人肺癌组织细胞中HIN-1基因甲基化分析

目的:HIN-1(High in normal-1)是新近鉴定的一个抑癌基因,在乳腺癌细胞中,HIN-1启动子区的高甲基化导致其表达沉默。本研究拟探讨肺癌组织和6种肺癌细胞株中抑癌基因HIN-1启动子区内的甲基化修饰水平。方法:甲基化特异性PCR(MSP)和Biosulfite(亚硫酸氢钠)测序法鉴定样品中HIN-1启动子内CpG甲基化状态,RT-PCR法检测5-aza-dc处理前后HIN-1在细胞内的表达水平。结果:在60例肺癌样本中,HIN-1启动子甲基化阳性样本为35例(阳性率58.3%),而5例正常肺组织样品均为阴性反应。采用RT-PCR法分析了肺癌细胞株H157,H358,H1299,A549,H727,DMS53内HIN-1基因的表达水平,发现前4种细胞株内HIN-1表达水平极低,也是在这4种肺癌细胞中,HIN-1基因启动子呈高甲基化状态。用去甲基化试剂5-aza-dc处理上述4种细胞96h后,所有这4种细胞内均出现HIN-1基因的重新活化。结论:抑癌基因HIN-1启动子高甲基化导致的HIN-1表达抑制可能是肺癌发生发展的重要因素。

Analysis of HIN - 1 Methylation in Tissues and Cell Lines of Human Lung Cancer

Objective:HIN-1(High in normal-1) is a newly identified tumor suppressor gene and was found to be silenced by the high methylation of HIN-1 promoter in the breast cancer cells.In this study,methylation status of the HIN-1 promoter in lung cancer tissues and 6 lung cancer cell lines were assayed.Methods:Methylation specific PCR(MSP) and Biosulfite sequencing were used to identify methylation status of CpG in the HIN-1 promoter;RT-PCR was performed to determine expression levels of HIN-1 in lung cancer cell lines before and after 5-aza-dc treatment for 96 hours.Results:Among 60 cases of primary lung cancer samples,strong methylation was detected in 37 cases(58.3%) and no methylation,however,was found in 5-cases of normal lung tissues.HIN-1 expression levels in lung cancer line H157,H358,H1299,A549,H727 and DMS53 were assayed by RT-PCR technique and results indicated that expression of HIN-1 was silenced in the H157,H358,H1299,A549 lines and interestingly,it is also these cell lines that have a high HIN-1 methylation status.Re-expression of HIN-1 occurred after treating these cell lines by 5-aza-dc,a demethylation reagent,for 96 hours.Conclusions:Tumor suppressor HIN-1 silencing by promoter methylation may promote lung cancer carcinogenesis.