Long Noncoding RNA Expression Profiling And Correlation Analysis In PBMCs From Patients With Rheumatoid Arthritis

Long noncoding RNAs（lncRNAs）,as a class of non coding RNA larger than 200bp,has attracted more and more attention in recent years due to its flexible regulation and functions at different levels of gene expression.LncRNA exists widely in mammalian cells,similar to mRNA,is usually transcribed by RNA polymerase Ⅱ.LncRNA is an active participant in the regulation of gene expression at all levels,including transcription,translation and post-translational modification through different ways,such as RNA-RNA,RNA-DNA,RNA-protein interaction.LncRNA is involved in chromatin modification,transcriptional regulation,splicing and post translation modification,affects cell and tissue growth,development,differentiation and apoptosis;played an important role in maintaining the body’s normal physiological functions,such as Xist,a key factor in the silencing of X chromosomal;H19 gene involved in imprinting;lincRNA-RoR,involved in cell differentiation of embryonic stem cells.LncRNA is involved in the differentiation and activation of immune cells,and plays an important role in the function of immune cells and initiating inflammatory response,meanwhile,lncRNA are emerging as critical regulators of gene expression in the immune system.The expression of Lnc-DC was upregulated in DC cells,Knockdown of lnc-DC during DC differentiation impaired the upregulation of DC-specific genes encoding products such as CD40,CD80,CD86 and CCR7 which was involved in antigen presentation,T cell activation and cell migration.Accordingly,DCs depleted of lnc-DC can not efficiently effect on CD4+ T cells or secrete inflammatory cytokines after pathogen stimulation.Differentiation of CD4+T cells into Th cell subsets is of great importance for initiating antigen specific adaptive immune response.Many studies had shown that lncRNA was an important participating factor in CD4+T cell differentiation.The expression profiles of T cell subsets revealed that lincR-Ccr2-5 ’AS was specifically expressed in Th2 cells,and after silencing lincR-Ccr2-5’ AS expression,the expression of about 1200 genes in Th2 cells was affected.LncRNARmrp,localizes to the nucleus of TH17 cells can promote assembly of the ROR γt-DDX5 complex at genomic loci of genes encoding critical TH17 cell effector molecules,including Il17a and Il17f.Furthermore,silence gene expression of Rmrp RNA in human Th 17 cells leads to decrease secretion of cytokines from TH17 cells,and mutant forms of Rmrp cause cartilage-hair hypoplasia,a congenital disease associated with immunological dysfunction.Treg cells are important for mediating immune suppression and controlling autoimmune diseases.The transcription factor FOXP3 is the main member of Treg cell differentiation,stability and function;and conserved non-coding sequence 3（CNS3）is an enhancer element of FOXP3.LncRNA FLICR（FOXP3 long intergenic non-coding RNA）,which is conserved and specifically expressed in Treg cells,is located near the FOXP3 gene.FLICR can fine-tune FOXP3 expression in cis by modifying chromatin accessibility at the CNS3-accessible region 5（AR5）region of FOXP3,thereby promots the differentiation of T cells to Treg cells and the function of Treg cells.LncRNA not only played an important role in promoting immune cell function,but also played an important role in restraining inflammatory response.lncRNA Lethe could prevent NF-kappa B aggregation in the promoter region of the target gene by binding to the p65（RelA）of NF-kappa B,thereby affecting the expression of a series of genes that rely on NF-kappa B transcription,and played an important role in limiting the excessive inflammatory response.LincRNA-EPS is expressed in macrophages and dendritic cells.By binding with hnRNPL,it affects the conformation and nucleosome location of chromosomes,and restricts the expression of immune response related factors.LncRNA13 inhibits the expression of immune related genes in a similar way with linc-EPS.In addition to the basic physiological functions of the immune system,IncRNA also participates in the development of immune related diseases,especially autoimmune diseases.So far,the most widely studied about autoimmune diseases are focus on the expression profiles,such as systemic lupus erythematosus,Sjogren’s syndrome,rheumatoid arthritis,ankylosing spondylitis,psoriatic arthritis and so on,a series of differential expressed IncRNA have been found.Much of the research on pathogenesis is mainly the correlation studies.For example,lncRNA NEAT1 can promote the expression of chemokines and inflammatory factors such as CXCL-10 and IL-6 through MAPK pathway.NEAT1 is highly expressed in peripheral blood cells of systemic lupus erythematosus（SLE）,and is positively correlated with disease activity,renal involvement and IFN score.It is speculated that NEAT1,which regulates innate immunity,may be involved in the pathogenesis of SLE.In the CD4+T cells of patients with Sjogren syndrome,the expression of TMEVPG1 was