Figure 5.

Decreased miR-206 expression promoted NF reprogramming into the CAF phenotype and enhanced IL-6 secretion by targeting Anxa2. (A) Expression of alpha-SMA and miR-206 in NFs was evaluated by FISH assay to observe the response of miR-206 expression to TGF-beta1 treatment (20 ng/mL for 12 hours). Scale bar=50 µm. (B) Relative changes in the alpha-SMA and miR-206 mRNA levels in response to TGF-beta1 treatment were detected by qPCR. (C) The protein expression of CAF markers, alpha-SMA and FAP in miR-206-mimic/CAFs and miR-206-inhibitor/NFs was detected by WB assay. (D) The binding site of miR-206 in the Anxa2 3' UTR was predicted, and the binding site was confirmed by dual-luciferase reporter assay. (E) Relative expression of Anxa2 in 3 pairs of NFs and CAFs was detected by qPCR. (F) Regulation of alpha-SMA and Anxa2 expression by miR-206 was analyzed by WB assay. (G) The correlation between miR-206 expression and Anxa2 expression in tissues was analyzed (P<0.05, R²=0.3726). (H) Anxa2 overexpression was confirmed by WB assay. (I) Alpha-SMA protein levels were evaluated after Anxa2 expression was upregulated in miR-206-mimic/CAFs. (J-M) The effects of Anxa2 in miR-206-mimic/CAFs on CAF proliferation, motility, collagen contraction and vascular formation were studied. (N) Fold changes in the IL-1beta, IL-6, IL-8, VEGF-alpha and CXCL12 levels in conditioned medium from miR-206-mimic/CAFs with Anxa2 upregulated expression. The data are shown as the mean ± SD, * P <0.05, ** P <0.01, *** P <0.001.