强直性脊柱炎患者外周血环状RNA的表达谱研究

Study on the expression profile of circular RNA in patients with ankylosing spondylitis

目的筛选AS患者PBMCs中差异表达的环状RNA(circRNA),分析其表达谱以探讨circRNAs在AS发病中的作用。方法采用circRNA微阵列芯片技术检测3例AS活动期(ASA)患者,3例AS稳定期(ASS)患者和3名健康对照者(HC)PBMCs中circRNAs的表达并通过倍数变化(FC)和P值进行筛选,找到差异表达的circRNAs;从差异表达靠前的circRNAs中随机选择4个circRNAs,采用实时荧光定量PCR(RT-qPCR)验证芯片结果;对差异表达的circRNAs进行基因本体论(GO)分析,京都基因与基因组百科全书(KEGG)分析,并通过微RNA(miRNA)靶标预测软件对circRNA/miRNA相互作用关系进行预测。采用t检验和Mann-Whitney U检验等方法对数据进行统计分析。结果芯片结果显示,ASA组较HC组共有800个显著差异表达的circRNAs(FC>1.5,P<0.05),其中466种上调,334种下调;ASS组较HC组共有1149个显著差异表达的circRNAs(FC>1.5,P<0.05),其中589种上调,560种下调;ASA组较ASS组间共有233个显著差异表达的circRNAs(FC>1.5,P<0.05),其中145种上调,88种下调。RT-qPCR验证结果提示,4个差异表达的circRNAs表达趋势与芯片结果一致。GO分析结果提示,这些差异表达的circRNAs主要参与无义介导的mRNA衰变、Rho GTPase酶结合等过程;KEGG分析结果在辅助性T细胞(Th)17细胞分化、趋化因子信号通路等处得到富集;miRNA靶标预测软件结果提示差异表达的circRNAs可能靶向结合miR-650、let-7b-5p等miRNAs发挥作用。结论和HC组相比AS患者PBMCs中存在差异表达的circRNAs,推测这些circRNAs可能参与AS的发病。

Objective To screen for circle RNA(circRNA)differentially expressed in peripheral blood mononuclear cells(PBMCs)of patients with ankylosing spondylitis(AS),and to analyze its expression profile to explore the role of circRNAs in the pathogenesis of AS.Methods CircRNA microarray chip technology was used to detect the expression of circRNAs in PBMCs of 3 patients with active AS,3 patients with stable AS and 3 healthy controls(HC),and then screening for differentially expressed circRNAs by fold change(FC)and P value.Then differentially expressed circRNAs among the circRNAs with the highest differential expression were selected randomly to verify the chip results by real-time fluorescence quantitative polymerase chain reaction(qPCR);Differentially expressed circRNAs were subjected to Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,and microRNA(miRNA)target prediction software was used to predict the circRNA/miRNA interaction relationship.Finally,the data were statistically analyzed by t test and Mann-Whitney U test.Results①Chip text results showed that there were 800 circRNAs with significantly different expression(FC>1.5,P<0.05)in active AS than HC,of which 466 were up-regulated and 334 were down-regulated;the stable AS had a total of 1149 significantly differentially expressed when compared with the HC(FC>1.5,P<0.05)circRNAs,of which 589 were up-regulated and 560 were down-regulated.233 circRNAs were significantly differentially expressed(FC>1.5,P<0.05)between active AS and stable AS,of which 145 were up-regulated and 88 were down-regulated.②The RT-qPCR verification results suggested that the expression trends of the four differentially expressed circRNAs were consistent with the results of the chip.③GO analysis results suggested that these differentially expressed circRNAs were mainly involved in nonsense-mediated mRNA decay,Rho GTPase binding and other processes.The analysis showed that the KEGG pathway were enriched in Th17 cell differentiation and chemokine signaling pathways.The results of miRNA target prediction software analysis suggested that differentially expressed circRNAs might play a role by targeting miR-650,let-7b-5p and other miRNAs.Conclusion Compared with HC group,there were differentially expressed circRNAs in Peripheral Blood Mononuclear Cells(PBMCs)of AS patients,The results of this study suggest that these circRNAs may be involved in the pathogenesis of AS.