SALL3甲基化与宫颈癌的相关性

目的检测正常宫颈组织、宫颈癌组织及宫颈癌细胞系中SALL3基因启动子区的甲基化状态,探讨其甲基化状态与SALL3基因表达的关系。方法采用甲基化特异性PCR(MS-PCR)分析正常宫颈、宫颈癌组织及宫颈癌细胞系中SALL3基因启动子区DNA的甲基化状态,RT-PCR检测宫颈癌细胞系、宫颈癌组织及正常宫颈组织中SALL3基因mRNA表达。结果宫颈癌和癌旁组织中的SALL3基因启动子区甲基化水平分别为0.60(IQR:0.73,秩均值:16.70)(P=0.046)和0.82(IQR:0.50,秩均值:17.15)(P=0.039),均显著高于正常宫颈组织的0.03(IQR:0.39,秩均值:8.44)。宫颈癌及癌旁组织中SALL3蛋白的表达分别为0.10(IQR:0.17,秩均值:8.40)(P=0.012)和0.26(IQR:0.21,秩均值:15.6)(P=0.000),均显著低于正常宫颈组织的0.57(IQR:0.73,秩均值:20.75)。给予0、5、10μmol/L5-Azacytidine去甲基化处理HeLa及SiHa细胞后,HeLa(F=28.704,P=0.001)和SiHa细胞(F=29.783,P=0.002)中SALL3mRNA的表达水平均随5-Azacytidine作用浓度的升高而升高。与高危型人乳头瘤病毒(HPV)阳性组织相比,HPV阴性宫颈组织中SALL3基因启动子区的甲基化水平明显降低(P=0.014),蛋白表达水平明显升高(P=0.021)。结论宫颈癌组织中SALL3基因启动子区的DNA高甲基化使其表达沉默,高危型HPV感染可能通过增加SALL3启动子区甲基化使其沉默促进宫颈癌的发生。

肝癌中Sall3基因甲基化的研究

目的:检测Sall3基因在肝癌细胞系和肝癌组织中的甲基化状况及其表达水平。方法:对7株肝癌细胞系、142对肝癌组织和6例正常肝组织进行Sall3基因启动子区域甲基化检测。甲基化研究使用甲基化特异性聚合酶链反应(methylation specific polymerase chain reaction,MSP),基因表达水平研究采用实时定量反转录PCR(real-time RT-PCR)。结果:MSP结果显示,7株肝癌细胞株中有6株Sall3基因的启动子区域为纯合甲基化,1株为纯合去甲基化;在6例正常肝组织中Sall3均为纯合去甲基化;在142对肝癌组织标本中,癌旁组织Sall3基因启动子区域甲基化率为18.31%,在癌组织中为64.79%。real-time RT-PCR结果显示,肝癌肿瘤细胞系Sall3mRNA的表达水平明显低于去甲基化的正常肝组织。结论:Sall3基因启动子甲基化在正常肝组织、肝癌组织(和肝癌细胞系)中有明显差异。

Aberrant methylation and downregulation of sall3 in human hepatocellular carcinoma

AIM: To investigated whether sall3 transcription was regulated by promoter CpG island hypermethylation in hepatocellular carcinoma (HCC). METHODS: The cell lines Huh7, HepG2, SK-HEP1, SM-MC7721, Bel7402, QGY7703 and a cohort of 38 HCC tissue specimens and corresponding nontumorous tissues were subjected to analysis for sall3 promoter CpG island methylation and mRNA transcription. sall3 promoter CpG island methylation levels were determined using the MassARRAY platform and mRNA transcription levels of the gene were detected by quantitative realtime polymerase chain reaction.RESULTS: The levels of sall3 mRNA were decreased by more than twofold in 33 of 38 tumor tissues compared to adjacent noncancerous tissues. Among these 33 tumor tissues with lower levels of sall3 mRNA, 24 showed higher levels of methylation. Based on these results, we hypothesized that the decrease in sall3 mRNA transcription level was likely due to promoter CpG island hypermethylation. Changes in sall3 mRNA transcription and promoter CpG island methylation were determined in the above six cell lines after treatment with 0, 0.1, 0.5 and 2.5 mmol 5-aza-2-deoxycytidine, a demethylating agent. Promoter CpG island methylation levels de- creased in a dose-dependent manner in all six cell lines, while the mRNA transcription level increased dose-dependently in Huh7, HepG2, SK-HEP1 and SMMC7721 cells and irregularly in Bel7402 and QGY7703 cells. CONCLUSION: These results indicated that promoter CpG island hypermethylation contributes to the downregulation of sall3 mRNA transcription in HCC.

SALL3基因启动子区域异常高甲基化肝癌患者预后不良

目的研究SALL3启动子高甲基化与原发性肝细胞癌(HCC)患者临床病理特征的关系。方法应用甲基化特异性PCR(MSP)技术分析283对HCC及其相邻非肿瘤组织中SALL3的甲基化状态,评估SALL3启动子甲基化与临床特征的相关性。结果肝细胞癌中SALL3启动子区甲基化率(38.87%)高于相邻癌旁组织(9.89%),差异有统计学意义(χ~2=64.43,P<0.000 1)。单因素分析显示,不同年龄、性别、HBV病毒感染状况、肿瘤大小的肝癌患者SALL3基因甲基化率差异无统计学意义(P>0.05),但不同肝癌细胞分化程度(χ~2=3.046,P=0.018 9)、临床分期(χ~2=4.684,P=0.030 4)、门静脉瘤栓侵犯(χ~2=14.930,P=0.000 1)的肝癌患者SALL3基因甲基化率差异有统计学意义。单因素Cox回归分析显示SALL3基因甲基化(OR=1.73,95%CI:1.28~2.33,P=0.002 6)、肝硬化(OR=1.56,95%CI:1.11~2.18,P=0.009 6)、临床分期(OR=1.63,95%CI:1.36~1.94,P=0.000 0)是患者5年生存的影响因素。多因数Cox回归分析证实SALL3基因甲基化(OR=1.41,95%CI:1.02~1.94,P=0.035 7)、临床分期(OR=1.73,95%CI:1.39~2.01,P=0.000 0)是影响HCC患者术后生存期的独立危险因素。SALL3启动子甲基化的HCC患者术后生存时间明显短于非甲基化HCC患者[(20.37±3.5)月vs.(30.77±5.5)月,P=0.002 4)]。结论 SALL3基因启动子区甲基化状态是检测原发性肝细胞癌和预后不良的潜在生物标志物。