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

Abstract

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. 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. 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.