The expression of survivin and its related genes in adipocyte-derived stem cell by demethylation

BACKGROUND: Survivin is thought to contribute to stem cell maintenance partly by a hypomethylation mechanism. This study attempted to elucidate the signal transduction pathway of adipocyte-derived stem cells (ASCs) by using a demethylating agent, 5-aza-2'-deoxycytidine (ADC), to analyze the survivin, MEK/ERK, c-Myc and p53 gene expression. METHODS: Demethylation in the ASCs was induced by 1 µM ADC treatment. RT-PCR for survivin mRNA was preformed, before and 24, 48 and 72 hours (hr) after ADC treatment. Western blotting analysis was performed for p53, survivin, unphosphorylated and phosphorylated (p)-MEK, and p-ERK. Immunohistochemistry for ERK and survivin was done to evaluate the localization of the proteins. RESULTS: ADC inhibited the population growth of the ASCs and it increased the number of apoptotic cells 24, 48, and 72 hr after treatment. ADC treatment slightly decreased the expression of survivin mRNA after 48 hr and its level was restored after 72 hr of treatment. Otherwise, the level of survivin protein gradually increased up to 48 hr and it was decreased at 72 hr. The levels of p-MEK and p53 were increased time-dependently. c-Myc and p-ERK were elevated after ADC treatment and their highest levels were seen 48 hr after treatment. The ADC treatment increased the nuclear expression of ERK and survivin in the ASCs. CONCLUSIONS: The overexpression of p-MEK/ERK, p53, and c-Myc increased the survivin protein expression of the demethylated ASCs. These results suggest that demethylation could alter the expression of survivin, and p53, c-Myc and the MAPK (MEK/ERK) pathway might play a role in survivin's regulation in ASCs.