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基于Methyl-Rad技术对结肠癌全基因组DNA甲基化图谱的研究

Genome-wide DNA methylation profile of colon cancer based on Methyl-RAD

摘要:

目的:分析结肠癌全基因组DNA差异甲基化位点(DMS)及差异甲基化基因(DMG)水平,寻找结肠癌相关甲基化基因。方法:在山东大学齐鲁医院结肠癌组织标本库中,随机数表法抽取5份结肠癌组织和配对癌旁组织,采用甲基化修饰依赖性内切酶测序法(Methyl-Rad)进行全基因组DNA甲基化测序,Edge R方法筛选DMS,并比较基因组不同功能元件上DMS的分布,对DMS绘制组间甲基化水平差异热图;进一步筛选DMG,对DMG进行Gene Ontology (GO)和Kyoto Encyclopedia of Genes and Genomes (KEGG)功能富集分析。同时,下载肿瘤基因组图谱(TCGA)数据库RNA测序数据分析差异表达RNA,将DMG和差异表达RNA共分析获得潜在的结肠癌相关甲基化基因,并采用实时荧光定量聚合酶链反应(RT-qPCR)和焦磷酸测序方法在30对结肠癌和癌旁组织中进行结肠癌相关甲基化基因的双向验证。基因的表达量和甲基化水平在结肠癌肿瘤组织和癌旁组织的差异采用配对样本的秩和检验进行统计学分析。结果:共筛选出132 999个 CCGG和8 487个CCWGG DMS,其中CCGG和CCWGG的DMS主要分布在内含子区,比例分别为50.47%(67 127/132 999)和48.79%(4 141/8 487)。共发现1 359个CCGG和1 052个CCWGG DMG,对DMG进行GO和KEGG功能富集分析。DMG和差异表达RNA共分析发现113个结肠癌相关基因,其中14个基因在结肠癌组织中呈高甲基化且低表达,99个基因呈低甲基化且高表达。RT-qPCR和焦磷酸测序实验证实MAFA-AS1和MS4A18在结肠癌组织中呈高甲基化且低表达,而SMAD1-AS2、ARHGEF3-AS1和LOC440084则呈低甲基化且高表达,与测序结果一致( P<0.05)。 结论:DNA甲基化为结肠癌提供了潜在的生物标志物,并为结肠癌发生发展的分子机制提供了新的思路。

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abstracts:

Objective:To analyze the differentially methylated sites (DMS) and differentially methylated genes (DMG) in the whole genome of colon cancer and identify colon cancer related DMG.Methods:Five pairs of colon cancer tissues and adjacent non-tumor tissues were selected by using the random number table from tissue sample bank of Qilu Hospital of Shandong University. Whole genome wide DNA methylation profiles were constructed using the Methyl-Rad technique. Edge R was used to screen DMS and the distributions of DMS on different elements were compared. The DMS were used to plot heat maps to distinguish tumor and non-tumor groups. Furthermore, the DMG were identified and submitted to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) function enrichment analysis. The RNA sequencing data for colon cancer was downloaded from The Cancer Genome Atlas(TCGA) database. The dysregulated expression RNA was co-analyzed with the DMGs to obtain colon cancer related genes. The RNA and methylation level of the top 5 colon cancer-related genes in the 30 pairs of colon cancer tumor tissues and adjacent non-tumor tissues were confirmed by real time quantitative PCR (RT-qPCR) and pyrosequencing assays.Results:A total of 132 999 CCGG and 8 487 CCWGG sites were identified as DMS. DMS were mainly located in the intron elements, accounting for 50.47% (67 127/132 999) and 48.79% (4 141/8 487) in CCGG and CCWGG DMS respectively. In addition, 1 359 CCGG and 1 052 CCWGG DMG were screened, and GO and KEGG pathway enrichment analysis were performed. Meanwhile, the RNA expression data of the 1 020 DMG which could be mapped in Ensemble v70 was downloaded and analyzed from TCGA database. After intersection analysis, 14 genes were found to be hypermethylated and down-regulated, and the remaining 99 genes were hypomethylated and upregulated. Among these genes, MAFA-AS1and MS4A18 were found to be hypermethylated and downregulated in colon cancer tumor tissues, while SMAD1-AS2, ARHGEF3-AS1 and LOC440084 were hypomethylated and upregulated, which confirmed our sequencing results ( P<0.05). Conclusion:The genome-wide DNA methylation analysis provides potential biomarkers, therapeutics targets and possible molecular mechanism for colon cancer.

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作者: 高玉 [1] 李杰 [2] 梁永媛 [2] 郑桂喜 [2]
作者单位: 青岛市市北区妇幼保健计划生育服务中心,青岛266032 [1] 山东大学齐鲁医院检验科,济南250012 [2]
期刊: 《中华检验医学杂志》2021年44卷2期 142-149页 ISTICPKUCSCDCA
栏目名称: 论著
DOI: 10.3760/cma.j.cn114452-20200629-00582
发布时间: 2021-04-06
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