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Objective:To investigate the differences in the expression profiles of cyclic RNA (circRNA) in peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) and its clinical significance.Methods:Venous blood were collected from 4 patients with RA (group T) and 4 healthy subjects (group C). The expression profiles of circRNA in PBMCs of the two groups were detected by Arraystar circRNA microarray, and the differentially expressed circRNA was analyzed by clustering analysis. The binding sites for interaction between differentially expressed circRNA and miRNA were predicted, and functional analysis such as geneontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed. quantitative real-time polymerase chain reaction (RT-qPCR) was used to verify the expression of partially differentially expressed circRNA in the two groups of PBMCs, and a circRNA-miRNA-mRNA regulatory network (ceRNA network) was constructed for the target circRNA with significantly differential expression. A receiver operating characteristic curve [receiver operating characteristic curve (ROC)] was established to analyze the potential diagnostic value of target circRNA. SPSS Statistics 23.0 and Graphpad Prism 8.0 were used to analyze the data, and the independent t test was used to analyze the difference between the two groups. Results:① Microarray results showed that, compared with group C, a total of 399 [fold of difference (FC)>1.5, and P<0.05] circRNA were abnormally expressed in PBMCs of group T; including 149 up-regulated and 250 down-regulated. ② Bioinformatics analysis: The prediction of the binding site of circRNA and miRNA suggested that the differentially expressed circRNA in RA might affect the inflammatory response by targeting miR-140-5p, miR-338-5p, and miR-9-5p. GO analysis showed that the differentially expressed circRNA was mainly involved in the intimal-binding organelles, protein metabolism and binding, etc. KEGG pathway analysis showed that most of the involved pathways were related to infection and human immune dysregulation. ③ The results of multi-sample RT-qPCR validation showed that the expression level of hsa_circRNA_009012 in group T was significantly higher than that in group C ( t=-4.417, P<0.01), the expression level of hsa_circRNA_101328 was significantly lower than that in group C ( t=-1.042, P<0.01), and the expression of hsa_circRNA_058230 had no significant change ( t=4.691, P>0.05). ④ ROC curve analysis indicated that hsa_circRNA_009012 had potential value in the diagnosis of RA [area under curve=0.96]. Conclusion:The expression of circRNA in PBMCs of patients with RA is imbalanced, and it may participate in the regulation of the development of RA. Among them, hsa_circRNA_009012 is expected to become a new biological marker for the diagnosis and treatment of RA.
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BACKGROUND: Cyclic RNA plasmacytoma variant translocation 1 (circPVT1) is involved in the senescence of fibroblasts, but the relationship of circPVT1 with nucleus pulposus senescence and its mechanism are still unclear. OBJECTIVE: To investigate the expression of circPVT1 in nucleus pulposus cell senescence and to explore its possible mechanism. METHODS: Human nucleus pulposus cells were cultured in vitro, and the senescence of nucleus pulposus cells was induced by ionizing radiation (5 Gy, 6 days). The expression of circPVT1 and let-7 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). CircPVT1 siRNA and anti-let-7 were transfected into normal nucleus pulposus cells, which were divided into control group, si-NC+anti-NC group, si-circPVT1+anti-NC group, si-NC+anti-let-7 group, and si-circPVT1+anti-let-7 group. The expressions of circPVT1 and let-7 mRNA were detected by qRT-PCR. Cell counting kit-8 assay was used to detect the inhibition of cell proliferation. Plate cell clone formation assay was used to detect colony formation. Cell senescence was detected by SA-β-gal staining. The expressions of p21, p27, let-7 target high mobility group protein A2 (HMGA2) and KRAS were detected by western blot assay. Double luciferase activity assay was used to verify the relationship between let-7 and target regulation of HMGA2 and KRAS. RESULTS AND CONCLUSION: (1) Compared with normal nucleus pulposus cells, the expression of circPVT1 was decreased, while let-7 expression and the positive rate of SA-β-gal staining were increased in the irradiated cells (P < 0.05). (2) Compared with the control group and si-NC+anti-NC group, the si-circPVT1+anti-NC group appeared to have decreased expression of circPVT1 mRNA, HMGA2 and KRAS proteins and number of clones formed as well as increased let-7 mRNA expression, p21, p27 protein expression, cell inhibition rate and positive rate of SA-β-gal staining (P < 0.05). However, opposite changes were found in the si-NC+anti-let-7 group in relative to the control group (P < 0.05). (3) The expression of circPVT1 mRNA, clone formation, and expressions of HMGA2 and KRAS proteins in the si-circPVT1+anti-let-7 group were higher than those in the si-circPVT1+anti-NC group, and lower than those in the si-NC+anti-let-7 group. Let-7 mRNA expression, cell inhibition rate, positive rate of SA-β-gal staining, and expressions of p21 and p27 proteins in the si-circPVT1+anti-let-7 group were lower than those in the si-circPVT1+anti-NC group, and higher than those in the si-NC+anti-let-7 group (P < 0.05). Double luciferase activity assay showed that HMGA2 and KRAS were the targets of let-7. These findings indicate that inhibition of circPVT1 can inhibit the aging of nucleus pulposus cells. The mechanism may be through binding let-7 to inhibit the targeting of HMGA2 and KRAS proteins.