CAJ | 학술논문

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.
목적 탐토인연선선양낭성암세포주중RUNX3기인계동자5'-CpG도갑기화급기표체정황.방법 이정량갑기화특이성PCR(qMSP)검측ACC-2、ACC-3화ACC-M세포재갑기전이매억제제5-담탈양포감(5-Aza-dc)처리전후RUNX3기인5'-CpG도갑기화상황,병분별용반전록-취합매련반응、면역형광화학화Western인적법분석RUNX3적표체변화.결과 RUNX3재ACC-2화ACC-3세포중약표체,재ACC-M중표체결실.경5-Aza-dc처리후,RUNX3재ACC-2화ACC-3세포중표체증가,재ACC-M중회복표체.격광공취초발현RUNX3단백주요정위재세포질중,경300 nmol/L5-Aza-dc처리72 h후,재ACC-2화ACC-3세포핵중출현표체,이재ACC-M세포중잉정위재세포질중.RUNX3기인계동자구역5'-CpG도재ACC-2、ACC-M화ACC-3중균위부분갑기화,기갑기화정도분별위50%、75%화33%.5-Aza-dc처리후,RUNX3기인재삼주세포주중균정현위비갑기화상태.결론 RUNX3갑기화시ACCs중RUNX3표체결실적주요원인지일,이RUNX3단백재ACCs세포질중적이위표체,야가능여RUNX3단백적공능피억제유관.
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.