TGF‐β1 Induced Methylation to Regulate SOCS3 Expression in Abnormal Activation of Vocal Fold Fibroblasts

ABSTRACT Objective To investigate the effect of TGF‐β1 on vocal fold fibroblasts (VFFs) phenotype and the expression of DNA methyltransferase 1 (DNMT1), and to reveal the putative mechanism by which TGF‐β1 regulates SOCS3 and its potential role in abnormal VFFs activation after vocal fold injury. Methods The normal VFFs group (Control), injured VFFs group (Injured), normal VFFs+TGF‐β1 group (Control+TGF‐β1), and injured VFFs+TGF‐β1 group (Injured+TGF‐β1) were set, and 5‐aza, a DNA methyltransferase inhibitor that reduces DNMT1 protein levels via covalent trapping and degradation, was added to block DNA methylation. Methylation‐specific PCR (MSP) was used to detect the methylation level of the SOCS3 promoter. The methylation level of the SOCS3 promoter, and the protein and mRNA expressions of Type I collagen (COL1), α‐smooth muscle actin (α‐SMA), DNMT1, SOCS3, JAK2, and STAT3 were detected before and after the addition of TGF‐β1 and 5‐aza. Results Compared to the Control group, the expressions of COL1, α‐SMA, and DNMT1 were elevated in the Injured, Control+TGF‐β1, and Injured+TGF‐β1 groups. After 5‐aza addition, no significant changes occurred in the Control group; however, the above markers were significantly decreased in the Injured, Control+TGF‐β1, and Injured+TGF‐β1 groups. The methylation level of the SOCS3 promoter was significantly increased, and SOCS3 expression was markedly downregulated in the Injured, Control+TGF‐β1, and Injured+TGF‐β1 groups, while the SOCS3 promoter was not significantly methylated in the Control group. After 5‐aza addition, the methylation level of the SOCS3 promoter was significantly decreased in the Injured, Control+TGF‐β1, and Injured+TGF‐β1 groups, and concomitantly, SOCS3 mRNA and protein expression were significantly upregulated. The expressions of JAK2 and STAT3 were significantly increased in the Injured, Control+TGF‐β1, and Injured+TGF‐β1 groups, whereas a significant decrease was observed after adding 5‐aza, with the changes positively correlated with SOCS3 upregulation. Conclusion TGF‐β1 is proposed to promote abnormal activation of VFFs, which may be associated with DNA methylation‐mediated regulation of SOCS3. Specifically, TGF‐β1 may downregulate SOCS3 expression via DNMT1‐mediated promoter DNA methylation, and this process is speculated to participate in abnormal VFFs activation by relieving the inhibitory effect of SOCS3 on the JAK2/STAT3 signaling pathway.