Expression And Clinical Significance Of Circular RNA In Peripheral Bloud Mononuclear Cells Of Patients With Gout

Background and objective:Circular RNAs(circRNAs)are non-coding RNAs with a single-stranded covalently closed-loop structure,and they exist widely and stably in eukaryotic cells.Evidence is mounting that circRNAs are potential diagnostic biomarkers and therapeutic targets for various diseases,however,knowledge of circRNAs in gout is limited.This study intends to analyze the expression profile of circRNA in peripheral blood mononuclear cells(PBMCs)of patients with primary gout through case-control analysis,and to preliminarily explore the possible mechanism of circRNA in the occurrence and development of gout.Methods:1.Peripheral venous blood samples from 5 patients with primary gout and 3 healthy controls(HC)were collected,and microarray gene chip technology was used to screen the differentially expressed circRNAs in their PBMCs.GO analysis and KEGG pathway analysis were performed on the significantly differentially expressed circRNAs(fold change>1.5,P<0.05).2.Peripheral venous blood samples,clinical data and laboratory indicators were collected from 45 patients with acute gout(AG),45 patients with intercritical gout(IG)and 60 HC,and the AG group and the IG group were collectively referred to as the Gout group;According to the fold change difference,4 significantly differentially expressed circRNAs(hsa＿circRNA＿103657、hsa＿circRNA＿000241、hsa＿circRNA＿100632、hsa＿circRNA＿405646)were selected,and real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect their relative expression in PBMCs of all 150 subjects;The circRNA-miRNA-mRNA network was established to predict the functions of circRNAs;Spearman correlation test was performed to evaluate the correlation between circRNAs and clinical variables of gout;Receiver operating characteristic curve was constructed to evaluate the diagnostic value of the identified circRNAs.Results:1.Microarray analysis showed that there were 279 differentially expressed circRNAs between Gout group and HC group(fold change>1.5 and P<0.05),of which 238 circRNAs were up-regulated in Gout group and 41 circRNAs were down-regulated in Gout group.2.GO analysis showed that the differential circRNAs were mainly related to the biosynthetic process of cyclic adenosine monophosphate,enzyme binding,adenylate cyclase activity and nuclear ubiquitin ligase complex.In the KEGG pathway analysis,the up-regulated circRNA in the gout group had enriched 34 pathways,and the down-regulated circRNA in the gout group had enriched 43 pathways.These differential genes were mainly involved in the process of autophagy,ubiquitin-mediated proteolysis,and the cGMP-PKG signaling pathway.3.In the stage of expanding the samples to detect the relative expression levels of 4 circRNAs by RT-qPCR,the expression levels of hsa＿circRNA＿103657 and hsa＿circRNA＿000241 in the Gout group were significantly higher than HC group(P<0.05),while the expression levels of hsa＿circRNA＿100632 and hsa＿circRNA＿405646 were not significantly different between the Gout group and the HC group(P>0.05).In addition,the expression levels of hsa＿circRNA＿103657 and hsa＿circRNA＿000241 in both the AG group and the IG group were significantly higher than those in the HC group(P<0.05),and there was no significant difference between the AG and IG groups(P>0.05).The expression levels of hsa＿circRNA＿100632 and hsa＿circRNA＿405646 were not statistically significant among the three groups(P>0.05)4.The circRNA-miRNA-mRNA network was constructed for hsa＿circRNA＿103657 and hsa＿circRNA＿000241 to predict and annotate their possible mechanisms.The top 5 miRNAs related to hsa＿circRNA＿103657 were hsa-miR-329-5p,hsa-miR-556-5p,hsa-miR-345-5p,hsa-miR-452-5p,hsa-miR-22-5p;the top 5 miRNAs associated with hsa＿circRNA＿000241 were hsa-miR-1303,hsa-miR-619-5p,hsa-miR-645,hsa-miR-4452,hsa-miR-5787.According to the principle of sequence pairing and the mechanism of endogenous competing RNAs,mRNAs that may be affected by hsa＿circRNA＿103657 and hsa＿circRNA＿000241 were predicted,and 10 miRNAs and 525 mRNAs were predicted to interact with them,respectively.In addition,the bioinformatics analysis also revealed that they were more likely to jointly participate in the PI3K-Akt signaling pathway to affect the occurrence and development of gout.5.In the Gout group,the expression level of hsa＿circRNA＿103657 was positively correlated with fasting blood glucose and triglyceride levels(r=0.419,P<0.001;r=0.263,P=0.013,respectively),and negatively correlated with high-density lipoprotein cholesterol levels(r=-0.210,P=0.048);the expression level of hsa＿circRNA＿000241 was positively correlated with triglyceride level(r=0.339,P=0.001).They were not associated with inflammatory indexes,serum uric acid levels,cholesterol,low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol(P>0.05)6.The area under curve(95%confidence interval)of hsa＿circRNA＿103657 and hsa＿circRNA＿000241 for the diagnosis of primary gout were 0.801(0.730-0.871;P<0.001)and 0.726(0.646-0.807;P<0.001),respectively.Conclusion:1.There are multiple abnormally expressed circRNAs in PBMCs of gout patients,and these differential circRNAs are involved in the pathogenesis of gout through various signaling pathways.2.The expression of hsa＿circRNA＿103657 was positively correlated with fasting blood glucose and triglyceride levels,and negatively correlated with high-density lipoprotein cholesterol levels,and the expression of hsa＿circRNA＿000241 was positively correlated with triglyceride levels.They may be involved in the regulation of lipid metabolism or glucose metabolism in gout.3.The level of hsa＿circRNA＿103657 in PBMCs of gout patients was significantly increased,and the area under the ROC curve of hsa＿circRNA＿103657 was more than 0.8,which is expected to be used as a biomarker for the diagnosis of gout.