Abstract
We investigated the relationship between promoter methylation and tracheal stem cell activation. We developed a model of rat tracheal epithelium regeneration after 5-fluorouracil (5-FU)-induced injury. Using immunohistochemistry and Western blotting, the expression levels of the stem cell pluripotency regulator Oct3/4 and differentiation marker CK14 were measured after 5-FU treatment. The methylation status of the Oct3/4, Nanog, and Sox2 promoters was investigated using methylation-specific PCR. Additionally, the effects of 5-azacytidine (5-azaC), a demethylating agent, on Oct3/4, Nanog, and Sox2 mRNA and protein expression were evaluated. Finally, we measured the activity of the maintenance and de novo DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. Our data indicate that Oct3/4, Sox2, and Nanog are transiently expressed in response to 5-FU-induced injury, and then they are gradually silenced as the cells differentiate. DNA methylation can result in silencing of gene expression, and it can determine whether tracheal stem cells are in an active or dormant state. Treatment with 5-FU reversed the methylation of the Oct3/4, Nanog, and Sox2 promoters, which corresponded to increases in Oct3/4, Nanog, and Sox2 mRNA and protein. Thus, both maintenance and de novo methyltransferases are involved in regulating tracheal stem cell dormancy and activation.
Highlights
Stem cells in adult animals normally exist in a state of dormancy in which they remain in the G0 phase of the cell cycle and do not undergo mitotic division [1,2,3]
Oct3/4 is transiently expressed in the rat tracheal epithelium after 5-FU-mediated injury
We investigated the expression of Oct3/4 in the rat tracheal epithelium after 5-FU-mediated injury by immunohistochemistry
Summary
Stem cells in adult animals normally exist in a state of dormancy in which they remain in the G0 phase of the cell cycle and do not undergo mitotic division [1,2,3]. Under specific conditions or in response to various factors in vivo, stem cells can be activated, re-enter the cell cycle, and differentiate into specific tissues [4,5,6]. These processes can occur during tissue regeneration in response to injury and other factors. The relationship between Oct3/4, Nanog, and Sox promoter methylation and tracheal stem cell activation was analyzed using methylation-specific PCR (MSPCR). Rat tracheal stem cells were treated with the demethylating agent 5-azacytidine (5-azaC), and changes in the expression of Oct3/4, Nanog, and Sox were quantified. Our results demonstrate that tracheal stem cell activation is controlled by epigenetic modifications
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