应用甲基化CpG岛扩增法结合代表性差异分析筛选结肠癌相关的甲基化DNA片段

目的 研究结肠癌癌旁粘膜和正常结肠组织间DNA甲基化分布的差异以及甲基化在结肠癌发生中的作用。方法 用甲基化CpG岛扩增(methylated CpG islands amplification，MCA)法富集基因组DNA甲基化序列，以癌旁粘膜DNA甲基化富集产物为检测子，正常结肠组织的甲基化富集产物为驱赶子进行代表性差异分析(representational difference analysis，RDA)获得DNA甲基化差异片段，经克隆、测序获得碱基序列．用生物信息学分析这些序列与相关基因结构的关系。结果 获得25个不同的差异甲基化序列，位于相关基因的5′端、外显子、内含子和3′端。其中1A01片段位于CECR7基因的第1外显子和LOC256866基因的5′端及第1外显子；1A12片段位于GR6基因的第1外显子前204 bp处。符合DNA甲基化对基因转录的调控规律。以1A12片段为探针，点杂交分析表明，该探针与4轮RDA和检测子均有杂交信号，而与驱赶子无杂交信号。结论 结肠癌癌旁组织和正常结肠组织的DNA甲基化存在差别，用MCA结合RDA方法可以有效分析这两种不同组织的甲基化分布差异．

Screening the differentially methylated DNA sequences of colorectal cancer by methylated CpG islands amplification coupled with representational difference analysis

OBJECTIVE: To screen the differentially methylated DNA sequences between mucosa adjacent to colorectal cancer (MACC) and normal colonic mucosa. METHODS: The methylated DNA sequences were enriched by methylation CpG islands amplification (MCA), and the differentially methylated DNA sequences between MACC and normal colonic mucosa were isolated by representational difference analysis (RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with BLAST program system in GenBank. With the separated fragment 1A12 as probe, dot blot was used to study its distribution between RDA products (No. 1-4 rounds), MACC (tester) and normal colonic mucosa(driver). RESULTS: Twenty-five differentially methylated DNA sequences were obtained. Preliminary studies indicated that 1A01 fragment was concerned with two different genes (LOC256866 and CECR7), it was located in the first exon of CECR7. 1A12 fragment was located in 5 flanking region of GR6 gene. By dot blot with 1A12 probe, hybridized signals were detected in MCA product of MACC and RDA products of No. 1-4 rounds, respectively. No signal was detected in MCA product of normal colonic mucosa. CONCLUSION: The differentially methylated DNA sequences can be isolated effectively between two different tissues with MCA coupled with RDA. Different methylated DNA fragments exist between MACC and normal colonic mucosa and these fragments may be concerned with colorectal cancer.