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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 methylation biomarkers that can early diagnose and predict metastasis-related genes in renal clear cell carcinoma. Methods: The cell lines of low metastatic and highly metastatic renal clear cell carcinoma established in vitro from fresh tissue specimens were collected and genomic DNA was extracted after pathological identification. Three different rounds of NotⅠ genomic subtractive hybridization were used to enrich differential methyl The sequence of the sequence, the sequence of the difference vector, the transformed host strain and the alpha screening, 40 sequences were selected for sequencing. Bioinformatics analysis was used to determine the promoter CpG island methylation sequence, and the unknown gene function was predicted. Results: 27 single clones were obtained after DNA sequencing methylation difference in high metastasis and low metastatic renal clear cell carcinoma genome; 5 of them have CpG islands, of which 2 correspond to the promoters of MYADM and LOC646024 genes Area. MYADM is associated with hematopoiesis and stem cell regeneration, whereas LOC646024 has 85% similarity to anti-tumor-associated UL16-binding protein 1 of NK cells. CONCLUSIONS: In this study, we obtained a variety of neoplastic methylation sequences from Chinese renal clear cell carcinoma with different metastatic potential and found that MYADM and LOC646024 candidate genes are associated with renal cell carcinoma metastasis.