title: Analysis of the genome-wide DNA methylation profile of side population cells in hepatocellular carcinoma.
abstract: BACKGROUND
  DNA methylation plays an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the DNA methylation profile of stem cells in hepatocellular carcinoma (HCC) remains unclear.
  
  
  OBJECTIVE
  To investigate the genome-wide DNA methylation profile of side population (SP) cells of HCC, a special subpopulation of cells enriched with cancer stem cells, by DNA methylation microarray analysis and to analyze the functions and signal pathways of the aberrantly methylated genes in SP cells.
  
  
  METHODS
  Side population cells were isolated from HCC cell lines Huh7 and PLC/PRF/5 using flow cytometry, and the tumorigenicity of these SP cells was assessed in NOD/SCID mice. The genome-wide DNA methylation status of SP cells and non-SP (NSP) cells was detected and compared by DNA methylation microarray analysis. Genes with differential methylation between SP and NSP cells were further analyzed for their functions and roles in related signaling pathways.
  
  
  RESULTS
  Subcutaneous inoculation of 1 × 10(3) SP cells yielded tumors in 60 % NOD/SCID mice, whereas no tumor was developed after the inoculation of 1 × 10(6) NSP cells. Genome-wide DNA methylation microarray analysis showed that 72 and 181 genes were hypermethylated and hypomethylated, respectively, in both Huh7 and PLC/PRF/5 SP cells as compared with their corresponding NSP cells. Analyses of signaling pathways revealed that hypermethylated and hypomethylated genes were related to four and eight pathways, respectively.
  
  
  CONCLUSIONS
  Hepatocellular carcinoma SP cells possessed a differential DNA methylation status compared with NSP cells, and the differentially methylated genes in SP cells were involved in 12 signaling pathways. Our results provide valuable clues for further investigations in elucidating the importance of epigenetic regulation in sustaining HCC SP cells and tumorigenesis.
authors.affiliation: Department of Hepatobiliary Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510080 Guangdong, People's Republic of China. zhaijingming06@163.com
authors.forename: Jing-Ming
authors.initials: JM
authors.lastname: Zhai
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Yin
authors.forename: Xun
authors.initials: X
authors.lastname: Hou
authors.forename: Xiao-Yi
authors.initials: XY
authors.lastname: Hao
authors.forename: Jian-Peng
authors.initials: JP
authors.lastname: Cai
authors.forename: Li-Jian
authors.initials: LJ
authors.lastname: Liang
authors.forename: Long-Juan
authors.initials: LJ
authors.lastname: Zhang
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D005819:Genetic Markers; D006801:Humans; D047468:Immunoprecipitation; D008113:Liver Neoplasms / Q000235:genetics*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction; D058985:Side-Population Cells
chemical_list: D014408:Biomarkers, Tumor; D005819:Genetic Markers