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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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Read distribution along the GPC3 gene and quantitative measurement of mRNA GPC3 by Fluidigm digital array assay.(A) Alignment of RNA-Seq sequence reads to GPC3 gene. Significantly higher read counts were detected for CD90+CSCs when compared with those of CD90+NTSCs, indicating the specificity of GPC3 in liver CD90+CSCs. For illustration purpose, only one exon of the gene was shown. (B) Each digital array chip can run twelve samples. The six samples of the right hand side of the chip were CD90+CSCs, and of the left hand side were the corresponding CD90+NTSCs. Digital array partitioned a RNA sample premixed with RT-PCR reagents into individual 765 RT-PCR reactions. In each partition, the red color indicated positive expression of GPC3 at mRNA level, whereas grey indicated no expression. The GPC3 mRNA level was quantified by counting the positive signals by the software. The mRNA expression of GPC3 was predominantly expressed in CD90+CSCs as compared with CD90+NTSCs (P<0.05).
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pone-0037159-g004: Read distribution along the GPC3 gene and quantitative measurement of mRNA GPC3 by Fluidigm digital array assay.(A) Alignment of RNA-Seq sequence reads to GPC3 gene. Significantly higher read counts were detected for CD90+CSCs when compared with those of CD90+NTSCs, indicating the specificity of GPC3 in liver CD90+CSCs. For illustration purpose, only one exon of the gene was shown. (B) Each digital array chip can run twelve samples. The six samples of the right hand side of the chip were CD90+CSCs, and of the left hand side were the corresponding CD90+NTSCs. Digital array partitioned a RNA sample premixed with RT-PCR reagents into individual 765 RT-PCR reactions. In each partition, the red color indicated positive expression of GPC3 at mRNA level, whereas grey indicated no expression. The GPC3 mRNA level was quantified by counting the positive signals by the software. The mRNA expression of GPC3 was predominantly expressed in CD90+CSCs as compared with CD90+NTSCs (P<0.05).

Mentions: We found that APOE, ESM-1, H19, ITIH1, PLVAP, PLK2, and LAMB1 were highly expressed in CD90+CSCs compared to CD90+NTSCs (P<0.01). The expression of GPC3 was unambiguously confined to CD90+CSCs and not detected in most of the CD90+NTSCs (Figure 4A). The absence of GPC3 in CD90+NTSCs was further demonstrated by Fluidigm digital array in human HCC tissues (P<0.05; Figure 4B).


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)

Read distribution along the GPC3 gene and quantitative measurement of mRNA GPC3 by Fluidigm digital array assay.(A) Alignment of RNA-Seq sequence reads to GPC3 gene. Significantly higher read counts were detected for CD90+CSCs when compared with those of CD90+NTSCs, indicating the specificity of GPC3 in liver CD90+CSCs. For illustration purpose, only one exon of the gene was shown. (B) Each digital array chip can run twelve samples. The six samples of the right hand side of the chip were CD90+CSCs, and of the left hand side were the corresponding CD90+NTSCs. Digital array partitioned a RNA sample premixed with RT-PCR reagents into individual 765 RT-PCR reactions. In each partition, the red color indicated positive expression of GPC3 at mRNA level, whereas grey indicated no expression. The GPC3 mRNA level was quantified by counting the positive signals by the software. The mRNA expression of GPC3 was predominantly expressed in CD90+CSCs as compared with CD90+NTSCs (P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351419&req=5

pone-0037159-g004: Read distribution along the GPC3 gene and quantitative measurement of mRNA GPC3 by Fluidigm digital array assay.(A) Alignment of RNA-Seq sequence reads to GPC3 gene. Significantly higher read counts were detected for CD90+CSCs when compared with those of CD90+NTSCs, indicating the specificity of GPC3 in liver CD90+CSCs. For illustration purpose, only one exon of the gene was shown. (B) Each digital array chip can run twelve samples. The six samples of the right hand side of the chip were CD90+CSCs, and of the left hand side were the corresponding CD90+NTSCs. Digital array partitioned a RNA sample premixed with RT-PCR reagents into individual 765 RT-PCR reactions. In each partition, the red color indicated positive expression of GPC3 at mRNA level, whereas grey indicated no expression. The GPC3 mRNA level was quantified by counting the positive signals by the software. The mRNA expression of GPC3 was predominantly expressed in CD90+CSCs as compared with CD90+NTSCs (P<0.05).
Mentions: We found that APOE, ESM-1, H19, ITIH1, PLVAP, PLK2, and LAMB1 were highly expressed in CD90+CSCs compared to CD90+NTSCs (P<0.01). The expression of GPC3 was unambiguously confined to CD90+CSCs and not detected in most of the CD90+NTSCs (Figure 4A). The absence of GPC3 in CD90+NTSCs was further demonstrated by Fluidigm digital array in human HCC tissues (P<0.05; Figure 4B).

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