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Functional analysis of microRNAs in human hepatocellular cancer stem cells.

Meng F, Glaser SS, Francis H, DeMorrow S, Han Y, Passarini JD, Stokes A, Cleary JP, Liu X, Venter J, Kumar P, Priester S, Hubble L, Staloch D, Sharma J, Liu CG, Alpini G - J. Cell. Mol. Med. (2012)

Bottom Line: Importantly, inhibition of let-7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR-181 led to a reduction in HSCs motility and invasion.Knocking down IL-6 and Twist in HSCs significantly reduced let-7 and miR-181 expression and subsequently inhibited chemoresistance and cell invasion.In conclusion, alterations of IL-6- and Twist-regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Scott & White Digestive Disease Research Center, Texas A&M Health Science Center College of Medicine and Scott & White Hospital, Temple, TX 76504, USA.

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Characterization of Cancer Stem Cells from human HCC tissues. (A) Real-time analysis of Oct 4, CD 133, Nestin, telom-erase, SSEA-4, AFP and CEA mRNA expression in HCC stem cell under self-renewal condition (HSC-SR), under differentiation (HSC-DF) and HepG2 HCC cells. All the markers are significantly up-regulated in HCC cancer stem cells. *P < 0.05 when compared with HepG2 group. (B) FACS analysis of HCC stem cells on day 1 before cell sorting. Results are given as the percentage of Oct-4 and CD133+ cells in the total population. In the histograms, the red line represents staining with Oct-4 or CD133 mAb, and the green lines represent the isotype control-matched mAb. (C) Cell lysates were obtained and immunoblotting analysis was performed for Oct-4, CD133, α-fetoprotein (AFP), CK-7, CK-19 and EpiCam using specific antibodies. The blots were stripped and reprobed for β-actin as a loading control and for quantitation. (D) StemTAG™ Alkaline Phosphatase assays performed at passage 4–6 HSCs and LSCs. HSC-SR and LSCs are maintained in an undifferentiated stage on specific culture dishes, as indicated by the high AP activity, thus confirming the self-renewal within these cells. (E) Formation of tumour spheres in HCC stem cells. In 1% FCS culture media 70–90% of the HSC-SR and HSC-DF became adherent after 7 days, with a minority of floating cells forming spheres composed of three to five cells. After an additional 7 days, the floating spheres were expanded to contain 15–20 HSC-SR cells, with bright appearance and sharp edge. (F) Immunocytochemistry for CD133 and AFP was performed in Normal liver stem cells (N-LSC), HepG2 cells, hepatocellular cancer stem cells growing under self-renewal conditions (HSC-SR) and differentiation conditions (HSC-DF). An increase in CD133 expression along with the enhanced expression of AFP is observed in HSC-SR and HSC-DF cells.
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fig01: Characterization of Cancer Stem Cells from human HCC tissues. (A) Real-time analysis of Oct 4, CD 133, Nestin, telom-erase, SSEA-4, AFP and CEA mRNA expression in HCC stem cell under self-renewal condition (HSC-SR), under differentiation (HSC-DF) and HepG2 HCC cells. All the markers are significantly up-regulated in HCC cancer stem cells. *P < 0.05 when compared with HepG2 group. (B) FACS analysis of HCC stem cells on day 1 before cell sorting. Results are given as the percentage of Oct-4 and CD133+ cells in the total population. In the histograms, the red line represents staining with Oct-4 or CD133 mAb, and the green lines represent the isotype control-matched mAb. (C) Cell lysates were obtained and immunoblotting analysis was performed for Oct-4, CD133, α-fetoprotein (AFP), CK-7, CK-19 and EpiCam using specific antibodies. The blots were stripped and reprobed for β-actin as a loading control and for quantitation. (D) StemTAG™ Alkaline Phosphatase assays performed at passage 4–6 HSCs and LSCs. HSC-SR and LSCs are maintained in an undifferentiated stage on specific culture dishes, as indicated by the high AP activity, thus confirming the self-renewal within these cells. (E) Formation of tumour spheres in HCC stem cells. In 1% FCS culture media 70–90% of the HSC-SR and HSC-DF became adherent after 7 days, with a minority of floating cells forming spheres composed of three to five cells. After an additional 7 days, the floating spheres were expanded to contain 15–20 HSC-SR cells, with bright appearance and sharp edge. (F) Immunocytochemistry for CD133 and AFP was performed in Normal liver stem cells (N-LSC), HepG2 cells, hepatocellular cancer stem cells growing under self-renewal conditions (HSC-SR) and differentiation conditions (HSC-DF). An increase in CD133 expression along with the enhanced expression of AFP is observed in HSC-SR and HSC-DF cells.

Mentions: Human hepatocellular CSCs and parental HCC cells were provided by Celprogen Inc. (San Pedro, CA, USA). Three groups of HSCs were chosen for the studies from seven sets on the basis of self-renewal and real-time polymerase chain reaction (PCR) tests for the specific markers (Fig. 1). The selected group of cells was cultured in Human Liver Cancer Stem Cell Undifferentiated Media to keep the stem cell phenotype (HSC-SR), and in Human Liver Cancer Stem Cell Differentiated Media for 10 passages for differentiated progenitor cell phenotype (HSC-DF). Malignant HCC cells (HepG2) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured as recommended by the supplier. Normal human liver stem cells were also obtained from Celprogen Inc.


