circRNA_17725 Promotes Macrophage Polarization towards M2 by Targeting FAM46C to Alleviate Arthritis

Abstract

Accumulating studies have implicated that circular RNAs (circRNAs) play vital roles in the pathogenesis of rheumatoid arthritis (RA). Dysregulation of macrophage polarization leads to immune homeostatic imbalance in RA. However, the altering effects and mechanisms of circRNAs on macrophages polarization and immune homeostatic balance remain largely unclear. We aimed to investigate the potential role of circRNA_17725 in RA. The high-throughput sequence was performed to identify the dysregulated circRNAs in RA. We confirmed the data by CCK-8, EdU, and Annexin V/PI staining to elucidate the proliferation and apoptosis. The expressions of M1/M2-associated markers were confirmed using real-time PCR and flow cytometry analysis. Luciferase reporter assay and RNA Binding Protein Immunoprecipitation (RIP) were used to demonstrate the underlying mechanism of circRNA_17725. The altering effect of circRNA_17725 on macrophages in vivo was evaluated using collagen-induced arthritis (CIA) mouse model. circRNA_17725 was demonstrated to be downregulated in peripheral blood mononuclear cells and CD14⁺ monocytes from RA cases in contrast to healthy controls. The negative association between circRNA_17725 and the disease activity indexes (CRP, ESR, and DAS28) was observed, suggesting a vital role of circRNA_17725 in RA disease activity. Besides, after a coexpression analysis based on high-input sequencing and the bioinformatics analysis in MiRanda and TargetScan databases, a circRNA_17725-miR-4668-5p-FAM46C competing endogenous RNA (ceRNA) network was hypothesized. A series of cytology experiments in vitro have implicated that circRNA_17725 could inhibit the proliferation but enhance the apoptosis of macrophages. Decreased expression of TNF-α, IL-1β, and MMP-9 were observed in the supernatant of circRNA_17725-overexpressed Raw264.7 macrophages, implicating the inhibitory effect of circRNA_17725 on macrophage inflammatory mediators. Furthermore, circRNA_17725 could promote macrophage polarization towards M2 by targeting miR-4668-5p/FAM46C as a miRNA sponge. Additionally, circRNA_17725-overexpressed macrophages alleviated arthritis and protected against joint injuries and bone destruction by inducing macrophage polarization towards M2 in collagen-induced arthritis (CIA) mice. This study has suggested that circRNA_17725 regulated macrophage proliferation, apoptosis, inflammation, and polarization by sponging miR-4668-5p and upregulating FAM46C in RA.

Figure 1

Expression of circRNA_17725 in the PBMCs and its association with the disease activity indexes of RA. (a) Expression of circRNA_17725 in the PBMCs from RA patients (^∗∗P < 0.01; RA/control: 35/28). (b) Pearson's correlation analysis: relationship of circRNA_17725 with CRP (n = 35). (c) Pearson's correlation analysis: relationship of circRNA_17725 with ESR (n = 35). (d) Pearson's correlation analysis: relationship of circRNA_17725 with DAS28 (n = 35).

Figure 2

Potential targets prediction of circRNA_17725. (a) Positive coexpression association between circRNA_17725 and FAM46C and other factors implicated by high-throughput sequencing and bioinformatics analysis. (b) Complementary base pairs of circRNA_17725 and miR-4668-5p, plus miR-4668-5p, and FAM46C. (c) A predicted circRNA_17725/miR-4668-5p/FAM46C ceRNA network (the red shape represents miR-4668-5p, the purple squares represent targeted mRNAs including FAM46C, and the green circle represents circRNA_17725).

Figure 3

Association between circRNA_17725, FAM46C, TNF-α, IL-10, and CD206 in RA. (a) Expression of circRNA_17725 in CD14⁺monocytes from RA patients in contrast to controls (^∗∗P < 0.01; RA/control: 40/30). (b) Expression of FAM46C in CD14⁺monocytes from RA patients in contrast to controls (^∗∗P < 0.01; RA/control: 40/30). (c) Pearson's correlation analysis: relationship of circRNA_17725 with FAM46C (n = 40). (d) Pearson's correlation analysis: relationship of circRNA_17725 with TNF-α (n = 40). (e) Pearson's correlation analysis: relationship of circRNA_17725 with IL-10 (n = 40). (f) Pearson's correlation analysis: relationship of circRNA_17725 with CD206 (n = 40).

