The role of SOX2 and OCT4 pluripotency markers in relation to TGF-β3- mediated in epithelial-mesenchymal transition during palatal fusion
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Citation
Abstract
BACKGROUND AND OBJECTIVE: We aimed to elucidate the relationship between TGF-β3 signaling and the expression of pluripotency markers during EMT in palatal fusion.
MATERIALS AND METHODS: Palatal samples were collected from the secondary palate of fetuses
from timed-pregnant C57BL mice at the E15 stage before palatal fusion. E15 palatal epithelial
cells were isolated using magnetic cell sorting with CD326 microbeads and treated with 1, 3,
or 10 ng/ml TGF-β3. To identify the role of TGF-β3, its expression was knocked down, and
downstream signaling was measured through Smad2, Smad3, Smad4, Snail, p38, and MMP13.
The epithelial cell barrier function was measured by transepithelial electrical resistance
(TEER). Next-generation RNA sequencing was performed to confirm the result of qRT-PCR.
To further identify the role of each pluripotency marker, we knocked down SOX2 and OCT4,
either alone or together, and treated cells with 1 ng/ml TGF-β3. E-cadherin, Fibronectin,
SOX2, and OCT4 expression in the epithelial lining were measured by immunofluorescence.
RESULTS: There was a significant decrease in E-cadherin and an increase in
fibronectin in response to1-3 ng/ml TGF-β3 treatment (p<0.05). TEER analysis demonstrateda significant decrease in resistance with 1 ng/ml TGF-β3 (p<0.05). Snail expression
was increased in response to all doses of TGF-β3 (p<0.05). MMP13 was increased with 1
ng/ml TGF-β3 (P<0.05). RNA sequencing confirmed the activation of EMT with 1 ng/ml TGF-
β3 and knockdown of TGF-β3. SOX2 and OCT4 signaling decreased from E14.5 to E15.5,
and TGF-β3 significantly reduced their expression.TGF-β3 knockdown decreased SOX2 and
increased OCT4 (p<0.05). TGF-β3 levels were significantly decreased by knockdown of
SOX2, OCT4, SOX2/OCT4, and SOX2/OCT4 in response to1 ng/ml TGF-β3 (p<0.05). The
most significant changes in E-cadherin and fibronectin were observed with SOX2/OCT4
knockdown in response to 1 ng/ml TGF-β3.
CONCLUSIONS: TGF-β3 at an optimal 1-3 ng/ml concentration, reduced epithelial marker and
increased mesenchymal marker expression, suggesting EMT in vitro. TGF-β3 downregulation
of SOX2 and OCT4 may be required for successful palatal fusion.
Description
2025
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Attribution-NonCommercial-NoDerivatives 4.0 International