Ge Ming-Hua, Ling Zhi-Qiang, Tan Zhuo, Chen Chao, Zheng Chuan-Ming, Xu Jia-Jie, Zhu Xin
Department of Surgical Oncology, Zhejiang Province Cancer Hospital, Hangzhou 310022, China.
Zhonghua Yi Xue Za Zhi. 2010 Dec 28;90(48):3426-30.
To investigate the methylation of 5'-CpG island and expression of RUNX3 in salivary gland adenoid cystic carcinoma cell lines.
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.
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.
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.
研究涎腺腺样囊性癌细胞系中RUNX3基因5'-CpG岛甲基化状态及RUNX3的表达情况。
分别采用逆转录-聚合酶链反应（RT-PCR）、激光扫描共聚焦显微镜（LSCM）及蛋白质免疫印迹法检测5-氮杂-2'-脱氧胞苷（5-Aza-dc）处理前后涎腺腺样囊性癌细胞系ACC-2、ACC-3及ACC-M中RUNX3基因及蛋白的表达。
ACC-2和ACC-3中RUNX3呈低表达，ACC-3细胞系中未检测到RUNX3表达。300 nmol/L 5-Aza-dc处理72小时后，ACC-2和ACC-3细胞中RUNX3表达增强，ACC-M细胞中RUNX3表达恢复。LSCM结果显示，RUNX3蛋白主要位于ACC细胞系的细胞质中。300 nmol/L 5-Aza-dc处理72小时后，ACC-2和ACC-3细胞中RUNX3阳性信号在细胞核及细胞质中均有分布，但ACC-M细胞中仅在细胞质中检测到微弱阳性信号。三个细胞系中均发现RUNX3基因启动子5'-CpG岛存在部分甲基化，ACC-2、ACC-M及ACC-3中CpG岛甲基化程度分别为50%、75%及33%。5-Aza-dc处理后，三个细胞系中RUNX3基因均呈去甲基化状态。
RUNX3基因甲基化在涎腺腺样囊性癌细胞系RUNX3表达沉默中起重要作用。RUNX3蛋白的细胞质异位定位可能与其在ACC细胞中的功能抑制相关。
