Identification Of Differential Expression Of Long Non-coding RNA In Patients With Rheumatoid Arthritis Associated Interstitial Lung Disease

Rheumatoid arthritis（RA）is a chronic,idiopathic and common autoimmune disease,with sex bias of at least 3:1 favoring females to males,mainly characterized by inflammatory joint fluid and synovium,erosion of the marginal bone,and degradation of articular cartilage together with systemic manifestations of immune responses.However,studies have shown that 50%of patients with RA have manifestations of extrarticular involvement,among them,interstitial lung disease（ILD）is a well-known complication of rheumatoid arthritis（RA）which often results in significant morbidity and mortality.The prevalence of rheumatoid arthritis-associated interstitial lung disease（RA-ILD）varies from 3%-67%in different series during last decade.The vast differences in these rates can be attributed to the populations being investigated,the criteria used for diagnosis,and the techniques used for detection[high-resolution computed tomography（HRCT）,chest radiography,lung function examination,etc].In the early stage,only about 10%of patients have clinical symptoms,most of the RA-ILD patients have no significant clinically symptoms.At 5 years after the first RA-ILD diagnosis,35%-39%of patients had died.Therefore,detection of this disease and intervention at an early stage is criticalIn the human genome,more than 90%is available for transcription;however,only 1%-2%encodes proteins.The majority of the remainder of the human genome is genes that do not have the potential to encode proteins;these genes are called noncoding RNA（ncRNA）.According to their length,ncRNA may be divided into long ncRNA（lncRNA）,which are considered to be>200 nt in length,or short ncRNA（sncRNA）containing less than 200 nucleotides.A lot of studies have shown that lncRNA plays a crucial role in post-transcriptional,transcriptional,and chromosomal gene regulation,respectively,and is involved in the development of autoimmune diseases（such as RA）and ILD.However,no studies have focused on the lncRNA profiles in RA-ILD.Our team aimed to explore the identification of differentially expressed lncRNA in peripheral blood mononuclear cells（PBMCs）from patients with RA-ILD.Part Ⅰ Characterizing the functional regulations between lncRNA-mRNA based on the expression profile of rheumatoid arthritis associated interstitial lung diseaseWe collected PBMCs from middle-aged female healthy controls,RA and RA-ILD patients,excluding those with known risk factors of RA-ILD,such as being elderly or,male,smoking,and having a history of other diseases.Then,a microarray analysis was applied to profile the lncRNA and mRNA levels.Analysis of the differential expression（DE）level of lncRNA and mRNA,the chromosomal enrichment of pathogenic genes,the effect of lncRNA on neighboring genes was performed,Pearson correlation test was performed to identify the lncRNA-mRNA association.Constructed the lncRNA-mRNA co-expression networks were used to identify the hub lncRNAs.The Gene Ontology（GO）and KEGG pathway were analyzed to investigate the potential role of differentially expressed genes in RA-ILD.The potential function of differentially expressed lncRNA in RA-ILD was analyzed,and preparation was made for screening some candidate lncRNAs for validation.Compared to the RA group,we obtained 5741 mRNAs（3115 and 2356 lncRNAs were identified to be up-and down-regulated,respectively）and 5250 lncRNAs（2965 and 2285 lncRNAs were identified to be up-and down-regulated,respectively）,which were significantly differentially expressed.It was found that the adjacent mRNAs（300kb）of 1677 lncRNAs in 5250 DE-lncRNAs were also differentially expressed;the differentially expressed lncRNAs had a relationship of adjacent mRNAs（100 kb）to a total of 3178 pairs.Compared to the HC group,we obtained 1728 mRNAs（733 and 995 lncRNAs were identified to be up-and down-regulated,respectively）and 1833 lncRNAs（1018 and 815 lncRNAs were identified to be up-and down-regulated,respectively）,which were significantly differentially expressed.It was found that the adjacent mRNAs（300kb）of 291 IncRNAs in 1833 DE-lncRNAs were also differentially expressed;the differentially expressed IncRNAs had a relationship of adjacent mRNAs（100 kb）to a total of 413 pairs.DE-mRNA and DE-lncRNA were not significantly enriched on a certain chromosome.GO analysis showed that the biological process of significant enrichment of genes up-regulated in PBMCs from patients with RA-ILD group compared to the RA group are mainly involved in cellular metabolic process,down-regulated genes are primarily involved in immune system processes.When compared to the HC group,up-regulated genes are mainly involved in neutrophil-mediated immunity and bone marrow leukocyte-mediated immunity;down-regulated genes are involved in T cell activation and T cell receptor signaling pathways.KEGG pathway analysis showed that,when compared to the RA group,up-regulated genes in PBMCs from patients with RA-ILD are mainly involved in NOD-like receptor signaling pathways and ubiquitin-mediated proteolysis,down-regulated genes are primarily involved in primary immunodeficiency and natural killer cell-mediated cytotoxicity.When compared to the HC group,up-regulated genes in PBMCs from patients with RA-ILD are mainly involved the IL-17 signaling pathway and the TNF signaling pathway,down-regulated genes are primarily involved in cholesterol metabolism and natural killer cell-mediated cytotoxicity.In order to further distinguish between the differentially expressed