a global public health problem.Chronic exposure of cigarette smoke,as a major risk factor of COPD,has attracted more attentions of clinicians and researchers.Yet,the exact cause of COPD is still unknown.It is a great need to find effective treatment toward COPD.Long non-coding RNAs（lnc RNAs）are kinds of RNAs with sequences length of more than 200 nucleotides,which can regulate the expression of protein-coding genes in many ways.In recent years,more and more evidence has shown that aberrant expression of lnc RNAs is associated with various human diseases,including COPD.For example,SCAL1 has been reported to be associated with cigarette smoke exposure and lung cancers.However,quite a few lnc RNAs associated with COPD are still unclear,and their biological functions are still unknown.Objectives This paper aims to investigate the expression profiles of lnc RNAs and protein-coding genes in the lung tissues of chronic cigarette smoke induced COPD mouse model,with the purpose of understanding the biological functions of these abnormally expressed lnc RNAs and their roles in the development of COPD.Methods We builded chronic cigarette smoke induced COPD mouse model by using LPS instillation and cigarette smoke.Control animals were housed in a clean environment without any intervention.Then,the lung function was measured in both two groups of mice.The bronchoalveolar lavage fluid was collected for inflammatory cell counting and measurement of Muc5 AC.The lung tissues were taken for pathological observation by H&E staining.RNA sequencing and bioinformatics analyses such as enrichment assays of GO,KEGG,and Chemicals were performed.The co-expression network of lnc RNAs and their associated protein-coding genes were constructed.The analysis of interaction between data of the present study and high-throughput data of patients with COPD from GEO datasets was made.Then a few of lnc RNAs and protein-coding genes,which were significantly differential expressed in RNA sequencing results,were selected for q RT-PCR validation in lungs of other chronic cigarette smoke induced COPD mouse model and control animals.Moreover,we detected the expression patterns of the human homologues of lnc RNAs and protein-coding genes above in 16 HBE cells and A549 cells with and without CSE treatments,as well as PBMCs from patients with COPD and healthy control persons.Results The results of lung function tests,Muc5 AC and the number of inflammatory cells of alveolar lavage fluid（BALF）,the average alveolar intercept and the lung pathologic analysis shown that the establishment of chronic cigarette smoke-induced COPD mouse model was built successfully by LPS and cigarette smoke.The bioinformatics analyses shown that there were 109 lnc RNAs and 260 m RNAs were significantly differential expressed in the lung tissues of chronic cigarette smoke-induced COPD mouse model versus control animals,when using fold change >2 and padj <0.05 as threshold criterion.GO analysis shown that,significantly differential expressed lnc RNAs associated protein-coding genes were mainly enriched in the cellular response to interferon beta（process of gene biology）,GTP binding（molecular function）and cytoplasm（cellular components）.KEGG analysis shown that these protein-coding genes were mainly related to the endoplasmic reticulum protein processing signaling pathway,as well as taurine and hypotaurine signaling pathway.There were 56 lnc RNAs,37 protein-coding genes and 61 connections betewwn lnc RNAs and their associated protein-coding genes in the network of lnc RNA and protein-coding genes.There were three significantly differential expressed protein-coding genes（IL1RL1,UCHL1 and GGT5）were found between chronic cigarette smoke-induced COPD mouse model and patients with COPD.QRT-PCR validation of 14 lnc RNAs（Fantom3_A930002I24,Fantom3₉530016F16,Fantom3_C130011B08,Fantom3_F830212L20,Fantom3₇420409G12,Fantom3₂810049O06,NR₁02714,Fantom3_D330021G15,NR₀33355,NR₀28593,Fantom3_A430043G10,NR₀33450,Fantom3₁200007C13,and Fantom3_D830009E10）and 14 protein-coding