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Isolation and stem cell property assessment of side population cells in hepatocellular carcinoma cell lines
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Abstract:
Objective: To isolate and characterize side population (SP) cells in human hepatocellular carcinoma (HCC) cells. Methods: Four HCC cell lines with a metastatic potential in an order of HCCLM3 > MHCC97-H>MHCC97-L>Huh7 were cultivated in routine culture conditions. Single cell suspensions were made for these lines and incubated with different concentrations of Hoechst33342 either alone or with 50 μmol/l verapamil. The proportion of SP cells in these suspensions was analyzed by flow cytometry. SP cells and main population (MP) cells in the single cell suspension of cell line HCCLM3 were sorted. The HCCLM3-derived SP cells and MP cells were then comparatively assessed for tumorsphere formation, migration and invasion in vitro, for tumorigenicity in vivo and for the expression of stem cell-related genes at the mRNA level. Results: The proportion of SP proportion was significantly different among HCCLM3 (16.9±18)%, MHCC97-H (8.4±0.7)%, MHCC97-L (4.6±0.5)% and Huh7 (1.0±0.2) (P<0.05). HCCLM3-derived SP cells but not MP cells displayed many characteristics of cancer stem cells including more sphere formation (253±5.1)% vs (11.2±2.6)%, P<0.05), higher invasion ability in vitro and stronger tumorigenicity in nude mice. The mRNA levels of ABCG2, CD13, CD44, Nanog, Sox2 and Klf4 in SP cells were 5.64, 2.26, 2.10, 378, 237, 2.92 times of those respectively in MP cells (all P<0.05). Conclusion: In the HCCLM3 cell line there is an enriched population of SP cells with liver cancer stem cell-like properties, which may be related to the highly metastatic potential of HCCLM3 cells.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 81160262, 81260331), and the National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2012ZX10002010001009)
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