CAJ | 학술논문

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,TIP30 mRNA和蛋白表达明显增强.随着5-Aza-dC去除时间的延长,TIP30 mRNA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4.96 μg/ml.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.
목적 탐토5-담잡포감(5-Aza-dC)대결직장암세포중TIP30기인표체적영향,병탐토기여불뇨밀정(5-Fu)민감성적관계.방법 5-Aza-dC처리결직장암HCT116세포,채용갑기화특이성PCR (MSP)방법검측TIP30기인계동자CpG도갑기화상태,역전록PCR검측TIP30 mRNA표체,Western blot법검측TIP30단백적표체,사갑기우담서람(MTT)법검측HCT116세포대5-Fu적민감성.결과 미경5-Aza-dC처리적HCT116세포중,TIP30기인계동자완전갑기화.5-Aza-dC처리HCT116세포3d후거제5-Aza-dC,HCT116세포TIP30기인비갑기화산물양성,계동자거갑기화.수착5-Aza-dC거제시간적연장,HCT116세포중TIP30기인계동자갑기화산물화비갑기화산물균위양성,즉계동자부분갑기화,부분비갑기화；거제5-Aza-dC제10천,TIP30기인계동자갑기화산물양성,TIP30기인계동자중신갑기화.미경5-Aza-dC처리적결직장암HCT116세포TIP30 mRNA화단백불표체.5-Aza-dC처리결직장암HCT116세포3d후거제5-Aza-dC,TIP30 mRNA화단백표체명현증강.수착5-Aza-dC거제시간적연장,TIP30 mRNA화단백표체수평축점강저,제10천체도최저수평.재5-Aza-dC처리전、5-Aza-dC처리후거제5-Aza-dC적제0、10천,5-Fu작용우HCT116세포적반수억제농도(IC50)분별위41.62、33.17화4.96 μg/ml.결론 TIP30기인표체차이가능여기계동자갑기화상태유관,차TIP30기인계동자갑기화차이여결직장암세포대화료약물적민감성상관.
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 cells.Methods The methylation profile of TIP30 gene in HCT116 colorectal cancer cells 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,d0 and d10 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 may affect the sensitivity of colorectal cancer cells to 5-Fu.