| BackgroundAnkylosing spondylitis(AS)is an inflammatory autoimmune disease that characterized by the inflammation of spine,sacroiliitis and attachment joints.Previous studies have shown that the pathogenesis of AS is related to genetic factors,especially Human leukocyte antigen(HLA)-B27 gene.However,these studies focus on encode-protein genes rather than non-cong RNAs,and the genetic background of autoimmune inflammatory diseases(e.g.AS)is very complicated,so they cannot help us fully understand the pathogenesis of AS.In recent years,increasing studies have found that long non-coding RNA(lnc RNA)and micro RNA(mi RNA)are abnormal in rheumatoid arthritis(RA),osteoarthritis(OA),systemic lupus erythematosus(SLE)and AS.Moreover,the competing endogenous RNA(ce RNA)hypothesis has roused people's wide concern.However,few studies have focused on lnc RNAs,mi RNAs,and their interactions are involved in the pathogenesis and progression of AS.Therefore,the purpose of this study was to investigate the role of lnc RNA H19,mi R22-5p and mi R675-5p in the pathogenesis of AS.MethodsThis study had three phases including the screening of differentially expressed(DE)lnc RNA in AS Peripheral blood mononuclear cells(PBMCs),the verification of DE lnc RNAs,and the subsequent functional reseacrches.(1)First,lnc RNA microarray was used to detect the expression of lnc RNAs in the PBMCs of 5 AS patients and 5 healthy controls(Age-and sex-matched with AS patients),and the DE lnc RNA profile was constructed.(2)Subsequently,four DE lnc RNAs o were detected by real-time quantitative PCR(q RT-PCR)in a cohort of 49 AS patients and 49 healthy controls(Age-and sex-matched with AS patients),and the target lnc RNAsof this study was determined.In the second cohort,we also tested the expression level of messenger RNA(m RNA).We used online software RNAhybrid predict binding sites for lnc RNA and m RNAs potentially associated with mi RNAs.(3)In vitro,we extracted primary PBMCs from 8 AS patients,and added phytohemagglutinin(PHA)to stimulate the number of cells.Adenovirus transfection and si RNA interference were used to change the expression of lnc RNA and mi RNA.Subsequently,q RT-PCR was used to measure the expression levels of lnc RNA,mi RNA,m RNA,and downstream molecules.Protein levels corresponding to m RNA were detected by Western blot.Enzyme-linked immunosorbent assay(ELISA)was used to detect cytokine levels.In addition,double luciferase reporter assay was used to detect the fluorescence activity of binding sites of lnc RNA and mi RNA,and mi RNA and m RNA.In statistical analysis,the Kolmogorov Smirnov test was used to determine whether the continuous variables are normally distributed or not.The student-t test was used for the comparison between groups of continuous variables that are normal distributions,while the mann-whitney U test was used for the comparison between groups of continuous variables that are not normal distributions.Categorical variables were compared by the Pearson chi-square test.Receiver operating characteristic(ROC)and area under curve(AUC)were used to evaluate whether DE lnc RNA could be used for the biomarker of AS.The Spearman rank correlation coefficient test was used to explore the correlation between lnc RNA and other molecules.SPSS 23.0 software was used for all statistical analysis,and Graph Pad Prism 5.01 software was used for generating graphics.P value less than 0.05 was considered statistically significant.ResultsAt the lnc RNA microarray phase,there were 3 males(60.0%)in AS and healthy control groups,and the age of AS patients was(22.6 ± 6.2),and healthy control group was(25.2 ± 6.6).At the q RT-PCR phase,the age of AS patients was(33.9 ± 11.7),and healthy control group was(31.1 ± 6.1).There were 32 cases(65.3%)of male patients in the AS group,and 25 cases(51.0%)of male individuals in the healthy control group.At the functional researches phase,the age of patients was(27.5 ± 5.9),including 5 male patients(62.5%).In the first two phases,the age and number of male patients in AS were not statistically significant compared to healthy controls.Lnc RNA Microarray revealed 154 DE lnc RNAs in AS patients,including 42 lnc RNAs were annotation(23 were up-regulated and 19 were down-regulated).q RT-PCR showed that the expression of H19 and LOC101929023 were up-regulated in AS compared to healthy controls,and ROC results showed that the area under the curve of H19(AUC= 0.653,P=0.009)was larger than that of LOC101929023(AUC= 0.637,P=0.020),so H19 was used for the next experiments.The online software RNAhybrid predicted binding sites for H19,TGF-? and VDR potentially related to mi R22-5p and mi R675-5p.In addition,RNA expression of VDR and IL-17 A were significantly up-regulated in AS patients,while TGF-?,IL-23 and TNF-? were not statistically significant.H19 was positively associated with the 5 m RNA expression levels.In vitro,the expression of mi R22-5p was significantly suppressed and the expression of mi R675-5p was amplified by Si-H19;the m RNA and protein expression of VDR was decreased by Si-H19,and the m RNA and cytokine levels of IL-17 A and IL-23 was also decreased by Si-H19.These results were confirmed by AD-H19.In addition,the m RNA and protein expression of VDR were amplified by mi R675-5p and mi R22-5p inhibitors,and the m RNA and cytokine levels of IL-17 A and IL-23 were also amplified by the two RNAs.In the dual-luciferase reporter assay,the luciferase activity of wild type H19 was suppressed by both mi R675-5p mimics and mi R22-5p mimics,and the luciferase activity of wild type VDR was also suppressed by both mi R675-5p mimics and mi R22-5p mimics.ConclusionA total of 42 DE annotated lnc RNAs were identified in this study,and the expression of lnc RNA H19 was up-regulated in AS patients.Mi R22-5p and mi R675-5p binding sites of H19,VDR and TGF-?.Therefore,H19 plays an important role in the pathogenesis of AS,and acts as a ce RNA in the mi R22-5p-VDR-IL-17A/IL-23 axis,or interact directly with mi R675-5p on the mir675-5p-VDR-IL-17A/IL-23 pathway to facilitate the progression of AS patients. |
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