Figure 4.

Rap1a/wnt pathway is required for TGFB3-AS1-mediated macrophage inflammation

(A) qRT-PCR and western blot were performed to determine the expression of Rap1a in the peripheral monocytes of CBS^+/+ and CBS^+/− mice fed with a 12-week regular or methionine diet (n = 6). (B) qRT-PCR and western blot were performed to detect the mRNA and protein expression of Rap1a in macrophages treated with different doses of Hcy (0, 50, 100, 200, and 500 μmol/L) for 24 h. (C) Rap1a mRNA and protein were analyzed by qRT-PCR and western blot after macrophages were transfected with si-Rap1a in the presence or absence of Hcy. (D) The contents of IL-1β, IL-6, and TNF-α in the supernatants of macrophages treated with si-Rap1a and Hcy were assayed by ELISA. (E) KEGG enrichment analysis of differentially expressed genes in macrophages treated with Hcy. (F–H) The expression of wnt3a, wnt5a, and β-catenin in the peripheral monocytes of CBS^+/+ and CBS^+/− mice were examined by qRT-PCR and western blot. (I and J) Western blot analysis of Rap1a, wnt3a, wnt5a, and β-catenin expression in macrophages co-transduced with TGFB3-AS1-siRNA or lenti-TGFB3-AS1 and Ad-Rap1a or si-Rap1a in the presence or absence of Hcy. (K and L) The contents of IL-1β, IL-6, and TNF-α in the supernatants of macrophages were co-transduced with TGFB3-AS-siRNA or lenti-TGFB3-AS1 and Ad-Rap1a or si-Rap1a in the presence of Hcy. ∗p < 0.05, ∗∗p < 0.01, compared with control, CBS^+/+ + Meth, Ad-vector or si-NC; #p < 0.05, compared with the group of 100 μmol/L Hcy, Ad-vector + 100 μmol/L Hcy, or si-NC + 100 μmol/L Hcy; ▵p < 0.05, compared with the group of Ad-Rap1a or si-Rap1a; ▿p < 0.05, compared with the group of Ad-vector + TGFB3-AS1-siRNA+ 100 μmol/L Hcy or si-NC + lenti-TGFB3-AS1 + 100 μmol/L Hcy.