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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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Prospective validation of RNA-Seq analysis using an independent cohort of 12 patients by qRT-PCR.Twenty-seven up-regulated genes and 15 down-regulated genes were selected for validation. The fold changes of selected genes measured by qRT-PCR were statistically significant (P<0.05). Gene expression difference was considered to be valid if the direction of change was the same (as estimated by RNA-Seq analysis). The percentage of concordance of qRT-PCR with the change of direction estimated by RNA-Seq analysis for the selected genes was 80%. *: The expression of GPC3 in CD90+NTSCs was not detected and its fold change could not be calculated. Further analysis by Fluidigm digital array confirmed the finding. **: The expression of BMPER in CD90+CSCs was not detected. Further analysis by Fludigim digital array confirmed the finding.
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pone-0037159-g003: Prospective validation of RNA-Seq analysis using an independent cohort of 12 patients by qRT-PCR.Twenty-seven up-regulated genes and 15 down-regulated genes were selected for validation. The fold changes of selected genes measured by qRT-PCR were statistically significant (P<0.05). Gene expression difference was considered to be valid if the direction of change was the same (as estimated by RNA-Seq analysis). The percentage of concordance of qRT-PCR with the change of direction estimated by RNA-Seq analysis for the selected genes was 80%. *: The expression of GPC3 in CD90+NTSCs was not detected and its fold change could not be calculated. Further analysis by Fluidigm digital array confirmed the finding. **: The expression of BMPER in CD90+CSCs was not detected. Further analysis by Fludigim digital array confirmed the finding.

Mentions: To eliminate potential bias as a result of pre-amplification and to further validate the RNA-Seq results, qRT-PCR of 27 up-regulated genes and 15 down-regulated genes was performed in 12 pairs of RNA samples prepared from CD90+CSCs and CD90+NTSCs derived from an independent batch of tumor and parallel non-tumor tissues, respectively. None of these samples underwent RNA amplification nor assayed for RNA-Seq analysis. A gene expression difference was considered to be valid if the trend of change of a gene measured by qRT-PCR agreed with that determined by the RNA-Seq analysis. Twenty-two out of 27 (81.5%) selected up-regulated genes were concordant with the trend estimated by RNA-Seq, whereas 12 out of 15 (80.0%) selected down-regulated genes were correlated with the down-regulated pattern estimated by RNA-Seq (Figure 3). This high concordant result suggested that RNA amplification did not introduce bias to the results of the gene expression profiling under study.


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)

Prospective validation of RNA-Seq analysis using an independent cohort of 12 patients by qRT-PCR.Twenty-seven up-regulated genes and 15 down-regulated genes were selected for validation. The fold changes of selected genes measured by qRT-PCR were statistically significant (P<0.05). Gene expression difference was considered to be valid if the direction of change was the same (as estimated by RNA-Seq analysis). The percentage of concordance of qRT-PCR with the change of direction estimated by RNA-Seq analysis for the selected genes was 80%. *: The expression of GPC3 in CD90+NTSCs was not detected and its fold change could not be calculated. Further analysis by Fluidigm digital array confirmed the finding. **: The expression of BMPER in CD90+CSCs was not detected. Further analysis by Fludigim digital array confirmed the finding.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037159-g003: Prospective validation of RNA-Seq analysis using an independent cohort of 12 patients by qRT-PCR.Twenty-seven up-regulated genes and 15 down-regulated genes were selected for validation. The fold changes of selected genes measured by qRT-PCR were statistically significant (P<0.05). Gene expression difference was considered to be valid if the direction of change was the same (as estimated by RNA-Seq analysis). The percentage of concordance of qRT-PCR with the change of direction estimated by RNA-Seq analysis for the selected genes was 80%. *: The expression of GPC3 in CD90+NTSCs was not detected and its fold change could not be calculated. Further analysis by Fluidigm digital array confirmed the finding. **: The expression of BMPER in CD90+CSCs was not detected. Further analysis by Fludigim digital array confirmed the finding.
Mentions: To eliminate potential bias as a result of pre-amplification and to further validate the RNA-Seq results, qRT-PCR of 27 up-regulated genes and 15 down-regulated genes was performed in 12 pairs of RNA samples prepared from CD90+CSCs and CD90+NTSCs derived from an independent batch of tumor and parallel non-tumor tissues, respectively. None of these samples underwent RNA amplification nor assayed for RNA-Seq analysis. A gene expression difference was considered to be valid if the trend of change of a gene measured by qRT-PCR agreed with that determined by the RNA-Seq analysis. Twenty-two out of 27 (81.5%) selected up-regulated genes were concordant with the trend estimated by RNA-Seq, whereas 12 out of 15 (80.0%) selected down-regulated genes were correlated with the down-regulated pattern estimated by RNA-Seq (Figure 3). This high concordant result suggested that RNA amplification did not introduce bias to the results of the gene expression profiling under study.

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