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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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Related in: MedlinePlus

Double immunofluorescence staining of CD90 and GPC3 in sorted PLC CD90+GPC3+ cells.The sorted cells were stained with fluorescein-conjugated anti-CD90 and anti-GPC3 antibodies. Nuclei were counterstained by DAPI. The merge image showed the expression of CD90 and GPC3 in both cytoplasm and cell membrane.
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pone-0037159-g007: Double immunofluorescence staining of CD90 and GPC3 in sorted PLC CD90+GPC3+ cells.The sorted cells were stained with fluorescein-conjugated anti-CD90 and anti-GPC3 antibodies. Nuclei were counterstained by DAPI. The merge image showed the expression of CD90 and GPC3 in both cytoplasm and cell membrane.

Mentions: PLC CD90+GPC3+ CSCs were sorted using FACS with the conjugated antibodies of FITC-GPC3 and PE-CD90 (Figure 7).We transfected PLC CD90+GPC3+ cells with either GPC3-specific siRNA or scrambled control. The efficacy of knockdown was determined by qRT-PCR at various time points. More than 90% inhibition of mRNA expression of GPC3 was achieved (Figure 8A). Consistently, by flow cytometry, the percentage of GPC3 expressing cells was reduced by more than 43% after GPC3 knockdown when compared to the negative control (Figure 8B). There was no remarkable change in the percentage of CD90+CSCs in PLC cells upon GPC3 transfection, implicating that GPC3 suppression has no effect on the liver CD90+CSCs.


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)

Double immunofluorescence staining of CD90 and GPC3 in sorted PLC CD90+GPC3+ cells.The sorted cells were stained with fluorescein-conjugated anti-CD90 and anti-GPC3 antibodies. Nuclei were counterstained by DAPI. The merge image showed the expression of CD90 and GPC3 in both cytoplasm and cell membrane.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351419&req=5

pone-0037159-g007: Double immunofluorescence staining of CD90 and GPC3 in sorted PLC CD90+GPC3+ cells.The sorted cells were stained with fluorescein-conjugated anti-CD90 and anti-GPC3 antibodies. Nuclei were counterstained by DAPI. The merge image showed the expression of CD90 and GPC3 in both cytoplasm and cell membrane.
Mentions: PLC CD90+GPC3+ CSCs were sorted using FACS with the conjugated antibodies of FITC-GPC3 and PE-CD90 (Figure 7).We transfected PLC CD90+GPC3+ cells with either GPC3-specific siRNA or scrambled control. The efficacy of knockdown was determined by qRT-PCR at various time points. More than 90% inhibition of mRNA expression of GPC3 was achieved (Figure 8A). Consistently, by flow cytometry, the percentage of GPC3 expressing cells was reduced by more than 43% after GPC3 knockdown when compared to the negative control (Figure 8B). There was no remarkable change in the percentage of CD90+CSCs in PLC cells upon GPC3 transfection, implicating that GPC3 suppression has no effect on the liver CD90+CSCs.

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