Correlation between circRNA0003353 in Peripheral Blood Mononuclear Cells and Immune Inflammation in Rheumatoid Arthritis Patients with Damp Heat Obstruction Syndrome

WANG Jie, LIU Jian, WEN Jian-ting, WANG Xin

Abstract

To investigate the expression of circRNA 0003353 in the peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients with dampness heat obstruction syndrome and to examine its effect on inflammatory response of fibroblast-like synoviocytes (FLS).  Methods  The PBMCs and serum samples of 55 RA patients with dampness heat obstruction syndrome and 30 healthy volunteers were collected. The expression of circRNA 0003353 and its correlation with clinical indexes were examined. The circRNA 0003353 overexpression plasmid and siRNA were constructed and transfected into RA-FLS cell line. RT-qPCR was used to determine the expression of circRNA 0003353 mRNA. The expressions of interleukin (IL)-4, IL-10 and IL-17 were examined by ELISA. The expressions of Janus kinase 2 (JAK2), p-JAK2, signal transducers and activators of transcription 3 (STAT3) and p-STAT3 were exmained by Western blot. CCK-8 assay was used to assess cell viability. Cell migration was assessed with Transwell migration assay.   Results  1) Compared with that of the normal group, the expression of circRNA 003353 in the PBMCs of RA patients with damp heat obstruction syndrome was significantly increased (P<0.05). 2) Pearson correlation analysis showed that circRNA 0003353 was positively correlated with erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), receptor activator of nuclear factor-κ B ligand (RANKL) and DAS28, and circRNA 0003353 was negatively correlated with IL-10 (P<0.05). 3) The findings on the association patterns showed that the increase in circRNA 0003353 was significantly correlated with the increase of ESR, IL-17, CRP and immunoglobulin (Ig) G. 4) Logistic regression analysis showed that circRNA 0003353 was a risk factor for RANKL, CRP and ESR. 5) RT-qPCR results showed that the expression of circRNA 003353 mRNA in pcDNA3.1-circRNA 0003353 group was significantly higher than that in pcDNA3.1-NC group (P<0.05), and that the expression of circRNA 003353 mRNA in si-circRNA 0003353 group was significantly lower than that in si-NC group (P<0.05). 6) ELISA and Western blot results showed that, compared with those of pcDNA3.1-NC group, the expression of IL-10 in pcDNA3.1-circRNA 0003353 group significantly decreased, the expression of IL-17 increased, and p-JAK2/JAK2 and p-STAT3/STAT3 ratios significantly increased (P<0.05). Compared with those of si-NC group, the expression of IL-10 in si-circRNA 0003353 group significantly increased, the expression of IL-17 and JAK2 decreased, and p-JAK2/JAK2 and p-STAT3/STAT3 ratios significantly decreased (P<0.05). 7) The results of CCK-8 and Transwell assays showed that the viability and migration of RA-FLS in pcDNA3.1-circRNA 0003353 group were higher than those in pcDNA3.1-NC group (P<0.05). Compared with those of si-NC group, the viability and migration ability of RA-FLS in si-circRNA 0003353 group decreased (P<0.05).   Conclusion  The expression of circRNA 0003353 is up-regulated in RA patients with damp heat obstruction syndrome, and it is involved in the pathogenesis of RA by activating the JAK2/STAT3 signaling pathway and promoting the inflammatory response.

 

Keywords: Rheumatoid arthritis, Dampness heat obstruction syndrome, circRNA, Fibroblast-like synoviocytes, Inflammation

 

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