and regulated genes in RA-ILD and RA group and the differentially expressed and regulated genes in RA-ILD and HC group,a co-expression network was used to perform correlation test between DE-lncRNAs and DE-mRNAs.Based on the RA-ILD and HC groups,the lncRNA-mRNA pairs with P value<0.05 and correlation coefficient>0.9 were selected.For the co-expressed DE-lncRNAs and DE-mRNAs,we constructed the co-expression network in RA-ILD and RA group,as well as in RA-ILD and HC group.We compared the node degree for each IncRNA between the two networks.These IncRNAs were ranked by the changed degree values,and the first 1%of lncRNA was screened.Finally,a total of 15 lncRNAs were obtained:TCONS_l2₀0030586,ENST00000551075,NR₀46241,ENST00000607625,TCONS_l2₀0006925,T206892,ENST00000602954,NR₀29401,ENST00000609151,uc001vrz.1,T112526,ENST00000507808,uc003qrv.1,T344288,ENST00000515150.The GO function enrichment of the mRNA set corresponding to lncRNA was found to be mainly focused on:isopentenyl transferase activity,transfer of alkyl or aryl（except methyl）groups,ribosome binding,thiol-dependent Sexual ubiquitin-specific protease activity in four aspects.Based on the differential expression level of lncRNA and mRNA,the chromosomal enrichment of pathogenic genes,the effect of IncRNA on neighboring genes,the analysis of GO and KEGG pathways,our team obtained the above results,which provided some ideas for the next step to select candidates lncRNAs for PCR and further study the related mechanism.Part Ⅱ Identification of differentially expressed IncRNA in PBMC of RA-ILD patients and analysis of its correlation with clinical indicatorsAccording to the consistency of the differential expression trend of lncRNA detected in the RA-ILD group,RA group and HC group,the fold-change degree of the differential expression,the type of lncRNA and whether it has been proved to be related to RA or ILD in the lncRNA disease database,6 lncRNAs were selected as candidates for PCR in total,including ENST00000429666,uc.471-,NR₀38935,ENST00000560199,ENST00000603415 and NR₀02819.We first used small samples（8 samples from each of the three groups）to preliminarily verify the selected 6 genes,and the results showed that the changes of ENST00000429666 and uc.471-were not significant.Therefore,we only verified NR₀38935,ENST00000560199,ENST00000603415 and NR₀02819 with expanded samples（32 samples from each of the three groups）.Considered that being male are a risk factor of RA-ILD,and RA-ILD can be classified as UIP（19 cases,including 13 cases of women,6 cases of men）and NSIP（13 cases,7 cases of women,6 cases of men）.According to the above factors,different groups were compared.PCR verification results showed that,no matter in which group comparison,the expression levels of NR₀02819,NR₀38935 and ENST00000603415 were significantly increased in the RA-ILD group,while the expression level of ENST00000560199 was significantly decreased.In addition to ENST00000603415,the other three IncRNAs were more differentially expressed in males than females.There was no significant statistical difference between 4 IncRNAs in RA-ILD subgroups.To further determine whether the differentially expressed lncRNAs have certain diagnostic significance for RA-ILD,we further calculated the ROC curve and AUC of NR₀02819,NR₀38935,ENST00000603415 and ENST00000560199.We found that,when as risk factors for RA-ILD,the area under the curve（AUC）values of NR₀02819,NR₀38935 and ENST00000603415 were 0.889,0.787（medium diagnostic accuracy）and 0.962（high diagnostic accuracy）,respectively.When as protective factors for RA-ILD,the AUC of ENST00000560199 was 0.863（medium diagnostic accuracy）.The optimized cutoff levels of NR₀02819,NR 038935,ENST00000603415 and ENST00000560199 were 3.576（with the sensitivity=0.844,and the specificity=0.937）,1.516（with the sensitivity=0.469,and the specificity=0.984）,1.847（with the sensitivity=0.813,and the specificity=0.969）,and 0.481（with the sensitivity=0.781,and the specificity=0.844）,respectively.In order to further analyze the possible role of differentially expressed lncRNAs in RA-ILD,we detected KL-6 of the RA-ILD group,which have been widely studied,that are more known RA-ILD related biomarkers.Furthermore,the RA-ILD related clinical indicators（such as ESR,CRP,RF,CCP）as well as lung function test results（FVC%,FEV1%,DLCO%）and widely recognized biomarker associated with RA or ILD（IL-17A,TNF-α,IL-6,IFN-γ）were analyzed.Correlation analysis results showed that NR₀02819 was positively correlated with KL-6,negatively correlated with FVC%,FEV1%,DLCO%and TNF-α,but not significantly correlated with IL-6,IFN-y,IL-17A,RF,CCP,ESR and CRP.NR₀38935 was positively correlated with KL-6,negatively correlated with FVC%,FEV1%,DLCO%,and not significantly correlated with IL-6,TNF-α,IFN-γ,IL-17A,RF,CCP,ESR and CRP.ENST00000560199 was negatively correlated with KL-6,positively correlated with FEV1%and DLCO%,not significantly correlated with FVC%,IL-6,TNF-α,IFN-γ,IL-17A,RF,CCP,ESR and CRP.ENST00000603415 was positively correlated with KL-6,negatively correlated with FVC%,not significantly correlated with KL-6,FEV1%,DLCO%,IL-6,TNF-α,IFN-γ,IL-17A,RF,CCP,ESR and CRP.Combined with the above results,literature review and gene database search analysis,our team believes that the differential expressions of lncRNA NR₀02819,NR₀38935,ENST00000603415 and ENST00000560199 may have a certain correlation with the severity of RA-ILD,and may play a certain role in the diagnosis of RA-ILD.Further large sample prospective studies and relevant mechanism researches still need to be performed.