Molecule Epidemiology Study On The Association Of Long Non-coding RNA With Rheumatoid Arthritis

BackgroudRheumatoid arthritis?RA?is a chronic,common autoimmune disease characterized by joint inflammation,synovitis,destruction of articular cartilage,together with systemic manifestations of inflammatory and immune response.RA could lead to joint swelling,pain,distortion,and ultimately cause function loss of patients'joint and stiffness,with a high disability rate.The pathogenesis underlying RA is complex,and remains largely unknown.Studies have found that several host factors?such as genetic susceptibilities,aberrant immune response,abnormal metabolic enzymes?and environmental factors?such as bacterial or viral infection?are involved in the development of RA.In recent years,the roles of non-coding RNA?ncRNA?in the development of diseases have attracted increasing attention.Studies have shown that ncRNA is involved in the pathogenesis of a variety of diseases.Long non-coding RNA?lncRNA?refers to RNAs longer than 200 nucleotides in length with little or no protein-coding capacity.LncRNA could play different biological functions through multiple mechanisms,participate in various biological processes,such as chromatin remodeling,selective splicing,protein transport,and modulates gene expression through epigenetics,transcriptional control,post-transcriptional processing as well as protein metabolism.Increasing studies have suggested that lncRNAs are involved in the regulation of autoimmunity-and inflammation-related processes,including nuclear factor-?B and Toll-like receptor signalling pathway,cytokine secretion,and immune cell proliferation and differentiation.Many lncRNAs have been confirmed to play key roles in the pathogenesis of RA,and their abnormal expression is closely related to the progression of the disease.Genome wide association studies?GWAS?have identified a large amount of single nucleotide polymorphisms?SNP?that were associated with the susceptibility of human disease including RA,and the majority of them were located in the non-coding intervals.However,there are few studies on the role of lncRNA gene variation in RA pathogenesis.Therefore,our study discussed the correlation between lncRNA and RA through lncRNA gene variation and expression.ObjectiveIn this study,we aimed to evaluate the association of four lncRNA?ANRIL,lnc-DC,MALAT1,ZFAS1?genes polymorphisms with susceptibility to RA patients.We also analyzed the differences in the expression levels of these lncRNA in PBMC from RA patients and healthy controls,and discussed the correlation between lncRNA expression levels and the main laboratory indicators,disease activity in RA patients.MethodsOur study employed case-control design,including the following three parts:?1?Association of lncRNA genes polymorphisms with RA susceptibilityA total of 1370 subjects including 660 RA patients and 710 healthy controls were consecutively enrolled for genotyping in this study.RA patients were selected from the department of the First Affiliated Hospital of Anhui Medical University and Anhui Provincial Hospital,and the diagnosis of these patients was according to the 1987American College of Rheumatology?ACR?revised criteria.Healthy controls were obtained from the health examination center of the First Affiliated Hospital and the Second Affiliated Hospital of Anhui medical university.RA patients and healthy controls included in this study were all Chinese Han people who were unrelated to each other.Six SNPs?rs1412830,rs7044859,rs944796,rs61271866,rs2518723,rs3217992?in ANRIL,two SNPs?rs7217280,rs10515177?in lnc-DC,five SNPs?rs619586,rs4102217,rs591291,rs11227209,rs35138901?in MALAT1,four SNPs?rs237742,rs73116127,rs6125607,rs6125608?in ZFAS1 were genotyped using improved multiple ligase detection reaction?iMLDR?.The genotype,allele,dominant and recessive model distribution frequencies of above SNPs between RA patients and healthy controls were analyzed in this study,and we also examined the association between the genotype,allele frequencies of these lncRNA and ACPA,RF in RA patients.Finally,we constructed the haplotype of each lncRNA gene by SHEsis software,and analyzed the distribution differences of these haplotypes in RA patients and healthy controls.?2?LncRNA expression levels in PBMC from RA patients and healthy controlsThe lncRNA?ANRIL,lnc-DC,MALAT1,ZFAS1?expressions in peripheral blood mononuclear cell?PBMC?from 120 RA patients and 120 healthy controls were detected by quantitative reverse transcription polymerase chain reaction?qRT-PCR?,and we analyzed the differences in lncRNA levels between RA patients and healthy controls.Then,the RA patients were classified into different groups:ACPA-positive patients,ACPA-negative patients,RF-positive patents,RF-negative patients.The differences in these lncRNA levels between ACPA-positive RA patients and ACPA-negative RA patients,as well as RA patients with RF-positive and RF-negative,were also detected.Finally,we also explored the correlations of lncRNA expression levels with other laboratory indicators,including C3,C4,erythrocyte sedimentation rate?ESR?,C-reactive protein?CRP?,disease activity of RA patients.?3?Associations between lncRNA genes polymorphisms with their levels in RA patientsIn this study,65 RA patients were included in both genotyping and qRT-PCR experiment.Therefore,we examine the associations between the respective genotype frequencies of these lncRNA genes with their expression levels in these patients,and preliminary investigated the effect of lncRNA polymorphism on their expression.In the genotyping experiment,the hardy-weinberg equilibrium?HWE?was used to test whether the genotype distribution of all SNP in the healthy control satisfies the law of genetic equilibrium.Logistic regression analysis was used to calculate the odds ratio?OR?and 95%confidence interval?CI?of all SNP,and adjust gender,age.The relative expression levels of lncRNA were calculated by comparison with housekeeping gene?