Figure 4

Location of circRNA_17725 in macrophages. (a) Real-time PCR: circRNA_17725 expression in Raw264.7 macrophages (^∗∗∗P < 0.001; n = 3). (b) FISH: location of circRNA_17725 in Raw264.7 macrophages (representative pictures; 20x).

Figure 5

circRNA_17725 regulated macrophage proliferation, apoptosis, and inflammation. (a) Flow cytometry: Lv-circRNA_17725 plasmids promoted the apoptosis of macrophages induced by H₂O₂ at 4 h (^∗∗P < 0.01; n = 3). (b) EdU: Lv-circRNA_17725 plasmids inhibited the proliferation of macrophages at 48 h (20x; ^∗∗P < 0.01; n = 3). (c) CCK-8: Lv-circRNA_17725 plasmids inhibited the proliferation of macrophages in a time-dependent way (^∗∗P < 0.01; n = 3). (d) ELISA: Lv-circRNA_17725 plasmids decreased TNF-α, IL-1β, and MMP-9 but increased IL-10 in cells cultural supernatant (^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001; n = 3).

Figure 6

circRNA_17725 promoted macrophage polarization towards M2. (a) Flow cytometry: circRNA_17725 promoted CD206⁺M2 and CD163⁺M2 cells while inhibited HLA-DR⁺M1 and CD68⁺M1 cells polarization (representative pictures). (b) Cell percentages of CD206⁺M2, CD163⁺M2, HLA-DR⁺M1, and CD68⁺M1 cells determined by flow cytometry (vs. Lv-control: ^∗P < 0.05, ^∗∗∗P < 0.001; vs. Lv-control: ^#P < 0.05, ^##P < 0.01; n = 3). (c) CD11c mRNA expression was decreased in circRNA_17725-overexpressed Raw264.7 cells (^∗∗P < 0.01; n = 3). (d) CD206 mRNA expression was increased in circRNA_17725-overexpressed macrophages (^∗∗P < 0.01; n = 3).

Figure 7

circRNA_17725 functioned as a ceRNA based on the circRNA_17725-miR-4668-5p-FAM46C network. (a) Real-time PCR: increased expression of FAM46C in Lv-circRNA-transfected Raw264.7 macrophages (^∗∗∗P < 0.001; n = 3). (b) Cell immunofluorescence: higher expression of FAM46C in Raw264.7 macrophages transfected by Lv-circRNA plasmids (representative pictures; 20x). (c) Real-time PCR: decreased miR-4668-5p in Lv-circRNA-treated cells (^∗∗P < 0.01; n = 3). (d) Luciferase reporter assay: miR-4668-5p mimics inhibited FAM46C expression in macrophages (^∗∗P < 0.01; n = 3). (e) RIP test: expression of circRNA_17725 in immunoprecipitations (^∗∗P < 0.01; n = 3). (f) RIP test: expression of miR-4668-5p in immunoprecipitations (^∗∗P < 0.01; n = 3).

Figure 8

circRNA_17725-overexpressed macrophage alleviated arthritis in CIA mice. (a) CIA mouse model construction flowchart (6 mice/group). (b) Arthritis score: circRNA_17725-overexpressed macrophage-treated CIA mice had lower arthritis score (vs. the WT group, ^∗∗∗P < 0.001; vs. the CIA-Lv-control group, ^##P < 0.01). (c) Representative pictures: less redness, swelling, and alleviated arthritis of CIA mice treated by circRNA_17725-overexpressed macrophages. (d) Representative pictures of HE staining of joint tissues from CIA mice treated by circRNA_17725-overexpressed macrophages (20x). (e) Representative Safranin O/Solid green staining pictures: less severe cartilaginous injury and bone damages in the joint tissues of CIA mice treated by Lv-circRNA-transfected macrophages (20x).

Figure 9

circRNA_17725 promoted M2 polarization in CIA mice. (a) Flow cytometry: CD14⁺CD163⁺ cells and CD14⁺CD11c⁺ cells infiltration in spleens from CIA mice (^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001; 6 mice/group, n = 3). (b) Real-time PCR: CD11c, CD163, and FAM46C mRNA expressions in spleen mononuclear cells of CIA mice (^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001; 6 mice/group, n = 3). (c) ELISA: decreased TNF-α and IL-1β in the plasm from Lv-circRNA-transfected macrophage-treated CIA mice (^∗∗P < 0.01, ^∗∗∗P < 0.001; 6 mice/group, n = 3).