Abstract
BackgroundAccumulating 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 FindingsCD90+ 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/SignificanceThe 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.
Highlights
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world with a high mortality rate [1]
We found that APOE, endothelial cell specific molecule-1 (ESM-1), H19, ITIH1, PLVAP, PLK2, and LAMB1 were highly expressed in CD90+cancer stem cells (CSCs) compared to CD90+non-tumorous stem cells (NTSCs) (P,0.01)
Hedgehog and Notch pathways which play critical roles in the selfrenewal process of CSCs. Because these developmental signaling cascades interact with other pathways in normal biological functions, concerns are raised that normal stem cells will be unavoidably damaged due to non-specificity of current anti-cancer therapies [32]
Summary
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world with a high mortality rate [1]. Lines of evidence have revealed the existence and importance of cancer stem cells (CSCs) in carcinogenesis in the past decades. CSCs are considered to be the root of cancers, and are responsible for tumor growth and differentiation of heterogeneous cell populations within tumors [6]. They have been demonstrated to be chemoresistant [7] and radioresistant [8]. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). We employed a more sensitive RNAsequencing (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
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