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Gene expression profiling of liver cancer stem cells by RNA-sequencing.

Ho DW, Yang ZF, Yi K, Lam CT, Ng MN, Yu WC, Lau J, Wan T, Wang X, Yan Z, Liu H, Zhang Y, Fan ST - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong, China.

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.

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.

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GPC3 expression and quantification of CD90+ cells in human liver tumor tissues.(A) Immunohistochemistry detected strong signals of GPC3 in liver tumor tissue, but negative staining for GPC3 was detected in the adjacent non-tumorous tissue (magnification×200). (B) Flow cytometry detected more CD45−CD90+ cells in tumor tissues (median, 0.645%; range, 0.06–4.59% of the gated cells) than that in adjacent non-tumorous tissues (median, 0.175%; range, 0.00–1.14%). (C) The number of CD45−CD90+cells was positively correlated with GPC3 expression level in the tumor tissues (Spearman correlation coefficient = 0.5997, P<0.0001).
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pone-0037159-g005: GPC3 expression and quantification of CD90+ cells in human liver tumor tissues.(A) Immunohistochemistry detected strong signals of GPC3 in liver tumor tissue, but negative staining for GPC3 was detected in the adjacent non-tumorous tissue (magnification×200). (B) Flow cytometry detected more CD45−CD90+ cells in tumor tissues (median, 0.645%; range, 0.06–4.59% of the gated cells) than that in adjacent non-tumorous tissues (median, 0.175%; range, 0.00–1.14%). (C) The number of CD45−CD90+cells was positively correlated with GPC3 expression level in the tumor tissues (Spearman correlation coefficient = 0.5997, P<0.0001).

Mentions: Among the genes identified by RNA-Seq, GPC3 was selected for further investigation because it is uniquely expressed in liver CD90+CSCs but absent in CD90+NTSCs. Immunohistochemistry revealed strong GPC3 expression in 34 out of forty-two HCC tumor tissues (80.9%), but no expression was detected in non-tumorous tissues (Figure 5A). This result was in concordance with the previous findings that GPC3 was differentially expressed in HCC [31].


Gene expression profiling of liver cancer stem cells by RNA-sequencing.

Ho DW, Yang ZF, Yi K, Lam CT, Ng MN, Yu WC, Lau J, Wan T, Wang X, Yan Z, Liu H, Zhang Y, Fan ST - PLoS ONE (2012)

GPC3 expression and quantification of CD90+ cells in human liver tumor tissues.(A) Immunohistochemistry detected strong signals of GPC3 in liver tumor tissue, but negative staining for GPC3 was detected in the adjacent non-tumorous tissue (magnification×200). (B) Flow cytometry detected more CD45−CD90+ cells in tumor tissues (median, 0.645%; range, 0.06–4.59% of the gated cells) than that in adjacent non-tumorous tissues (median, 0.175%; range, 0.00–1.14%). (C) The number of CD45−CD90+cells was positively correlated with GPC3 expression level in the tumor tissues (Spearman correlation coefficient = 0.5997, P<0.0001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351419&req=5

pone-0037159-g005: GPC3 expression and quantification of CD90+ cells in human liver tumor tissues.(A) Immunohistochemistry detected strong signals of GPC3 in liver tumor tissue, but negative staining for GPC3 was detected in the adjacent non-tumorous tissue (magnification×200). (B) Flow cytometry detected more CD45−CD90+ cells in tumor tissues (median, 0.645%; range, 0.06–4.59% of the gated cells) than that in adjacent non-tumorous tissues (median, 0.175%; range, 0.00–1.14%). (C) The number of CD45−CD90+cells was positively correlated with GPC3 expression level in the tumor tissues (Spearman correlation coefficient = 0.5997, P<0.0001).
Mentions: Among the genes identified by RNA-Seq, GPC3 was selected for further investigation because it is uniquely expressed in liver CD90+CSCs but absent in CD90+NTSCs. Immunohistochemistry revealed strong GPC3 expression in 34 out of forty-two HCC tumor tissues (80.9%), but no expression was detected in non-tumorous tissues (Figure 5A). This result was in concordance with the previous findings that GPC3 was differentially expressed in HCC [31].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong, China.

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.

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.

Show MeSH
Related in: MedlinePlus