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

Abstract

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 NotⅠgenomic 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.