5-氮杂胞苷调控TIP30基因表达对人结直肠癌细胞氟尿嘧啶敏感性的影响  

Effects of 5-Aza-dC on 5-Fu chemosensitivity by modulating TIP30 gene expression in human colorectal cancer cells

作　　者：陈小兵[1] 陈贝贝[1] 李剑[2] 王新锋[1] 马一杰[1] 罗素霞[1] 吕慧芳[1] 

机构地区：[1]河南省肿瘤医院内科,郑州450008 [2]河南省肿瘤医院普外科,郑州450008

出　　处：《中华肿瘤杂志》2013年第11期824-827,共4页Chinese Journal of Oncology

基　　金：基会项目：河南省医学科技重大攻关项目（2011010012）

摘　　要：【摘要】目的探讨5-氮杂胞苷（5-Aza—dC）对结直肠癌细胞中TIP30基因表达的影响，并探讨其与氟尿嘧啶（5-Fu）敏感性的关系。方法5-Aza—dC处理结直肠癌HCT116细胞，采用甲基化特异性PCR（MSP）方法检测TIP30基因启动子CpG岛甲基化状态，逆转录PCR检测TIP30 mRNA表达，Western blot法检测TIP30蛋白的表达，四甲基偶氮唑蓝（MTT）法检测HCT116细胞对5-Fu的敏感性。结果未经5-Aza—dC处理的HCT116细胞中，TIP30基因启动子完全甲基化。5-Aza—dC处理HCT116细胞3d后去除5-Aza—dC，HCT116细胞TIP30基因非甲基化产物阳性，启动子去甲基化。随着5-Aza—dC去除时间的延长，HCT116细胞中TIP30基因启动子甲基化产物和非甲基化产物均为阳性，即启动子部分甲基化，部分非甲基化；去除5-Aza-dC第10天，TIP30基因启动子甲基化产物阳性，TIP30基因启动子重新甲基化。未经5-Aza—dC处理的结直肠癌HCT116细胞TIP30 mRNA和蛋白不表达。5-Aza—dC处理结直肠癌HCT116细胞3d后去除5-Aza—dC，TIP30mRNA和蛋白表达明显增强。随着5-Aza-dC去除时间的延长，TIP30mRNA和蛋白表达水平逐渐降低，第10天达到最低水平。在5-Aza-dC处理前、5-Aza-dC处理后去除5-Aza-dC的第0、10天，5-Fu作用于HCT116细胞的半数抑制浓度（IC50）分别为41．62、33．17和4．96μg／m1。结论TIP30基因表达差异可能与其启动子甲基化状态有关，且TIP30基因启动子甲基化差异与结直肠癌细胞对化疗药物的敏感性相关。Objective To investigate the effect of 5-Aza-2＇-deoxycytidine （5-Aza-dC） on TIP30 gene expression and the relationship between TIP30 expression and the sensitivity to 5-fluouracil （5-Fu） in colorectal cancer ceils. Methods The methylation profile of TIP30 gene in HCT116 colorectal cancer ceils was determined by methylation-specific PCR. The levels of TIP30 mRNA and protein were determined by RT-PCR and Western blot after the 5-Aza-dC treatment. MTT assay was used to detect the chemosensitivity of HCT116 cells to 5-Fu. Results TIP30 gene displayed complete DNA methylation in the HCT116 cells without 5-Aza-dC pretreatment. After the 5-Aza-dC treatment for 3 days, only demethylating PCR amplification product was detected and TIP30 gene showed DNA demethylation. With the prolongation of the time of removal of 5-Aza-dC treatment, methylated and demethylated PCR amplification products were observed and TIP30 gene displayed both DNA methylation and DNA demethylation in the colorectal cancer cells. At the day 10 after removal of 5-Aza-dC, methylating PCR amplification product appeared and TIP30 gene showed DNA methylation. No expressions of TIP30 mRNA and protein were detected in the HCT116 cells untreated with 5-Aza-dC. After the treatment of 5-Aza-dC for 3 d and then removed the 5-Aza-dC, the expressions of TIP30 mRNA and protein were increased obviously. With the prolonged time after 5-Aza-dC removal, the expressions of TIP30 mRNA and protein decreased and reached the lowest level on day 10. The IC50 values of 5-Fu were 41.62, 33.17 and 4.96 μg/ml in the HCT116 cells pretreated with 5-Aza-dC, dO and dl0 with the drug removal after drug treatment for 3 d, respectively. Conclusions The results of this study show that the expression of TIP30 gene may be associated with its DNA methylation status and mayaffect the sensitivity of colorectal cancer cells to 5-Fu.

关 键 词：结直肠肿瘤 5-氮杂胞苷 DNA甲基化 氟尿嘧啶 敏感性 

分 类 号：R735.3[医药卫生—肿瘤]