Expression Profile Of CircRNA In Rheumatoid Arthritis With Osteoporosis

Background and objective:Circular RNA（circRNA）is a class of endogenous RNA molecules with closed circular structure,which has many important biological functions such as acting as microRNA（miRNA）sponge,regulating transcription,influencing protein function,and even being translated into protein.It is closely related to the occurrence of human diseases.Long-term rheumatoid arthritis（RA）will lead to a variety of complications,and bone involvement is one of the main extrarticular complications of RA.Therefore,rheumatoid arthritis combined with osteoporosis（OP）is common in clinical practice,resulting in reduced quality of life and increased mortality.Studies on circRNA in osteoporosis and rheumatoid arthritis have been widely carried out,but the pathogenesis of osteoporosis in rheumatoid arthritis is not completely clear at present,and studies on circRNA in rheumatoid arthritis combined with osteoporosis are limited.Therefore,it is of great significance to explore the role of circRNA in the pathogenesis of rheumatoid arthritis patients with osteoporosis.By exploring the role of circRNA in the pathogenesis of rheumatoid arthritis patients with osteoporosis and its chip expression profile,this study aims to explore the early screening indicators and potential clinical significance of RA patients with OP,and provide new clues and ideas for the early diagnosis of RA patients with OP.Methods:1.Differential circRNAs were analyzed by chip technology,and diagnostic markers of RA combined with OP were searched.（1）Sample collection chip making and bioinformatics analysis:A case-control research was used in this study to collect peripheral blood mononuclear cells（PBMC）from patients with simple rheumatoid arthritis（RA control group）and patients with rheumatoid arthritis complicated with osteoporosis（OP+RA experimental group）,and 10 PBMC specimens were collected（5 in the experimental group and 5 in the control group）.Chip technology was used for screening differentially expressed circRNAs.In-depth bioinformaticsanalysis of differentially expressed circRNAs using GO/KEGG,and 7 circRNAs with differentially expressed circRNAs（multiple change>1.6,P<0.05）,real-time fluorescence quantitative PCR（qRT-PCR）was used to verify the chip results.（2）Differential circRNAs clinical sample detection:Peripheral blood of 35 RA group and 35 RA+OP group were selected,PBMC was isolated and total RNA was extracted,and the expression of differentially expressed circRNAs in the two groups was verified by qRT-PCR.The circRNAs with statistical difference between the experimental group and the control group were screened by Mann-Whitney nonparametric test.（3）ROC curve drawing of different circRNAs:ROC curve drawing was conducted according to sensitivity,specificity,positive prediction rate and negative prediction rate to screen out possible biomarkers of rheumatoid arthritis combined with osteoporosis.2.Clinical data and inspection findings of 70 patients verified by qRT-PCR were collected and analyzed for correlation with differentially expressed circRNAs.Results:1.Results of chip scanning,circRNAs verification and ROC curve analysis:（1）circRNA chip analysis results:There were 95 significantly differentially expressed circRNAs in the experimental group compared with the control group,including 38 up-regulated circRNAs and 57 down-regulated circRNAs.GO analysis results suggested that these differentially expressed circRNAs mainly existed in the plasma membrane and nuclear plasma,and were involved in monocarboxylic acid binding,positive regulation of atypical Wnt signaling pathways,and primary cell metabolism.KEGG pathway analysis demonstrated that these circRNAs were proliferator-activated after RNA polymerase and peroxisome proliferators Receptors（PPARs）signaling pathway,intracellular macrophage,ubiquitin-mediated proteolysis,etc.The results of miRNA target prediction software suggest that the above differentially expressed circRNAs may target to hsa-mir-6882-3p,hsa-mir-543,hsa-mir-892a and other miRNAs and play a role by regulating human bone marrow part-time stem cells.Seven differentially expressed circRNAs were randomly screened out（up-regulated:hsa_circRNA₀027234,hsa_circRNA₀025016,hsa circRNA 002487,hsa_circRNA₀052522;Down-regulated:hsa_circRNA₄103627,hsa_circRN A₀102101,hsa_circRNA₀000122）were tested by qRT-PCR,and the results showed that the expression trend was in accordance withthat of chip.（2）The PBMC specimens of RA group and RA+OP group were tested by qRT-PCR with expanded sample capacity,and the test results showed that the expression direction three differentially expressed circRNAs（hsa_circRN A₀102101,hsa_circRNA₀027234,and hsa_circRNA₀025016）was consistent with the chip results,and hsa_circRNA₀102101 was down-regulated.hsa_circRNA₀027234 and hsa_circRNA₀0025016 were up-regulated（P<0.05）hsa_circ₀103627,hsa_circRNA₀052522,hsa_circRNA₀000122 and hsa_circRNA₀002487 had no statistical significance（P>0.05）.（3）ROC curve analysis The areas under ROC curve of hsa_circRNA₀025016,hsa_circRNA₀102101 and hsa_circRNA₀027234 were 0.700（P<0.05）,0.340（P<0.05）,0.612（P>0.05）.2.Analysis results of patient-related clinical data:（1）Lymphocyte count（LY）,globin（GLOB）and age in RA+OP group were significantly higher than those in RA+OP group（P<0.05）.（2）Sperman correlation test indicates that hsa_circRNA₀025016 is positively correlated with anti-cyclic Citrullinated peptide Antibody（CCP）（correlation coefficient R=0.310,P=0.009）,and HSA_circ₀102101 was positively correlated with GLOB and neutrophil granule count（GR）（correlation coefficients r=0.251,P=0.036;r=0.290,P=0.015）;hsa_circ₀027234 was positively correlated with high sensitivity C Reactive protein（hs-CRP）and GR（correlation coefficients r=0.354,P=0.003;r=0.243,P=0.043）,other laboratory indicators had no statistical significance.Conclusion:1.Gene chip suggests that there were differentially expressed circRNAs in peripheral blood PBMC of RA combined with OP patients,and they may participate in the pathogenesis of rheumatoid arthritis combined with osteoporosis by regulating cellular transcription and participating in signal transduction pathways.2.The expression of hsa_circRNA₀025016 in PBMC of patients with rheumatoid arthritis combined with osteoporosis was significantly increased,and the area under ROC curve was 0.7,which is expected to be used as a biomarker for the diagnosis of rheumatoid arthritis combined with osteoporosis.3.The expression of hsa_circ₀025016 was positively correlated with CCP antibody,the expression of hsa_circ₀102101 was positively correlated with GLOB and GR,and the expression of hsa_circ₀027234 was positively correlated with hs-CRP and GR.They may participate in the pathogenesis of rheumatoid arthritis combined with osteoporosis by regulating inflammatory factors.Hsa_circ₀025016 combined with CCP antibody is expected to improve diagnostic efficiency.