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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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High prevalence of CD90+GPC3+ cells in CD90+CSCs derived from human HCC cell lines and liver tumors.A significant increase in the number of CD90+GPC3+ cells were detected within CD90+ cell population of PLC and MHCC97L cells. (A) In PLC cells, 95.3% of CD90+ cells co-expressed GPC3. (B) In MHCC97L cells, 99.0% of CD90+ cells co-expressed GPC3. (C) Analysis of a representative pair of human liver tissues indicated that only 4.5% of CD90+ population expressed GPC3 in non-tumorous tissues, while 89.9% of CD90+ cells expressed GPC3 in the matched tumorous tissues (median, 86.4%; range, 54.2–91.0%; n = 5). These results demonstrated that GPC3 is distinctly expressed in liver CD90+CSCs.
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pone-0037159-g006: High prevalence of CD90+GPC3+ cells in CD90+CSCs derived from human HCC cell lines and liver tumors.A significant increase in the number of CD90+GPC3+ cells were detected within CD90+ cell population of PLC and MHCC97L cells. (A) In PLC cells, 95.3% of CD90+ cells co-expressed GPC3. (B) In MHCC97L cells, 99.0% of CD90+ cells co-expressed GPC3. (C) Analysis of a representative pair of human liver tissues indicated that only 4.5% of CD90+ population expressed GPC3 in non-tumorous tissues, while 89.9% of CD90+ cells expressed GPC3 in the matched tumorous tissues (median, 86.4%; range, 54.2–91.0%; n = 5). These results demonstrated that GPC3 is distinctly expressed in liver CD90+CSCs.

Mentions: To validate the specificity and abundance of GPC3 in CD90+CSCs, two-color flow cytometry was used to measure the expression of total GPC3 (both cell membrane and cytoplasm) [31] in CD90+CSCs derived from two HCC cell lines. GPC3 was distinctly expressed in CD90+ cells derived from PLC cell line (95.3%, Figure 6A) and MHCC97L cell line (99.0%, Figure 6B). This result indicated predominant expression of GPC3 in liver CSCs. Further study on human HCC tissues also demonstrated that GPC3 was highly expressed in liver CD90+CSCs (median, 86.4%; range, 54.2–91.0%; n = 5; Figure 6C).


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)

High prevalence of CD90+GPC3+ cells in CD90+CSCs derived from human HCC cell lines and liver tumors.A significant increase in the number of CD90+GPC3+ cells were detected within CD90+ cell population of PLC and MHCC97L cells. (A) In PLC cells, 95.3% of CD90+ cells co-expressed GPC3. (B) In MHCC97L cells, 99.0% of CD90+ cells co-expressed GPC3. (C) Analysis of a representative pair of human liver tissues indicated that only 4.5% of CD90+ population expressed GPC3 in non-tumorous tissues, while 89.9% of CD90+ cells expressed GPC3 in the matched tumorous tissues (median, 86.4%; range, 54.2–91.0%; n = 5). These results demonstrated that GPC3 is distinctly expressed in liver CD90+CSCs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351419&req=5

pone-0037159-g006: High prevalence of CD90+GPC3+ cells in CD90+CSCs derived from human HCC cell lines and liver tumors.A significant increase in the number of CD90+GPC3+ cells were detected within CD90+ cell population of PLC and MHCC97L cells. (A) In PLC cells, 95.3% of CD90+ cells co-expressed GPC3. (B) In MHCC97L cells, 99.0% of CD90+ cells co-expressed GPC3. (C) Analysis of a representative pair of human liver tissues indicated that only 4.5% of CD90+ population expressed GPC3 in non-tumorous tissues, while 89.9% of CD90+ cells expressed GPC3 in the matched tumorous tissues (median, 86.4%; range, 54.2–91.0%; n = 5). These results demonstrated that GPC3 is distinctly expressed in liver CD90+CSCs.
Mentions: To validate the specificity and abundance of GPC3 in CD90+CSCs, two-color flow cytometry was used to measure the expression of total GPC3 (both cell membrane and cytoplasm) [31] in CD90+CSCs derived from two HCC cell lines. GPC3 was distinctly expressed in CD90+ cells derived from PLC cell line (95.3%, Figure 6A) and MHCC97L cell line (99.0%, Figure 6B). This result indicated predominant expression of GPC3 in liver CSCs. Further study on human HCC tissues also demonstrated that GPC3 was highly expressed in liver CD90+CSCs (median, 86.4%; range, 54.2–91.0%; n = 5; Figure 6C).

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