IL-6-miR-210 Suppresses Regulatory T Cell Function and Promotes Atrial Fibrosis by Targeting Foxp3.

Figures

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  Fig. 1. Negative correlation between the levels of IL-6 and IL-10 in patients with AF. (A) ELISA results of IL-6 and IL-10 in peripheral whole blood of AF patients and normal subjects. (B) ELISA results of IL-6 and IL-10 in right atrial tissue of AF patients and normal subjects. (C) FACS analysis of Treg cells (FoxP3) in peripheral whole blood of AF patients and normal subjects. (D) FACS analysis of Treg cells (FoxP3) in right atrial tissue of AF patients and normal subjects.*P < 0.05 vs nomal controls.

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  Fig. 2. IL-6 supresses the function of Tregs and promotes fibrosis of mechanocyte. (A) Western blot result of Foxp3 in CD4+ T cells. (B) ELISA results of IL-10 in CD4+ T cells. (C) FACS analysis of Treg cells (FoxP3) in CD4+ T cells. (D) Vimentin immunostaining of mechanocyte in atrial tissue of mice. (E) Western blot result of α-SMA, collagen I and collagen III in CD4+ T cells co-cultured with atrium mechanocyte. *P < 0.05, **P < 0.01 vs control; #P <

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  Fig. 3. IL-6 inhibits Treg cell function via regulating miR-210 by targeting Foxp3. (A) The qRT-PCR result of miR-210 in serum and right atrial tissue of AF patients. (B) The mRNA level of miR-210 in CD4+ T cells stimulated by IL-6 and PBS. (C) Schematic of the putative miR- 210 target site in human Foxp3 3′-UTR and the seven mutated nucleotides are colored red. WT, wild-type; Mut, mutant; NC, negative control. (D) The mRNA level of Foxp3 in CD4+ T cells transfected with miR-210 mimic and miR-210 inhibitor. (E) The protein level of Foxp3 in CD4+ T cells transfected with miR-210 mimic and miR-210 inhibitor. (F) ELISA result of IL-10 in CD4+ T cells transfected with miR-210 inhibitor and stimulated by IL-6 and PBS. (G) Western blot result of Foxp3 in CD4+ T cells transfected with miR-210 inhibitor stimulated by IL-6 and PBS. (H) Western blot result of α-SMA, collagen I and collagen III in transfected CD4+ T cells co-cultured with atrium mechanocyte. *P < 0.05, **P < 0.01 vs normal or mimic NC or inhibitor NC; #P < 0.05, ##P < 0.01 vs inhibitor NC or IL-6 + inhibitor NC or Ang II + mimic NC; &P <

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  Fig. 4. IL-6 promotes expression of miR-210 via regulating HIF-1. (A) Western blot result of HIF-1α in right atrial tissue of AF patients and normal subjects. (B) The protein level of HIF-1α in CD4+ T cells stimulated by IL-6 and PBS. (C) The mRNA level of miR-210 in CD4+ T cells transfected with HIF-1α siRNA and stimulated by IL-6 and PBS. (D) ELISA result of IL-10 in CD4+ T cells transfected with HIF-1α siRNA and stimulated by IL-6 and PBS. (E) FACS analysis of Treg cells (FoxP3) in CD4+ T cells transfected with HIF-1α siRNA and stimulated by IL-6 and PBS. (F) Western blot result of Foxp3 in CD4+ T cells in CD4+ T cells transfected with HIF-1α siRNA and stimulated by IL-6 and PBS. (G) Western blot result of α-SMA, collagen I and collagen III in transfected CD4+ T cells co-cultured with atrium mechanocyte. *P < 0.05, **P < 0.01 vs normal or control or si-Ctrl; #P < 0.05, ##P < 0.01 vs IL-6 + si-Ctrl or Ang II + si-Ctrl.

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  Fig. 5. Effects of Treg cells modified with miR-210 on mice with AF. Animal model of mouse atrial fibrosis was established, and miR- 210 antogomir and control were transfected into Treg cells. (A) H&E staining result in each group. Scale bars = 20 µM. (B) Masson staining result in each group. Scale bars = 20 µM. (C) The cardiomyocyte area in each group. (D) The fibrosis area in each group. (E) The ratio of heart weight (HW)/body weight (BW) in each group. (F) Western blot result of α-SMA, collagen I and collagen III in each group. *P < 0.05, **P < 0.01 vs control; # P < 0.05 vs model; & P < 0.05, && P < 0.01 vs antogomir NC.