Expression Of Long Noncoding RNA IFNG-AS1 In Patients With Rheumatoid Arthritis And Study On Its Regulatory Factors

Objective: To study the alteration of long-non-coding RNA(Lnc RNA)-IFNG-AS1 expression in peripheral blood mononuclear cells(PBMCs)of patients with rheumatoid arthritis(RA),to investigate the correlation between lnc-IFNG-AS1 and IFN-γ,T-bet and clinical diagnostic criteria of RA,and to provide a new way to study the pathogenesis of RA disease.Methods: 1.The expression of lnc-IFNG-AS1 and IFNG mRNA in PBMCs from RA patients,osteoarthritis(OA)patients and healthy volunteers were measured by real-time quantitative PCR(q RT-PCR),the correlation between the level of lnc-IFNG-AS1 and IFNG mRNA in PBMCs from patients with RA was analyzed.2.The relative expression level of T-bet mRNA in PBMCs from RA patients,OA patients and healthy volunteers was measured by q RT-PCR,the correlation between the level of T-bet mRNA and lnc-IFNG-AS1 in PBMCs from patients with RA was analyzed.3.CD4+T cells were purified from PBMCs of healthy volunteers by immunomagnetic beads,the relative expression levels of T-bet mRNA and lnc-IFNG-AS1 were measured by q RT-PCR after T-bet si RNA or NC transfection for 24 hours,to investigate the effect of T-bet on lnc-IFNG-AS1 in CD4 + T cells.4.The levels of T-bet mRNA,lnc-IFNG-AS1 and IFNG mRNA in PBMCs were significantly correlated with rheumatoid factor(RF),C-reactive protein(CRP),erythrocyte sedimentation rate(ESR)levels,the relative expression levels of T-bet mRNA,lnc-IFNG-AS1 and IFNG mRNA were compared between the into anti-cyclic citrullinated peptide antibody(anti-CCP-Ab)positive and negative group of RA patients.Results:1.The relative expression of lnc-IFNG-AS1 was significantly increased in PBMCs from RA patients compared with healthy volunteers(0.35±0.25vs0.13±0.10,P <0.001),the relative expression of IFNG mRNA was significantly increased(0.93±0.56vs0.66±0.42,P <0.05);the relative expression of lnc-IFNG-AS1 in PBMCs was significantly elevated in OA paitents compared with healthy volunteers(0.25±0.09vs0.13±0.10,P <0.01),the relative expression of IFNG mRNA was no significantly elevated in OA paitents compared with healthy volunteers(0.73±0.48vs0.66±0.42,P> 0.05).A significant positive correlation was observed between the relative expression of lnc-IFNG-AS1 and IFNG mRNA in PBMCs from paitents with RA(r=0.5467,P=0.0015),while there was no significant correlation in PBMCs from paitents with OA.The relative expression of lnc-IFNG-AS1 in PBMCs from paitents with RA was analyzed by receiver operating characteristic curve(ROC curve).The area under the roc curve(AUCR0C)was 0.815,and the standard Error(SE)was 0.053,P <0.01,95% confidence interval(CI)is 0.711 ~ 0.919,the best cutoff value is 0.102.2.Compared with healthy volunteers,the relative expression of T-bet mRNA in PBMCs from RA patients was significantly increased(0.77±0.45vs0.49±0.22,P <0.01),while there was no statistically significant from patients with OA(0.66±0.32vs0.49±0.22,P>0.05).A strong positive correlation was observed between the expression of lnc-IFNG-AS1 and T-bet mRNA in PBMCs from RA patients(r=0.6923,P <0.0001),while there was no significant correlation between the expression of lnc-IFNG-AS1 and T-bet mRNA in PBMCs from OA patients(r=0.0872,P> 0.05).3、 Compared with negative control(NC)group,circulating CD4 + T cells was transfected with 25 n M,50 n M and 100 n M si RNA-T-bet.After incubation for 24 hours,the relative expression of T-bet mRNA in CD4 + T cells of healthy volunteers was significantly decreased(0.84 ± 0.14vs1.26 ± 0.17,0.68 ±0.10vs1.26 ± 0.17,0.37 ± 0.13vs1.26 ± 0.17,P <0.001),and the relative expression of lnc-IFNG-AS1 was also significantly decreased(1.17±0.27vs1.99±0.32,0.97±0.21vs1.99±0.32,0.40±0.15 vs 1.99±0.32,P <0.001),there was a dose-dependent of inhibition by si RNA-T-bet.4、 A significantly positive correlation was observed between the relative expression of lnc-IFNG-AS1 in PBMCs and CRP levels(r=0.4751,p=0.0069),ESR(r=0.3821,p=0.0309)or RF levels(r= 0.5118,p=0.0106)in peripheral blood of RA patients;the relative expression of lnc-IFNG-AS1 in PBMCs from serum anti-CCP-Ab positive group patients of RA was significantly elevated than that in negative group(0.48 ±0.28 vs 0.21 ± 0.14,P <0.05).Conclusions: 1、Compared with healthy volunteers,the relative expression of lnc-IFNG-AS1 and IFNG mRNA was significantly observed in RA patients.The relative expression level of lnc-IFNG-AS1 in peripheral blood were positively correlated with RF,CRP or ESR levels,our results indicate that that lnc-IFNG-AS1 may reflect the severity of the disease to a certain extent.2、The area under the ROC curve(AUCR0C)of the relative expression of lnc-IFNG-AS1 in PBMCs from RA patients was 0.815,the results indicate that lnc-IFNG-AS1 has potential clinical value in the diagnosis of RA.3、Compared with healthy volunteers,the relative expression of T-bet mRNA in RA patients was significantly observed.A positively correlated was found between the relative expression of T-bet mRNA and the relative expression of lnc-IFNG-AS1.The relative expression of lnc-IFNG-AS1 was significantly decreased after si RNA-T-bet transfection in circulating CD4 + T cells.,it is suggested that the expression of lnc-IFNG-AS1 can be regulated by T-bet molecules in RA patients.