Gene Expression Profiling of Liver Cancer Stem Cells by RNA-Sequencing

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.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

“…A simple explanation is the increased coverage with ultradeep sequencing methods and coverage of any cell type including tumors, many developmental stages, as well as (tumor) cell lines numerous times over (Carninci 2009;Ritz et al 2011;Haas et al 2012;Ho et al 2012;Shah et al 2012;Eswaran et al 2013;Hu et al 2013;Schonberg et al 2013;Yoshihara et al 2013). With all this overabundance, there is a debate raging between those that claim almost any identified transcript and snippet of RNA to be functional (Mattick 2003;Lee et al 2009;Carninci 2010;Kishore et al 2010;Clark et al 2011;Bernstein et al 2012;Kapranov and St Laurent 2012;Khayrullina et al 2012;Gebetsberger and Polacek 2013;Mattick and Dinger 2013) and more conservative voices (Brosius 2005c;Huttenhofer et al 2005;Robinson 2010;van Bakel et al 2010;Graur et al 2013).…”

Efficacy of Using Cancer Stem Cell Markers in Isolating and Characterizing Liver Cancer Stem Cells

“…Currently, identification of CSCs is achieved through several approaches, including (1) flow cytometry separation using CSCs surface markers [13]; (2) detection of side population by the Hoechst 33342 exclusion assay [14]; (3) in vitro floating tumor sphere formation in serum-free medium [15] coupled with xenograft tumor formation in immune-deficient mice [16,17]; and (4) other assays such as aldehyde dehydrogenase (ALDH) activity assay [18] and immunohistochemistry analysis [15]. Several LCSC markers have been reported, including CD133, CD90, CD44, epithelial cell-adhesion molecule (EpCAM), CD13, OV6, and ALDH [19,20]. However, the reliability of each of these markers in identifying true LCSCs varies with LCSCs sorted from different laboratories showing high heterogeneity [21].…”

“…These cells possessed stem cell properties and were able to stably form tumors in nude mice [20]. Similarly, CD133 + and CD44 + cells displayed stem cell properties and a greater tumorigenic ability compared with CD133 -and CD44 -cells [19,20]. So far, no universal markers for LCSCs have been identified, and isolating LCSC on the basis of single markers is problematic.…”

Expression of Glypican-3 in Mouse Embryo Stem Cells and its Derived Hepatic Lineage Cells Treated with Diethylnitrosamine in vitro

“…In D-galactosamine and 2-AAF/PH treated hepatocarcinogenesis model, GPC-3 expression was highly induced in oval cells (Grozdanov et al, 2006). Recent report showed that GPC-3 was markedly elevated in CD90(+) liver cancer stem cells (CSCs) and was highly expressed in human liver tumor tissues but absent in adjacent non-tumorous liver tissues (Ho et al, 2012). These data represented that GPC-3 expression showed various patterns in hepatic tumors and could be different in liver tumor models.…”