Gene expression profiling of liver cancer stem cells by RNA-sequencing

PLoS One. 2012;7(5):e37159. doi: 10.1371/journal.pone.0037159. Epub 2012 May 14.


Background: Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90(+) liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90(+) cells sorted from tumor (CD90(+)CSCs) with parallel non-tumorous liver tissues (CD90(+)NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis.

Methodology/principal findings: CD90(+) cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90(+) cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90(+)CSCs and CD90(+)NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90(+)CSCs and CD90(+)NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90(+)CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90(+)CSCs compared to CD90(+)NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90(+)CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90(+)CSCs in liver tumor tissues.

Conclusions/significance: The identified genes, such as GPC3 that are distinctly expressed in liver CD90(+)CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Base Sequence
  • Carcinoma, Hepatocellular / genetics*
  • Carcinoma, Hepatocellular / immunology
  • Carcinoma, Hepatocellular / metabolism
  • Carcinoma, Hepatocellular / pathology
  • Cell Line, Tumor
  • Cell Proliferation
  • DNA Primers / genetics
  • Female
  • Gene Expression Profiling
  • Gene Knockdown Techniques
  • Glypicans / antagonists & inhibitors
  • Glypicans / genetics
  • Glypicans / metabolism
  • Humans
  • Liver Neoplasms / genetics*
  • Liver Neoplasms / immunology
  • Liver Neoplasms / metabolism
  • Liver Neoplasms / pathology
  • Male
  • Middle Aged
  • Neoplastic Stem Cells / immunology
  • Neoplastic Stem Cells / metabolism*
  • Neoplastic Stem Cells / pathology
  • RNA, Neoplasm / genetics
  • RNA, Neoplasm / metabolism
  • RNA, Small Interfering / genetics
  • Thy-1 Antigens / metabolism
  • Tumor Stem Cell Assay


  • DNA Primers
  • GPC3 protein, human
  • Glypicans
  • RNA, Neoplasm
  • RNA, Small Interfering
  • Thy-1 Antigens