Differential Expression Analysis Of CircRNA In Peripheral Blood PBMC Of Patients With Gout And Screening Of Potential Molecular Markers

Objective:In this study,differentially expressed circRNAs in primary gout were screened by gene chip technology,and the role of circRNA in gout was analyzed.To explore the potential clinical significance of differentially expressed circRNA in gout and its feasibility as a molecular marker for gout diagnosis by expanding the sample size and combining clinical data.Methods:1.In this study,a case-control study was conducted to collect 3 cases of acute gout patients（active gout,AG）,3 cases of intermittent gout patients（inactive gout,IG）and 3 cases of normal controls（normal control,HC）,and the differentially expressed circRNAs in peripheral blood PBMC were screened by gene chip technology.Furthermore,The circRNAs with significant differential expression were analyzed by GO and KEGG for in-depth bioinformatics.Seven differentially expressed circRNAs were randomly selected and the chip results were verified by real-time fluorescence quantitative PCR（qRT-PCR）.2.（1）Enlarge the sample size.The expression levels of circRNAs in the three groups have been verified by qRT-PCR.Mann-Whitney non-parametric test was used to screen out circRNAs with statistically different expressions between the disease group and the normal control group,and related clinical data were collected.（2）Correlation analysis between differentially expressed circRNAs and clinical data of gout patients.（3）The ROC curve was used to analyze the above circRNAs as a diagnostic tool for gout.Results:1.CircRNA microarray scanning results:（1）Compared with the HC group,there were 93 and 14 circRNAs with significantly differently expressed circRNAs up-regulated in the AG group and IG group,respectively,and 23 and 27 with down-regulated significantly differently expressed circRNAs;Compared with the IG group,there were 86 circRNAs were significantly up-regulated.and 19 circRNAs were down-regulated in the AG group.（2）In-depth biological information analysis of differential circRNAs,GO analysis found that compared with the normal control group,differential circRNAs are mainly concentrated in the positive regulation of cells and biological processes such as protein transcription.In terms of cell components,it is mainly related to the inner membrane of the cell and endometrium-bound organelles.Molecular functions are mainly related to the regulation of DNA transcription.KEGG pathway analysis found that differentially expressed circular RNAs are mainly involved in the MAPK inflammatory signaling pathway and monocytes express FcyR Phagocytic immune response.（3）Seven differentially expressed circRNAs were randomly selected and verified by RT-qPCR.The results indicated that their expression trends were consistent with the microarray results.2.The expression levels of the seven verified circRNAs in peripheral blood PBMCs were measured by qRT-PCR in enlarged samples.The results showed that hsa_circRNA₁04633、hsa_circRNA₀08961、hsa_circRNA₀08337、hsa_circRNA₁00599 was significantly higher in the AG and IG groups than in the HC group[hsa_circRNA₁04633:1.91（2.21）vs 2.34（1.33）vs 1.17（0.65）;hsa_circRNA₀08961:2.16（6.64）vs 2.42（13.92）vs 0.66（0.62）;hsa_circRNA₀08337:1.51（2.01）vs 1.77（1.14）vs 0.96（0.66）;hsa_circRNA₁00599:1.40（1.77）vs 2.25（1.81）vs 1.17（0.89）;both P<0.05],There was no significant difference between the AG group and the IG group（P>0.05）;the expression of hsa_circRNA₁01323 in the AG group and the IG group was significantly higher than that in the HC group,and the AG group was significantly higher than the IG group[hsa_circRNA₁01323:1.80（2.34）vs 1.60（1.48）vs 0.89（0.5）);P<0.05],hsa_circRNA₄06281 expression in AG group was significantly lower than that in HC group[hsa_circRNA₄06281:0.90（1.44）vs 1.67（1.26）vs 1.76（2.72）;P<0.05],There was no significant difference between AG group and IG group,IG group and HC group（P>0.05）;hsa_circRNA₀09158 expression was higher in AG group and IG group than in HC group[hsa_circRNA₀09158:1.05（0.96）vs 1.06（1.12）vs 1.02（0.67）],but the difference was not statistically different.3.Correlation analysis with laboratory indexes of gout patients found that the differentially expressed hsa_circRNA₁04633,hsa_circRNA₀08961,hsa_circRNA₀09158,hsa_circRNA₀08337、hsa_circRNA₁00599,hsa_circRNA₄06281,and hsa_circRNA₁01323 were all related to uric acid（correlation coefficients r and p=0.041;R=0.449,p=0.001;r=0.404,p=0.002;r=0.412,p=0.002;r=0.337,p=0.013;r=0.393,p=0.003）;hsa_circRNA₀08337、hsa_circRNA₁00599、hsa_circRNA₁01323 is positively correlated with 24-hour uric acid（correlation coefficients r and p values are r=0.39,p=0.004;r=0.351,p=0.009;r=0.32,p=0.018）;In addition,hsa_circRNA₁01323 was positively correlated with LDLC（r=0.479,p=0.006）.4.By ROC curve analysis,the results showed that the area under the ROC curve（AUC）of hsa_circRNA₀08961、hsa_circRNA₀08337、hsa_circRNA₁04633、hsa_circRNA₁00599、hsa_circRNA₁01323 and hsa_circRNA₄06281 were 0.839、0.833、0.802、0.723、0.634,0.734,0.634.In order to further improve the diagnostic value,the top three circRNAs with highest AUC values（ie:hsa_circRNA₀08961、hsa_circRNA₀08337,hsa_circRNA₁04633）were analyzed by ROC curve.The AUC is 0.868（0.791-0.945）,which is higher than the diagnostic efficiency of a single circRNA.The expression of hsa_circRNA₁01323 in AG group was higher than that in IG group and HC group.Considering it as a potential marker of inflammation,ROC curve analysis showed that its AUC value was 0.711.Conclusion:1.Gene chip indicated that there were differentially expressed circRNAs in peripheral blood PBMCs of gout patients,which may be involved in the development of gout,and further bioinformatics analysis found that differentially expressed circRNAs may be involved in the inflammation of gout.2.The abnormally expressed hsa_circRNA₁04633、hsa_circRNA₀08961、hsa_circRNA₀08337、hsa_circRNA₁00599、hsa_circRNA₁01323 was up-regulated in gout patients,and hsa_circRNA₄0628 was down-regulated in gout patients.These differentially expressed circRNAs may be involved in gout uric acid metabolism.3.hsa_circRNA₀08961、hsa_circRNA₀08337、hsa_circRNA₁04633 is expected to be a biomolecular marker for the diagnosis of gout.