基因组消减杂交技术筛选肾透明细胞癌转移相关甲基化基因

Screening for clear cell renal cell carcinoma metastasis-associated methylations by genome subtractive hybridization

目的:筛选能够早期诊断和预测肾透明细胞癌转移相关基因的甲基化生物标志。方法:应用本室从临床新鲜组织标本体外培养建立的低转移性和高转移性肾透明细胞癌细胞株.病理学鉴定后提取基因组DNA.应用3轮NotⅠ基因组消减杂交的方法富集差异甲基化序列,差异序列连接载体,转化宿主菌和α筛选后.选取40个序列进行测序。应用生物信息学分析确定启动子CpG岛甲基化序列,并对未知基因功能进行预测。结果:DNA测序后获得了27个单一克隆在高转移和低转移性肾透明细胞癌基因组中存在甲基化差异;其中5个序列具有CpG岛,其中2个对应于基因MYADM和LOC646024的启动子区。MYADM与血细胞成熟、干细胞再生有关;而LOC646024与NK细胞抗肿瘤相关的UL16结合蛋白1有85%的相似度。结论:本研究从不同转移能力的中国人肾透明细胞癌基因组中获得多种转移相关性的新甲基化序列.发现MYADM和LOC646024候选基因与肾癌转移相关。

Objective： To screen for methylated biomarkers for the early diagnosis and prediction of clear cell renal cell cancer （ccRCC） metastasis. Methods： Highly and lowly metastatic ccRCC cell lines,which were established using fresh surgical specimens in our laboratory,were used in this study. Genomic DNA was extracted after pathological identification. Methylated genomic fragments were enriched by 3 cycles of Not Ⅰ genomic DNA subtractive hybridization and were linked to vectors,which was then used for transformation,followed by a selection. Forty positive clones were randomly selected for DNA sequencing. Bioinformatics analysis was used to identify methylation of CpG islands and to predict the function of unknown genes. Results： DNA sequencing revealed 27 independent clones with different methylations between the highly and lowly metastatic ccRCC. Five of the 27 clones contained CpG islands, and 2 of the 5 fragments contained CpG islands in the promoter regions of genes MYADM and LOC646024. MYADM was associated with maturation of hematopoietic cells and regeneration of stem cells. LOC646024 shared 85% homology with UL16 binding protein 1; the latter was related to tumor killing function of NK cells. Conclusion： Novel methylated sequences have been discovered from Chinese ccRCC cells with different potentials for metastasis. Methylation of 2 candidate genes,MYADM and LOC646024,is indicated to be involved in ccRCC metastasis. Our findings are valuable for the biomarker exploration to predict metastasis as well as molecular epidemiological research.