Song Deye, Ni Jiangdong, Xie Hongming, Ding Muliang, Wang Jun
Department of Orthopaedics, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, P.R. China.
Exp Ther Med. 2014 May;7(5):1071-1076. doi: 10.3892/etm.2014.1571. Epub 2014 Feb 21.
This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into , then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the expression levels of the tumor suppressor gene PTEN in the MG-63 osteosarcoma cell line . The expression levels of mRNA and the cellular apoptotic rate were also increased. The elevated activation and expression levels of the PTEN gene may be associated with the low methylation levels of the CG site that binds to the transcription factors Sp1 and Myc in the PTEN gene promoter, and they promote the combination of the transcription factors and the gene promoter.
本研究采用MG-63骨肉瘤细胞系，探讨甲基化抑制剂5-氮杂胞苷（5-Zac）引起的磷酸酶和张力蛋白同源物（PTEN）基因启动子去甲基化及PTEN基因表达水平的变化，以及二者之间的关联。将不同浓度的5-Zac（0、5和10μmol/L）加入MG-63细胞培养基中，细胞培养72小时。对细胞进行以下操作：蛋白质免疫印迹分析检测PTEN蛋白；逆转录-聚合酶链反应（PCR）检测PTEN基因的mRNA转录水平；流式细胞术检测细胞凋亡率；亚硫酸氢盐处理每组细胞的DNA。对PTEN启动子基因以及转录因子特异性蛋白1（Sp1）和Myc进行PCR扩增并转化，然后挑选多个克隆进行测序，检测扩增的PTEN启动子片段的甲基化状态。用浓度为0、5和10μmol/L的5-Zac培养MG-63细胞72小时后，每组PTEN蛋白表达水平逐渐升高，呈浓度依赖性：0μmol/L组与5和10μmol/L组相比，P<0.05；5μmol/L组与10μmol/L组相比，P=0.007。PTEN基因的mRNA表达水平显著升高。0、5和10μmol/L组的凋亡率分别为0.69±0.42%、2.50±0.30%和6.59±0.62%，两两之间差异有统计学意义（P<0.01）。三组亚硫酸氢盐DNA测序结果显示，5-Zac处理后，CG位点与转录因子的结合受去甲基化影响。三组的平均去甲基化率有统计学差异。综上所述，甲基化抑制剂5-Zac导致MG-63骨肉瘤细胞系中抑癌基因PTEN的表达水平显著升高。mRNA表达水平和细胞凋亡率也升高。PTEN基因激活和表达水平升高可能与PTEN基因启动子中与转录因子Sp1和Myc结合的CG位点低甲基化水平有关，且促进了转录因子与基因启动子的结合。
