Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong, China.
PLoS One. 2012;7(5):e37159. doi: 10.1371/journal.pone.0037159. Epub 2012 May 14.
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.
越来越多的证据表明肿瘤的生长和癌症的复发是由癌症干细胞驱动的。我们之前的工作已经证明了肝癌（HCC）中存在 CD90（+）肝癌干细胞（CSC）。然而，这些细胞的特征仍然知之甚少。在这项研究中，我们采用了更敏感的 RNA 测序（RNA-Seq）来比较从肿瘤（CD90（+）CSC）中分离的 CD90（+）细胞与平行的非肿瘤性肝组织（CD90（+）NTSC）的基因表达谱，并阐明潜在靶基因在肝癌发生中的作用。
方法/主要发现：使用荧光激活细胞分选法分别从肿瘤和相邻的非肿瘤性人肝组织中分离 CD90（+）细胞。对 3 例 HCC 患者的 CD90（+）细胞的扩增 RNA 进行 RNA-Seq 分析。在 CD90（+）CSC 和 CD90（+）NTSC 之间建立了差异基因表达谱，并通过相同的扩增 RNA 进行定量实时 PCR（qRT-PCR）验证，然后在 12 例 HCC 患者的独立队列中进一步确认。在 CD90（+）CSC 和 CD90（+）NTSC 之间有 500 个基因表达差异（119 个上调和 381 个下调基因）。基因本体分析表明，CD90（+）CSC 中过表达的基因与炎症、耐药性和脂质代谢有关。在差异表达的基因中，糖蛋白聚糖-3（GPC3），糖蛋白家族的一个成员，在 CD90（+）CSC 中明显高于 CD90（+）NTSC。免疫组织化学显示，GPC3 在 42 个人类肝肿瘤组织中高度表达，但在相邻的非肿瘤性肝组织中不存在。流式细胞术表明，GPC3 在肝癌细胞系和人肝肿瘤组织中的肝 CD90（+）CSC 和成熟癌细胞中高度表达。此外，GPC3 的表达与肝肿瘤组织中 CD90（+）CSC 的数量呈正相关。
结论/意义：在肝 CD90（+）CSC 中明显表达的鉴定基因，如 GPC3，可能是 HCC 治疗的有前途的基因候选物，而不会对正常肝干细胞造成损害。