significantly upregulated,and the expression level was positively correlated with SSA,erythrocyte sedimentation rate and IgG.In rheumatoid arthritis,lncRNA-C5T1 can regulate the expression of RA susceptible gene C5,and lncRNA-C5T1 transcription starts in the 3 ’non coding region of C5.After knockdown of lncRNA-C5T1,the expression level of C5 decreases.LOC100506036 was highly expressed in T cells of RA patients,after silencing the gene,the expression level of RA associated gene NFAT1（nuclear factor of activated T cells 1）and SMPD1 decreased which can speculate that LOC100506036 participates in the inflammatory process of RA by regulating gene expression of SMPD1 and NFAT1.In the synovial synovium of RA patients,135 lncRNAs were found differential expressed by chip technique analysis,and 62 IncRNA were upregulated whereas 73 lncRNAs were downregulated.The expression of lncRNA in peripheral blood mononuclear cells of patients with RA has not yet been reported.Rheumatoid arthritis is a typical chronic inflammatory disease involving joints.It mainly causes progressive joint destruction and related complications of blood vessels,bones and metabolism.In the domestic incidence of 0.3-0.5%,the disease course is prolonged and the rate of disability is high,which seriously affects the quality of life of the patients.Rheumatoid arthritis can occur at all ages,mainly involving women.Rheumatoid arthritis primarily encroachs on synovial tissues.In the joint involved with RA,abnormal proliferation of synovial tissue,infiltration of inflammatory cells and destruction of bone and cartilage tissue can be seen.In the whole course of RA,the three pathophysiological process of synovial proliferation,inflammation,autoimmunity,interact with each other,link throughout the pathogenesis of RA,forming a perplexing network and resulting in no clear conclusion on the pathogenesis of RA.Until 1980s,with the understanding of the function of cytokine,the RA was considered a T cell-mediated autoimmune disease.Based on this theory,several biological therapy targeting to T cells（CTLA-4）and cytokines（such as IL-6R/TNF/IL-17/IL-1）was carried out,and achieved good curative effect,consolidate the position of the theoretical in the pathogenesis of RA.At present,it is generally believed that the loss of immune tolerance and the imbalance of autoimmune regulation lead to the disorder of autoimmune system,which eventually leads to a series of inflammatory reactions in the joints.The genetic and environmental factors play an important role in the process of disease.Based on the above research background,in this study,we examined the lncRNA expression profile in peripheral blood mononuclear cells（PBMCs）of female rheumatoid arthritis patients using microarray analysis.Bioinformatics methods were used to analyze differentially expressed genes in GO and KEGG pathways,so as to found out the biological pathways that differentially expressed genes might participate.Combined with the detected susceptibility loci and the related information of lncRNA,the differential expression of lncRNA was quantitatively verified and analyzed,then we will further explore RA associated differentially expressed lncRNA and explore its role in the pathogenesis of RA,in order to identify new biomarkers used in diagnosis,disease monitoring and prognosis and hence achieve transformation from laboratory results to clinical applications.Part Ⅰ LncRNA expression profiling and functional analysisin PBMCs from patients with rheumatoid arthritisObjective:To identify the differentially expressed IncRNA and mRNA in RA patients using microarray analysis,and explore the possible pathways of aberrantly expressed mRNA that might involved in the pathogenesis of RA by GO and KEGG analysis.Method:Between July 2016 and December 2016,27 female RA patients with RA were recruited from the Department of Rheumatology of Liaocheng People’s Hospital,and 27 corresponding healthy controls Healthy controls were enrolled from volunteers of the health examination center of Liaocheng People’s Hospital.The 27 female RA patients and corresponding healthy controls were randomly divided into three groups according to age.The peripheral blood mononuclear cells were separated by density gradient centrifugation.After being dissolved by Trizol,each group of samples were mixed with equivalent PBMCs.The three pairs of samples were prepared for lncRNA microarray analysis using an Arraystar human LncRNA microarray V4.0 and Array images were acquired by Agilent Feature Extraction software（version 11.0.1.1）.Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software package.Differentially expressed lncRNAs and mRNAs between the two groups were identified through P<0.05 and fold-change>2.0.Cluster analysis was used to analyze the expression of IncRNA and mRNA in differential expression by scatter plot,volcano map and cluster analysis,so as to establish differential expression profiles of lncRNA and mRNA.The distribution of differentially expressed IncRNA and mRNA in chromosomes and the distribution of different times of differential variation were statistically analyzed,and the biological information of differentially expressed IncRNA and mRNA was further understood at different levels.Bioinformatics technology was used to analyze mRNA with significant difference in expression between GO（gene ontology）and KEGG Pathway（Kyoto Encyclopedia of Genes and Genomes）,in order to find out the biological pathways that differentially expressed genes might participate.Result:Arraystar Human IncRNA Microarray V4.0.Approximately 40,173 lncRNAs and 20,730 coding transcripts can be detected by this third-generation IncRNA microarray.Based on the cutoff criteria of a fold change>2.0 and a P value<0.05,a total of 2,099 lncRNAs and 2,307 mRNAs were significantly differentially expressed in all three pairs.Among the 2,099 differentially expressed lncRNAs,683 were upregulated and 1,416 were downregulated.Among the 2,307 differentially expressed mRNAs,331 were upregulated and 1976 were downregulated.n the differential expression of IncRNA and mRNA,the rates of downregulation were 67.46%and 77.85%,respectively.The distribution of differentially expressed IncRNA and mRNA in chromosomes was found to be basically the same.The number of abnormal expression on chromosome 2 was the largest,which was more than 200,and the number of abnormal expression on chromosome 22 was the least.According to the analysis of fold changes,the proportion of IncRNA and mRNA between 2-3 times of the difference is more than 50%,and more than 10 times is less than 1%.The results showed that the majority of DE-lncRNAs and DE-mRNAs are downregulated.By GO and KEGG database analysis,differential expression of mRNA is involved in many biological processes related to RA,such as MHC molecules and receptors,acute phase reaction protein,cell adhesion molecule and ubiquitination mediated protein decomposition.Conclusion:We identify the aberrantly expressed IncRNA and mRNA in RA patients and the majority of DE-lncRNAs and DE-mRNAs are downregulated,which indicates that deficiency of gene expression or the degration of RNA might play an important role in the mechanism of RA.With the analysis of GO and KEGG,several pathways that differentially expressed mRNA involved might associated with RA.Part Ⅱ Verification and correlation analysis of differentlyexpressed lncRNA in RAObjective:This part is to validate the microarray data by real-time quantitative polymerase chain reaction（RT-qPCR）and make a correlated analysis with clinical characteristics IL-6,TNF-alpha,disease activity indicators and simplified disease activity index（SDAI）,in order to find biomarkers that can be uses for assessing and diagnosing of RA.Method:Between July 2016 and December 2016,36 RA patients with RA were recruited from the Department of Rheumatology of Liaocheng People’s Hospital,and 24 corresponding healthy controls Healthy controls were enrolled from volunteers of the health examination center of Liaocheng People’s Hospital.Based on the folds of changes in expression（fold changes>3,P<0.05）,the length of each lncRNA（length<2500 bp）,whether the lncRNAs have definite sequences,and whether the context of genes are associated with RA,we selected 6 candidate lincRNAs for validation by real-time quantitative polymerase chain reaction（RT-qPCR）in the validation cohort consisting of 36 RA patients and 24 healthy controls.By statistical software GraphPad Prism5,non parametric Mann-Whitney test was used for statistical analysis,Spearman correlation analysis was carried out between the expression levels of significantly differentially expressed lncRNAs and clinical characteristics:the serum levels of IL-6 and TNF-a,the Simplified Disease Activity Index（SDAI）.Result:RT-qPCR results showed that ENST00000456270 and NR₀02838 were increased in RA PBMCs,whereas the expression levels of NR₀26812,uc001zwf.1 and ENST00000566394 were decreased.The changes were statistically significant for 4 lncRNAs（ENST00000456270,NR₀02838,NR₀26812 and uc001zwf.1）,only the change of one lncRNA,ENST00000566394,was not statistically significant（p=0.6052）.All the five aberrantly expressed lncRNAs,showed the same trends in qPCR and microarray analysis.The expression level of ENST00000412896 was too low（CT>33）in both RA and normal controls to be analyzed by qPCR.The expression levels of NR₀02838 and ENST00000456270 were strongly correlated with the Simplified Disease Activity Index（SDAI）（r=0.5191,p=0.0020 and r=0.8347,P<0.0001）,and the expression level of ENST00000456270 was significantly associated with the serum levels of IL-6（r=0.5653,P=0.0003）and TNF-α（r=0.4332,P=0.0083）,while NR₀26812 and uc001zwf.1 had no significant correlation with IL-6,TNF-a and SDAI.Additionally,the correlation of NR₀0283 8 with IL-6 and TNF-α was not significant.Conclusion:The result of RT-qPCR was concoindence with the result of microarray,which confirmed the reliability of screening differentially expressed by microarray analysis.ENST00000456270,could serve as a potential biomarker for assessing and diagnosing RA patients.