Galamb Orsolya, Kalmár Alexandra, Barták Barbara Kinga, Patai Árpád V, Leiszter Katalin, Péterfia Bálint, Wichmann Barnabás, Valcz Gábor, Veres Gábor, Tulassay Zsolt, Molnár Béla
Orsolya Galamb, Barnabás Wichmann, Gábor Valcz, Zsolt Tulassay, Béla Molnár, Molecular Medicine Research Group, Hungarian Academy of Sciences, H-1088 Budapest, Hungary.
World J Gastroenterol. 2016 Dec 21;22(47):10325-10340. doi: 10.3748/wjg.v22.i47.10325.
To analyze colorectal carcinogenesis and age-related DNA methylation alterations of gene sequences associated with epigenetic clock CpG sites.
DNA methylation analysis of 353 epigenetic clock CpG sites published by Steve Horvath was performed using methylation array data for a set of 123 colonic tissue samples [64 colorectal cancer (CRC), 42 adenoma, 17 normal; GEO accession number: GSE48684]. Among the differentially methylated age-related genes, secreted frizzled related protein 1 () promoter methylation was further investigated in colonic tissue from 8 healthy adults, 19 normal children, 20 adenoma and 8 CRC patients using bisulfite-specific PCR followed by methylation-specific high resolution melting (MS-HRM) analysis. mRNA expression of age-related "epigenetic clock" genes was studied using Affymetrix HGU133 Plus2.0 whole transcriptome data of 153 colonic biopsy samples (49 healthy adult, 49 adenoma, 49 CRC, 6 healthy children) (GEO accession numbers: GSE37364, GSE10714, GSE4183, GSE37267). Whole promoter methylation analysis of genes showing inverse DNA methylation-gene expression data was performed on 30 colonic samples using methyl capture sequencing.
Fifty-seven age-related CpG sites including hypermethylated , , and hypomethylated MGP, PIPOX were differentially methylated between CRC and normal tissues ( < 0.05, Δβ ≥ 10%). In the adenoma normal comparison, 70 CpG sites differed significantly, including hypermethylated , , , and hypomethylated , ( < 0.05, Δβ ≥ 10%). In MS-HRM analysis, the SFRP1 promoter region was significantly hypermethylated in CRC (55.0% ± 8.4 %) and adenoma tissue samples (49.9% ± 18.1%) compared to normal adult (5.2% ± 2.7%) and young (2.2% ± 0.7%) colonic tissue ( < 0.0001). DNA methylation of promoter was slightly, but significantly increased in healthy adults compared to normal young samples ( < 0.02). This correlated with significantly increased mRNA levels in children compared to normal adult samples ( < 0.05). In CRC tissue the mRNA expression of 117 age-related genes were changed, while in adenoma samples 102 genes showed differential expression compared with normal colonic tissue ( < 0.05, logFC > 0.5). The change of expression for several genes including , , and , followed the same pattern in aging and carcinogenesis, though not for all genes (., MGP).
Several age-related DNA methylation alterations can be observed during CRC development and progression affecting the mRNA expression of certain CRC- and adenoma-related key control genes.
分析与表观遗传时钟CpG位点相关的基因序列的结直肠癌发生及年龄相关的DNA甲基化改变。
使用一组123个结肠组织样本（64个结直肠癌（CRC）、42个腺瘤、17个正常样本；GEO登录号：GSE48684）的甲基化芯片数据，对Steve Horvath公布的353个表观遗传时钟CpG位点进行DNA甲基化分析。在差异甲基化的年龄相关基因中，使用亚硫酸氢盐特异性PCR随后进行甲基化特异性高分辨率熔解（MS-HRM）分析，进一步研究8名健康成年人、19名正常儿童、20个腺瘤和8名CRC患者的结肠组织中分泌型卷曲相关蛋白1（SFRP1）启动子甲基化情况。使用153个结肠活检样本（49名健康成年人、49个腺瘤、49个CRC、6名健康儿童）的Affymetrix HGU133 Plus2.0全转录组数据（GEO登录号：GSE37364、GSE10714、GSE4183、GSE37267）研究年龄相关的“表观遗传时钟”基因的mRNA表达。使用甲基捕获测序对30个结肠样本进行显示DNA甲基化与基因表达数据呈负相关的基因的全启动子甲基化分析。
57个年龄相关的CpG位点在CRC和正常组织之间存在差异甲基化，包括高甲基化的 、 、 以及低甲基化的MGP、PIPOX（P<0.05，Δβ≥10%）。在腺瘤与正常组织比较中，70个CpG位点有显著差异，包括高甲基化的 、 、 、 以及低甲基化的 、 （P<0.05，Δβ≥10%）。在MS-HRM分析中，与正常成人（5.2%±2.7%）和年轻（2.2%±0.7%）结肠组织相比，SFRP1启动子区域在CRC（55.0%±8.4%）和腺瘤组织样本（49.9%±18.1%）中显著高甲基化（P<0.0001）。与正常年轻样本相比，健康成年人中 启动子的DNA甲基化略有但显著增加（P<0.02）。这与儿童中 mRNA水平相比正常成人样本显著增加相关（P<0.05）。在CRC组织中，117个年龄相关基因的mRNA表达发生改变，而在腺瘤样本中，与正常结肠组织相比，102个基因显示出差异表达（P<0.05，logFC>0.5）。包括 、 、 和 等几个基因的表达变化在衰老和癌变过程中遵循相同模式，尽管并非所有基因（如MGP）都是如此。
在CRC发生发展过程中可观察到一些年龄相关的DNA甲基化改变，影响某些CRC和腺瘤相关关键调控基因的mRNA表达。
