Objective: To investigate the methylation of 5'-CpG island and expression of RUNX3 in salivary gland adenoid cystic carcinoma cell lines.
Methods: RT-PCR (reverse transcription-polymerase chain reaction), laser scanning confocal microscope (LSCM) and Western blot were used to detect the expression of RUNX3 gene and protein in salivary gland adenoid cystic carcinoma cell lines, ACC-2, ACC-3, and ACC-M, before/after a treatment of 5-Aza-dc respectively.
Results: A weak expression of RUNX3 was found in ACC-2 and ACC-3. And no expression of RUNX3 was found in ACC-3 cell line. After a treatment of 300 nmol/L 5-Aza-dc for 72 hours, the expression of RUNX3 in ACC-2 and ACC-3 cells was enhanced, and in ACC-M was restored. LSCM results showed that the RUNX3 protein was located mainly in the cytoplasm of ACC cell lines. After a treatment of 300 nmol/L 5-Aza-dc for 72 h, both nuclear and cytoplasmic location of RUNX3 positive signals were found in the ACC-2 and ACC-3 cells. However, a weak positive signal was still only found in the cytoplasm of ACC-M cells. Partial methylation in promoter 5'-CpG island of RUNX3 gene was found in all three cell lines. And the methylation degree of CpG island was 50%, 75% and 33% in ACC-2, ACC-M and ACC-3 respectively. After a treatment of 5-Aza-dc, the RUNX3 gene showed unmethylated status in all three cell lines.
Conclusions: The methylation of RUNX3 plays an important role in the silencing of RUNX3 expression in ACC cell lines. The cytoplasmic mislocalization of RUNX3 may be correlated with the inhibition of its function in ACC cells.