title: Gene expression profiling of liver cancer stem cells by RNA-sequencing.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: David W Y
authors.initials: DW
authors.lastname: Ho
authors.forename: Zhen Fan
authors.initials: ZF
authors.lastname: Yang
authors.forename: Kang
authors.initials: K
authors.lastname: Yi
authors.forename: Chi Tat
authors.initials: CT
authors.lastname: Lam
authors.forename: Michael N P
authors.initials: MN
authors.lastname: Ng
authors.forename: Wan Ching
authors.initials: WC
authors.lastname: Yu
authors.forename: Joyce
authors.initials: J
authors.lastname: Lau
authors.forename: Timothy
authors.initials: T
authors.lastname: Wan
authors.forename: Xiaoqi
authors.initials: X
authors.lastname: Wang
authors.forename: Zhixiang
authors.initials: Z
authors.lastname: Yan
authors.forename: Hang
authors.initials: H
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Sheung Tat
authors.initials: ST
authors.lastname: Fan
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D017931:DNA Primers / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D053673:Glypicans / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D014411:Neoplastic Stem Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D018800:Thy-1 Antigens / Q000378:metabolism; D014410:Tumor Stem Cell Assay
chemical_list: D017931:DNA Primers; C105937:GPC3 protein, human; D053673:Glypicans; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D018800:Thy-1 Antigens