应用甲基化基因检测技术分析前列腺癌中HIC1的甲基化状态

Detection of HIC1 promoter methylation in prostate cancer using MSP and BSP methods

背景与目的:癌高甲基化基因1(hypermethylated in cancer 1,HIC1)因表观遗传甲基化修饰导致其在多种癌细胞和组织中表达沉默,可能与肿瘤的发生、发展有关。而这一现象在前列腺癌研究中的报道甚少。该研究采用甲基化基因检测技术对前列腺癌中HIC1启动子进行甲基化状态分析。方法:采用甲基化特异性聚合酶链式反应(methylation-specific polymerase chain reaction,MSP)和亚硫酸盐测序PCR(bisulfate sequencing PCR,BSP)技术,对前列腺癌细胞PC3、C4-2B和正常前列腺上皮细胞Pr EC,以及前列腺癌组织标本和正常前列腺穿刺标本,进行HIC1启动子甲基化分析;并使用去DNA甲基化酶5-Aza-Cd R处理PC3、C4-2B和Pr EC,经反转录PCR(reverse transcription-PCR,RT-PCR)和蛋白[质]印迹法(Western blot)分析HIC1在基因水平和蛋白质水平上的变化。结果:MSP分析结果发现,在PC3、C4-2B和Pr EC中HIC1甲基化率分别为78.23%、72.15%和10.63%,差异有统计学意义(P<0.05)。对36例人前列腺癌实体瘤和36例人前列腺正常穿刺组织进行BSP测序,统计显示,甲基化率分别是80.30%和31.56%。5-Aza-Cd R处理后的PC3、C4-2B和Pr EC经RT-PCR和Western blot分析发现,与未处理对照相比,HIC1转录水平和蛋白水平表达均显著上调。结论:在前列腺癌中,抑癌基因HIC1启动子呈高度甲基化状态并出现表达沉默或下调,可作为前列腺癌的早期预测指标和潜在的治疗靶点。

Background and purpose： Hypermethylated in cancer 1 （HIC1） is silenced in multiple cancer cells and tissues by DNA methylation of epigenetic modification, which may modulate the initiation and progression of tumors. However, there are few reports about this phenomenon in prostate cancer. This study aimed to investigate the status of HIC1 promoter methylation in prostate cancer using methylation methods. Methods： Methylation-specific polymerase chain reaction （MSP） and bisulfate sequencing PCR （BSP） were used to detect the methylation status of HIC1 promoter in prostate cancer cell lines PC3 and C4-2B, prostate normal cell line PrEC, primary Chinese PCa tissues and the respective healthy control cases. HIC1 expression level was respectively determined by reverse transeription-PCR （RT-PCR） and Western blot assays in PC3, C4-2B and PrEC cells treated with 5-Aza-CdR. Results： We found that the percentages of HIC1 promoter methylation were 78.23%, 72.15% and 10.63% in PC3, C4-2B and PrEC cells by MSP analyses. Moreover, the levels of methylated HIC1 promoter in 36 primary Chinese PCa tissues compared with the respective healthy control cases were 80.30% vs 31.56%. Expressions of HIC1 mRNA and protein level were restored in PC3 and C4-2B cells after 5-Aza-CdR treatment. Conclusion： These findings demonstrate that HIC1 promoter region is hypermethylated in prostate cancer, which results in silence or downregulation of HIC1. The status ofHIC1 methylation can be a valuable marker in the early stage of prostate cancer and a potential therapeutic target.