Han Xu, Tan Zhi-Jun, Guo Ren-de, Li Zhao-Jin, Wang Yu-Liang
Department of General Surgery, Tianjin First Central Hospital, Tianjin, China.
Zhonghua Zhong Liu Za Zhi. 2013 Jan;35(1):17-21. doi: 10.3760/cma.j.issn.0253-3766.2013.01.004.
To investigate the effect of demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human pancreatic cancer cell line MiaPaca2 and the expression and methylation of tumor suppressor gene RUNX3.
Human pancreatic cancer cell line MiaPaca2 cells were treated with different concentrations of 5-Aza-CdR. Morphological changes of MiaPaca2 cells were observed by light microscopy. The activity of cell proliferation was analyzed by MTT assay. The changes of RUNX3 mRNA expression were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Changes of RUNX3 gene methylation was detected by methylation-specific polymerase chain reaction.
MiaPaca2 cells were treated with 2.5, 5, 10 and 20 µmo1/L 5-Aza-CdR, respectively. The inhibition rates of MiaPaca2 cells treated for 24 h were (9.17 ± 2.15)%, (10.75 ± 2.04)%, (12.57 ± 1.64)% and (18.70 ± 1.51)%, respectively. The inhibition rates were (14.94 ± 1.68)%, (18.60 ± 1.57)%, (22.84 ± 1.58)% and (33.24 ± 1.53)%, respectively, after 48 h treatment; (21.46 ± 1.60)%, (28.62 ± 1.72)%, (35.14 ± 1.64)% and (45.06 ± 1.47)%, respectively, after 72 h treatment; and (26.35 ± 1.71)%, (34.48 ± 1.69)%, (40.05 ± 1.60)% and (49.99 ± 1.61)%, respectively, after 96 h treatment. The differences between inhibition rates of each experimental and control groups (0.00 ± 0.00)% were statistically significant (P < 0.05). At the same time, the inhibition rates of different concentration groups showed significant differences (P < 0.05). At 48 h, 72 h and 96 h, the inhibition rates of each pair concentration groups showed significant differences (P < 0.05). 5-Aza- CdR inhibited the growth of MiaPaca2 cells, and the higher the concentration, the stronger the inhibition after 24 h. 5-Aza-CdR also reversed the methylation status of RUNX3 gene, and restored the expression of RUNX3 mRNA with a dose-effect relationship.
The methylation of RUNX3 gene is significantly related with the occurrence and development of pancreatic cancer, and abnormal methylation of RUNX3 gene may contribute to the loss of RUNX3 mRNA expression. 5-Aza-CdR may effectively cause reversion of RUNX3 methylation, and treatment with 5-Aza-CdR can reactivate the gene expression and inhibit the cell growth. This may provide a new way for diagnosis and treatment of pancreatic cancer.
探讨去甲基化药物5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对人胰腺癌细胞系MiaPaca2生长及抑癌基因RUNX3表达和甲基化的影响。
用不同浓度的5-Aza-CdR处理人胰腺癌细胞系MiaPaca2细胞。通过光学显微镜观察MiaPaca2细胞的形态变化。采用MTT法分析细胞增殖活性。用半定量逆转录-聚合酶链反应（RT-PCR）检测RUNX3 mRNA表达的变化。用甲基化特异性聚合酶链反应检测RUNX3基因甲基化的变化。
分别用2.5、5、10和20 μmol/L的5-Aza-CdR处理MiaPaca2细胞。处理24 h后，MiaPaca2细胞的抑制率分别为(9.17 ± 2.15)%、(10.75 ± 2.04)%、(12.57 ± 1.64)%和(18.70 ± 1.51)%。处理48 h后，抑制率分别为(14.94 ± 1.68)%、(18.60 ± 1.57)%、(22.84 ± 1.58)%和(33.24 ± 1.53)%；处理72 h后，分别为(21.46 ± 1.60)%、(28.62 ± 1.72)%、(35.14 ± 1.64)%和(45.06 ± 1.47)%；处理96 h后，分别为(26.35 ± 1.71)%、(34.48 ± 1.69)%、(40.05 ± 1.60)%和(49.99 ± 1.61)%。各实验组与对照组（0.00 ± 0.00）%的抑制率差异有统计学意义（P < 0.05）。同时，不同浓度组的抑制率差异有统计学意义（P < 0.05）。在48 h、72 h和96 h时，各配对浓度组的抑制率差异有统计学意义（P < 0.05）。5-Aza-CdR抑制MiaPaca2细胞的生长，24 h后浓度越高抑制作用越强。5-Aza-CdR还逆转了RUNX3基因的甲基化状态，并恢复了RUNX3 mRNA的表达，呈剂量-效应关系。
RUNX3基因甲基化与胰腺癌的发生发展密切相关，RUNX3基因的异常甲基化可能导致RUNX3 mRNA表达缺失。5-Aza-CdR可能有效引起RUNX3甲基化的逆转，用5-Aza-CdR治疗可重新激活基因表达并抑制细胞生长。这可能为胰腺癌的诊断和治疗提供一种新方法。
