A study of rat vocal-fold cells found a recurring pattern of changes accompanying injury-related activation: cells from injured animals, and cells exposed to TGF-β1, had higher levels of COL1, α-SMA and DNMT1, while methylation of the SOCS3 promoter, the gene’s regulatory region, was higher and SOCS3 expression was lower. When researchers added 5-aza, the activated cultures showed lower COL1, α-SMA and DNMT1, while the normal Control phenotype was not significantly changed. The same exposure was associated with lower SOCS3 promoter methylation and higher SOCS3 expression. The pattern is suggestive, but it does not establish cause and effect.
What was tested
The researchers used 40 adult male Sprague-Dawley rats weighing 300 to 350 grams. Twenty underwent unilateral vocal-fold injury, with five rats in each group. Cells from uninjured and injured animals were grown as vocal-fold fibroblast cultures, then compared as normal VFFs, injury-derived VFFs, and normal or injured VFFs exposed to exogenous TGF-β1. The researchers also added 5-aza to each basic group to inhibit methylation.
To follow the molecular changes, the team measured methylation in the SOCS3 promoter with methylation-specific PCR. It also measured protein and messenger RNA levels before and after TGF-β1 and 5-aza exposure. The results were summarized as means with standard errors and tested with one-way analysis of variance followed by Tukey pairwise comparisons. A probability value below 0.05 was treated as statistically significant.
The molecular pattern
COL1, α-SMA and DNMT1 were higher in the injured and TGF-β1-treated groups than in Control cultures. DNMT1 was higher at both the protein and messenger RNA levels in each injured or TGF-β1-treated group, and its expression showed a positive association with TGF-β1 expression. The reported association had a probability value below 0.01, but the study design cannot show which change came first.
The SOCS3 findings moved in the opposite direction. Its promoter was more methylated in injured and TGF-β1-treated cultures than in Control cultures, while SOCS3 expression was lower; the Control promoter was not significantly methylated. In activated groups, 5-aza exposure was associated with lower promoter methylation and higher SOCS3 messenger RNA and protein expression.
JAK2 and STAT3 showed a matching pattern. Their protein, messenger RNA and phosphorylation measures were higher in activated groups and lower after 5-aza, alongside higher SOCS3 expression and a less fibrotic phenotype. The study did not directly manipulate SOCS3 alone, so these coordinated changes do not show that SOCS3 controlled JAK2/STAT3.
A proposed chain, not a proven one
The matching results led the authors to propose a chain of linked events. In their interpretation, injury-associated TGF-β1 may be linked to DNMT1-mediated SOCS3 promoter methylation, reduced SOCS3, and JAK2/STAT3 activation. That is a proposed model, not a demonstrated mechanism.
But the evidence stops short of proving that chain. The study lacked direct TGF-β1 neutralization, DNMT1-specific silencing, and SOCS3 gain- or loss-of-function experiments, leaving the contribution of each step untested. 5-aza is a broad-spectrum methyltransferase inhibitor, so global demethylation or other nonspecific effects cannot be excluded. The mechanistic work was conducted in vitro and had no in vivo validation.
The paper therefore offers a molecular lead for further testing, not a validated treatment pathway. Direct tests that isolate TGF-β1, DNMT1 and SOCS3, followed by in vivo work, are needed to determine whether the proposed chain has physiological relevance.
Paper data and sources
Original title: TGF‐β1 Induced Methylation to Regulate SOCS3 Expression in Abnormal Activation of Vocal Fold Fibroblasts
Authors: Wang H, Zhao A, Li X et al.
Journal/Repository: World journal of otorhinolaryngology - head and neck surgery
Status: Peer-reviewed
First online: 2026-10-11
DOI: Not available
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