genes（Uchl1,Per3,Nqo1,Ahrr,Hlf,Ggt1,Nr1d2,Serpina3 f,Ggt5,Dnaja1,Hspa5,Fkbp4,Hspa1 a,and Il1rl1）in chronic cigarette smoke-induced COPD mouse model and control animals shown that these lnc RNAs and protein-coding genes shared the same expression tendencies both in RNA sequencing and q RT-PCR.Moreover,results shown that 8 lnc RNAs（Fantom3_F830212L20,Fantom3₇420409G12,NR₀28593,NR₀33450,Fantom3_C130011B08,Fantom3₂810049O06,NR₁02714 and Fantom3_D330021G15）human homologous and 7 protein-coding genes（UCHL1,NR1D2,PER3,HLF,AHRR,NQO1,and SERPINA3）shared the same expression tendencies both in chronic cigarette smoke-induced COPD mouse model and 16 HBE cells with CSE treatment;7 lnc RNAs（Fantom3_C130011B08,Fantom3₇420409G12,Fantom3_D830009E10,NR₀33450,NR₀33355,NR₁02714,and Fantom3_F830212L20）human homologous and 7 protein-coding genes（NR1D2,NQO1,AHRR,GGT5,HSPA5,FKBP4 and UCHL1）shared the same expression tendencies both in chronic cigarette smoke-induced COPD mouse model and A549 cells with CSE treatment;2 lnc RNAs（NR₁02714 and NR₀28593）human homologues and 7 protein-coding genes（UCHL1,DNAJA1,GGT5,FKBP4,IL1R1,HSPA1A,and HSPA5）shared the same expression tendencies both in chronic cigarette smoke-induced COPD mouse model and peripheral blood PBMCs from patients with COPD.Results also shown that,the level of GSSG in plasma,Fantom3_F830212L20 human homologous molecules and NQO1 in PBMCs of patients with COPD were higher than those of healthy controls.The amounts of cigarette smoke of patients with COPD were positively correlated with their levels of GSSG in plasma,as well as the expression levels of Fantom3_F830212L20 human homologous molecules and NQO1 in PBMCs.The levels of GSSG,Fantom3_F830212L20,Nqo1 m RNA and Nqo1 protein in lung tissues of mice with tobacco smoke exposure for 7 days and mice with tobacco smoke exposure for 3 months were higher than those of control animals.The expression levels of Fantom3_F830212L20,Nqo1 m RNA and protein were up-regulated along with increased CSE concentration（0-1%）.Furthermore,the expression levels of Fantom3_F830212L20 Nqo1 m RNA were markedly elevated at the early stage of CSE exposures,and decreased gradually in the late stage of CSE exposures（0-24h）.However,the level of Nqo1 protein increased along with increased CSE exposures（0-24h）.Knockdown of Fantom3_F830212L20 in mle12 cells inhibited intracellular GSH levels,as well as Nqo1 at the m RNA and protein levels.Conclusions 1.A total of 109 lnc RNAs and 260 m RNAs were significantly differentially expressd in lung tissues of chronic cigarette smoke-induced COPD mouse model as compared with control animals.The biological functions of significantly differential expressed lnc RNAs associated protein-coding genes,are mainly related to the endoplasmic reticulum protein processing signaling pathway,as well as taurine and hypotaurine signaling pathway.2.Lnc RNA NR₁02714（or NR₁02714 human homologues）and its relevant protein-coding gene UCHL1,shared the same expression tendencies in lungs of chronic cigarette smoke-induced COPD mouse model,16 HEB cells and A549cells with CSE treatments,as well as peripheral blood PBMCs from patients with COPD.This result suggests that NR₁02714（or NR₁02714 human homologues）and its relevant protein-coding gene UCHL1,might be involved in the development of COPD.3.The overexpression of Fantom3_F830212L20 both in COPD mouse model and mle12 cells with CSE treatment.Fantom3_F830212L20 might be able to protect cells against cigarette smoke-induced oxidative stress by regulating the expression of Nqo1 in mle12 cells.4.The expression levels of Fantom3_F830212L20 human homologous molecules and NQO1 m RNA in PBMCs of patients with COPD are higher than those of healthy controls.The amounts of cigarette smoke of patients with COPD are positively correlated with the levels of GSSG in plasma,as well as the RNA levels of Fantom3_F830212L20 human homologous molecules and NQO1 in PBMCs.The relationship of Fantom3_F830212L20 human homologous molecules and NQO1 are needed to be determined.