目的 研究人肾癌ACHN细胞中胰岛素样生长因子2( IGF2)基因的印迹状态及甲基化诱导药物TCF1001对IGF2启动子hP4区域甲基化状态的影响,并进一步探讨靶向诱导IGF2启动子hP4甲基化对ACHN细胞IGF2印迹状态的影响.方法 采用聚合酶链反应-限制性内切酶切片段长度多态性分析(PCR-RFLP)方法鉴定ACHN细胞IGF2基因组杂合状态,并对其印迹状态进行分析,判断IGF2基因是否处于基因印迹丢失(LOI)状态.实验组用甲基化诱导药物TCF1001,对照组用CT010,空白组用PBS处理ACHN细胞,提取基因组DNA后,采用亚硫酸氢盐测序法研究其对ACHN细胞IGF2启动子hP4区域甲基化水平的影响,同时采用PCR-RFLP法研究其对IGF2基因印迹状态的影响.结果 人肾癌ACHN细胞IGF2基因处于LOI.TCF1001可有效地靶向诱导IGF2启动子hP4区域发生部分甲基化. IGF2启动子hP4甲基化可逆转IGF2基因的LOI.结论 TCF1001可靶向诱导ACHN细胞IGF2基因启动子hP4甲基化, hP4甲基化可逆转人肾癌细胞IGF2基因的LOI,为肾癌的基因治疗提供理论依据.
Objective To investigate the imprinting status of insulin-like growth factor 2 (IGF2) in renal cell carcinoma ACHN cell line, as well as the effect of methylated drug TCF1001 on methylation status of IGF2 hP4 promoter. Furthermore, the relationship between hP4 promoter methyl-ation and imprinting status change of IGF2 gene was also examined. Methods The heterozygote status of IGF2 gene was detected by polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP). The imprinting status of IGF2 was evaluated by reverse transcription - polymerase chain reaction (RT-PCR) and PCR-RFLP. ACHN cells were then treated with either the methylated drug TCF1001 or control drugs (CT010 or PBS). Bisulfite sequencing was used to study the methyla-tion status of IGF2 hP4 promoter region. Meanwhile, PCR-RFLP was for analyzing the variation of IGF2 imprinting status. Results It was found that IGF2 of ACHN cells was in the status of loss of im-printing (LOI). TCF1001 could effectively induce methylation of IGF2 hP4 promoter. Furthermore, the LOI of IGF2 could be reversed when IGF2 hP4 promoter was partially methylated. Conclusions IGF2 hP4 promoter can be targeted methylated by TCF1001. IGF2 hP4 promoter targeted methylation can partially reverse the LOI of IGF2, which provides the theoretical basis of renal cell carcinoma ge-netic therapy.