结肠癌发生中基因启动子和外显子 DNA 甲基化变化 - mRNA 表达和肿瘤突变改变。
Gene promoter and exon DNA methylation changes in colon cancer development - mRNA expression and tumor mutation alterations.
机构信息
Molecular Medicine Research Group, Hungarian Academy of Sciences, Szentkirályi str 46, Budapest, H-1088, Hungary.
2nd Department of Internal Medicine, Semmelweis University, Szentkirályi str 46, Budapest, H-1088, Hungary.
出版信息
BMC Cancer. 2018 Jun 27;18(1):695. doi: 10.1186/s12885-018-4609-x.
BACKGROUND
DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes.
METHODS
Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed.
RESULTS
According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations.
CONCLUSIONS
DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.
背景
DNA 突变随机且零星地发生在与生长相关的基因中，主要发生在胞嘧啶上。胞嘧啶的去甲基化可能会通过自发脱氨导致遗传不稳定性。目的是对结直肠癌（CRC）相关基因进行全基因组甲基化和靶向突变分析，并对 TP53 通路基因进行 mRNA 表达分析。
方法
采用长散布核元件-1（LINE-1）BS-PCR 联合焦磷酸测序法，估计结直肠正常-腺瘤-癌序列中全基因组 DNA 甲基化水平。对 6 个正常相邻组织（NAT）、15 个腺瘤组织（AD）和 9 个 CRC 组织进行甲基化捕获测序。进行总体定量甲基化分析、选择高甲基化/低甲基化基因、突变区域的甲基化分析和 TP53 通路基因启动子分析。评估了 12 个 CRC 相关基因（APC、BRAF、CTNNB1、EGFR、FBXW7、KRAS、NRAS、MSH6、PIK3CA、SMAD2、SMAD4、TP53）的突变情况。还分析了 TP53 通路基因的 mRNA 表达。
结果
根据 LINE-1 甲基化结果，在正常-腺瘤-癌序列中观察到整体低甲基化。在 top50 差异甲基化区域（DMRs）中，AD-N 比较中 TP73、NGFR、PDGFRA 基因呈高甲基化，FMN1、SLC16A7 基因呈低甲基化。在 CRC-N 比较中，DKK2、SDC2、SOX1 基因呈高甲基化，而 ERBB4、CREB5、CNTN1 基因呈低甲基化。在某些突变热点区域检测到显著的 DNA 甲基化改变。在腺瘤中，TP53 基因体被高甲基化所修饰。在腺瘤中，APC、TP53 和 KRAS 突变分别发生在 30%、15%和 21%，在 CRC 中分别发生在 29%、53%和 29%。在几个发生启动子甲基化改变的 TP53 通路基因中观察到 mRNA 表达变化。
结论
在结直肠的发展过程中，可以通过大量的启动子和基因体区域观察到具有连续表型效应的 DNA 甲基化。
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