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

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.

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.

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.

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.