Functional analysis of microRNAs in human hepatocellular cancer stem cells.

Meng F, Glaser SS, Francis H, DeMorrow S, Han Y, Passarini JD, Stokes A, Cleary JP, Liu X, Venter J, Kumar P, Priester S, Hubble L, Staloch D, Sharma J, Liu CG, Alpini G - J. Cell. Mol. Med. (2012)

Characterization of Cancer Stem Cells from human HCC tissues. (A) Real-time analysis of Oct 4, CD 133, Nestin, telom-erase, SSEA-4, AFP and CEA mRNA expression in HCC stem cell under self-renewal condition (HSC-SR), under differentiation (HSC-DF) and HepG2 HCC cells. All the markers are significantly up-regulated in HCC cancer stem cells. *P < 0.05 when compared with HepG2 group. (B) FACS analysis of HCC stem cells on day 1 before cell sorting. Results are given as the percentage of Oct-4 and CD133+ cells in the total population. In the histograms, the red line represents staining with Oct-4 or CD133 mAb, and the green lines represent the isotype control-matched mAb. (C) Cell lysates were obtained and immunoblotting analysis was performed for Oct-4, CD133, α-fetoprotein (AFP), CK-7, CK-19 and EpiCam using specific antibodies. The blots were stripped and reprobed for β-actin as a loading control and for quantitation. (D) StemTAG™ Alkaline Phosphatase assays performed at passage 4–6 HSCs and LSCs. HSC-SR and LSCs are maintained in an undifferentiated stage on specific culture dishes, as indicated by the high AP activity, thus confirming the self-renewal within these cells. (E) Formation of tumour spheres in HCC stem cells. In 1% FCS culture media 70–90% of the HSC-SR and HSC-DF became adherent after 7 days, with a minority of floating cells forming spheres composed of three to five cells. After an additional 7 days, the floating spheres were expanded to contain 15–20 HSC-SR cells, with bright appearance and sharp edge. (F) Immunocytochemistry for CD133 and AFP was performed in Normal liver stem cells (N-LSC), HepG2 cells, hepatocellular cancer stem cells growing under self-renewal conditions (HSC-SR) and differentiation conditions (HSC-DF). An increase in CD133 expression along with the enhanced expression of AFP is observed in HSC-SR and HSC-DF cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3116063&req=5

fig01: Characterization of Cancer Stem Cells from human HCC tissues. (A) Real-time analysis of Oct 4, CD 133, Nestin, telom-erase, SSEA-4, AFP and CEA mRNA expression in HCC stem cell under self-renewal condition (HSC-SR), under differentiation (HSC-DF) and HepG2 HCC cells. All the markers are significantly up-regulated in HCC cancer stem cells. *P < 0.05 when compared with HepG2 group. (B) FACS analysis of HCC stem cells on day 1 before cell sorting. Results are given as the percentage of Oct-4 and CD133+ cells in the total population. In the histograms, the red line represents staining with Oct-4 or CD133 mAb, and the green lines represent the isotype control-matched mAb. (C) Cell lysates were obtained and immunoblotting analysis was performed for Oct-4, CD133, α-fetoprotein (AFP), CK-7, CK-19 and EpiCam using specific antibodies. The blots were stripped and reprobed for β-actin as a loading control and for quantitation. (D) StemTAG™ Alkaline Phosphatase assays performed at passage 4–6 HSCs and LSCs. HSC-SR and LSCs are maintained in an undifferentiated stage on specific culture dishes, as indicated by the high AP activity, thus confirming the self-renewal within these cells. (E) Formation of tumour spheres in HCC stem cells. In 1% FCS culture media 70–90% of the HSC-SR and HSC-DF became adherent after 7 days, with a minority of floating cells forming spheres composed of three to five cells. After an additional 7 days, the floating spheres were expanded to contain 15–20 HSC-SR cells, with bright appearance and sharp edge. (F) Immunocytochemistry for CD133 and AFP was performed in Normal liver stem cells (N-LSC), HepG2 cells, hepatocellular cancer stem cells growing under self-renewal conditions (HSC-SR) and differentiation conditions (HSC-DF). An increase in CD133 expression along with the enhanced expression of AFP is observed in HSC-SR and HSC-DF cells.
Mentions: Human hepatocellular CSCs and parental HCC cells were provided by Celprogen Inc. (San Pedro, CA, USA). Three groups of HSCs were chosen for the studies from seven sets on the basis of self-renewal and real-time polymerase chain reaction (PCR) tests for the specific markers (Fig. 1). The selected group of cells was cultured in Human Liver Cancer Stem Cell Undifferentiated Media to keep the stem cell phenotype (HSC-SR), and in Human Liver Cancer Stem Cell Differentiated Media for 10 passages for differentiated progenitor cell phenotype (HSC-DF). Malignant HCC cells (HepG2) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured as recommended by the supplier. Normal human liver stem cells were also obtained from Celprogen Inc.

Bottom Line: Importantly, inhibition of let-7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR-181 led to a reduction in HSCs motility and invasion.Knocking down IL-6 and Twist in HSCs significantly reduced let-7 and miR-181 expression and subsequently inhibited chemoresistance and cell invasion.In conclusion, alterations of IL-6- and Twist-regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Scott & White Digestive Disease Research Center, Texas A&M Health Science Center College of Medicine and Scott & White Hospital, Temple, TX 76504, USA.

Show MeSH
Related in: MedlinePlus