人涎腺腺样囊性癌细胞系中RUNX3基因5＇-CpG岛甲基化及蛋白表达

RUNX3 expression and its methylation of 5＇-CpG island in salivary gland adenoid cystic carcinoma cell lines ACC-2, ACC-3 and ACC-M

目的 探讨人涎腺腺样囊性癌细胞株中RUNX3基因启动子5＇-CpG岛甲基化及其表达情况.方法 以定量甲基化特异性PCR（qMSP）检测ACC-2、ACC-3和ACC-M细胞在甲基转移酶抑制剂5-氮脱氧胞苷（5-Aza-dc）处理前后RUNX3基因5＇-CpG岛甲基化状况,并分别用反转录-聚合酶链反应、免疫荧光化学和Western印迹法分析RUNX3的表达变化.结果 RUNX3在ACC-2和ACC-3细胞中弱表达,在ACC-M中表达缺失.经5-Aza-dc处理后,RUNX3在ACC-2和ACC-3细胞中表达增加,在ACC-M中恢复表达.激光共聚焦发现RUNX3蛋白主要定位在细胞质中,经300 nmol/L5-Aza-dc处理72 h后,在ACC-2和ACC-3细胞核中出现表达,而在ACC-M细胞中仍定位在细胞质中.RUNX3基因启动子区域5＇-CpG岛在ACC-2、ACC-M和ACC-3中均为部分甲基化,其甲基化程度分别为50%、75%和33%.5-Aza-dc处理后,RUNX3基因在三株细胞株中均呈现为非甲基化状态.结论 RUNX3甲基化是ACCs中RUNX3表达缺失的主要原因之一,而RUNX3蛋白在ACCs细胞质中的异位表达,也可能与RUNX3蛋白的功能被抑制有关.

Objective To investigate the methylation of 5＇-CpG island and expression of RUNX3 in salivary gland adnoid 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 adenoic cystic carcinoma cell lines, ACC-2, ACC3, 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 of5-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.