-actin in the same sample as internal control,and expressed using2^-??Ctmethod normalized to endogenous control.Database was established by Epi Data 3.1,statistical analysis was performed with the SPSS 23.0,and scatter plot was drawn using GraphPad Prism version 5.0.P value?two-sided?of less than 0.05was considered as statistically significant,however,in the statistical analysis of genotype frequency,?needed to be corrected and was considered as 0.003?0.05/16?after correction.Results?1?Association of lncRNA genes polymorphisms with RA susceptibilityThe observed genotype frequencies of rs7044859 in the healthy control was not conform to HWE,thus we excluded this SNP in finally analysis.Our results demonstrated that there were no significant associations between ANRIL?rs1412830,rs944796,rs61271866,rs2518723,rs3217992?,lnc-DC?rs7217280,rs10515177?,MALAT1?rs619586,rs4102217,rs591291,rs11227209,rs35138901?,ZFAS1?rs237742,rs73116127,rs6125607,rs6125608?gene polymorphism and RA genetic susceptibility,respectively?all P>0.003?.Our study also analyzed the correlation between the above SNP and RA genetic susceptibility in the dominant,recessive model,and the results showed that there was no statistically significant correlation?all P>0.003?.To examine the potential genetic association between the genotype,allele frequencies of ANRIL,lnc-DC,MALAT1,ZFAS1 genes and ACPA and RF in RA patients,the results showed that the rs944796 G allele,rs2518723 T allele frequencies were significantly increased in RA patients with ACPA-positive when compared to patients with ACPA-negative?all P<0.05?,while rs3217992 T allele frequency was reduced?P=0.039?.In this study,no significiant association was found between lncRNA SNP genotype,allele frequency and RF in RA patients?all P>0.05?.Six main haplotypes?CCATC,CCTCC,CCTCT,CCTTC,CGTTC,TCATC?for ANRIL,three main haplotypes?AG,GA,GG?for lnc-DC,six main haplotypes?ACTCT,AGCCT,AGTCC,AGTCT,GGTCT,GGTGT?for MALAT1 and five main haplotypes?CACA,CGCA,CGCG,CGTA,TGCA?for ZFAS1 were detected by SHEsis software in this study.The results demonstrated that the haplotype CGTA frequency was significantly higher in RA patients than normal controls?P=0.036,OR=1.191,95%CI:1.012-1.402?,while the haplotype frequency distribution difference of other lncRNA genes between RA patients and healthy control was not statistically significant?P>0.05?.?2?LncRNA expression levels in PBMC from RA patients and healthy controlsThe expression levels of ANRIL,lnc-DC,MALAT1,ZFAS1 in PBMC were significantly reduced in RA patients than normal controls?Z=-5.244,P<0.001;Z=-8.169,P<0.001;Z=-10.138,P<0.001;Z=-7.650,P<0.001?.The differences in these lncRNA levels between ACPA-positive RA patients and ACPA-negative RA patients,as well as RA patients with RF-positive and RF-negative RA patients,were not statistically significant?all P>0.05?.The correlation of ANRIL,lnc-DC,MALAT1,ZFAS1 expression levels with other clinical parameters of RA patients were also analyzed,and the results showed that the expression level of ZFAS1 was significantly negatively associated with CRP in RA patients?r_s=-0.278,P=0.002?.There was no significant correlation between lncRNA expression level and C3,C4 and ESR levels in RA patients?all P>0.05?.DAS28 was used to evaluate the disease activity of RA patients in this study,and no statistically significant correlation was found between the expression levels of these lncRNA and DAS28 scores of RA patients?all P>0.05?.Finally,our study investigated the effect of clinical medication on the expression level of lncRNA in RA patients,and fonud that glucocorticoids,disease-modifying antirheumatic drugs?DMARDs?,biologics had no effect on the expression level of lncRNA in PBMC of RA patients.?3?Associations between lncRNA genes polymorphisms with their levels in RA patientsThe association between the dominant model of MALAT1 gene rs4102217?GG vs.CG+CC?and MALAT1 level was statistically significant?P=0.019?,and the expression level of MALAT1 was decreased in PBMC of RA patients with rs4102217GG genotype.However,there was no statistical significance between other lncRNA genes polymorphisms and their expression levels?all P>0.05?.Conclusions?1?Five SNP in ANRIL gene,two SNP in lnc-DC gene,five SNP in MALAT1gene and four SNP in ZFAS1 gene were not associated with RA susceptibility.In addition,there were significant associations between ANRIL gene rs944796,rs2518723 and rs3217992 polymorphisms and ACPA in RA patients.?2?Compared with healthy controls,ANRIL,lnc-DC,MALAT1,ZFAS1expression levels were reduced in PBMCs from RA patients.Moreover,the expression level of ZFAS1 was negatively correlated with the CRP in RA patients.?3?The expression MALAT1 level in PBMC of RA patients was affected by rs4102217 polymorphism.