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Isolation of stem-like cells from human hepatocellular carcinoma PLC/PRF-5 cells and screening for their specific miRNAs
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Abstract:
Objective:To isolate stem-like cells from hepatocellular carcinoma cell line PLC/PRF-5 and to study their miRNA profile. Methods：〖WTBZ〗ABCG2+ and ABCG2- PLC/PRF-5 cells were isolated from the PLC/PRF-5 cell line by magnetic activated cell sorting (MACS) method, and further identified by flow cytometry. The colony formation ability in soft agar and tumor formation ability in NOD/SCID mice of ABCG2+ and ABCG2- PLC/PRF-5 cells were observed. miRNA chip was adopted to screen the differentially expressed miRNAs between ABCG2+ and ABCG2- PLC/PRF-5 cells; and real-time PCR assay was used to confirm the results of miRNA chip. Results：〖WTBZ〗The purity of ABCG2+PLC/PRF-5 cells isolated by MACS method was (84.20±4.52)%. The colony number and size formed by ABCG2+ PLC/PRF-5 cells were more and larger than those formed by ABCG2- cells (47.17±10.50 vs 23.33±7.31, P<0.05). 1×104 ABCG2+ cells could form tumors with at least 5×105 cells needed for ABCG2- cells. The size of tumors generated by 5×105 ABCG2+ cells was larger than that by the ABCG2- cells (\[3.73±1.19\] cm3 vs \[0.72 ±0.57\] cm3, P<0.01). There are 20 miRNAs differentially expressed between ABCG2+ and ABCG2- cells, with 13 up-regulated and 7 down-regulated. Real-time PCR assay was applied to further verify the differential expression of hsa-miR-30a and hsa-miR-630, and the results were in accordance with those of miRNA chip. Conclusion:ABCG2+ PLC/PRF-5 cells in hepatocellular carcinoma cell line PLC/PRF-5 have the properties of cancer stem cells. Twenty miRNAs are differentially expressed between ABCG2+ and ABCG2- PLC/PRF-5 cells, which might play important roles in the carcinogenesis of hepatocellular carcinoma.
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Project Supported:
Project supported by the Natural Science Foundation of Fujian Province (No.2008J0077)
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