The Preliminary Study Of Cancer Stem-like Cells In The Hepatocellular Carcinoma Cell Lines

Objectives: Hepatocellular carcinoma (HCC) is one of the most common solid malignanies in China. It's possible that hepatocellular carcinoma contains cancer stem cells responsible for its malignancy. The purposes of this study areâ‘´to isolate and identify cancer stem-like cells from the PLC/PRF-5 cell line.⑵to study the relationship between ABCG2 and tumorigenic ability.⑶to investigate the differentially expressed miRNA in PLC/PRF-5 cell line,which are useful for further studies on specific functions of miRNA in pathogenesis of HCC.Methods:â‘´The expression of ABCG₂⁺ in the PLC/PRF-5 cell line was examined by flow cytometry. ABCG₂⁺ were isolated from the PLC/PRF-5 cell line using magnetic activated cell sorting(MACS)method.The purity was analyzed by flow cytometry. The proliferation and the ability of colony forming in soft agar of ABCG₂⁺ and ABCG2- cells were observed. ABCG₂⁺ and ABCG2- populations were inoculated into the axillary fossa of NOD/SCID mices at the injection dose 5×10³,1×10⁴,5×10⁴,5×10⁵ respectively.The mice were monitored for palpable tumor formation and were sacrificed after 6 weeks to assess tumor formation rate.⑵MicroRNA microarray was used to investigate the differentially expressed miRNAs in the PLC/PRF-5 cell line. ABCG₂⁺ cells and ABCG2- cells total RNAs were extracted by mirVana RNA Isolation Kit.Hybridizations were made by applying the cell miRNAs to mammalian miRNAs chip.Data analysis was proceeded by Agilent Scan Control software version A.7.0 and Agilent Feature Extraction (FE) software version 9.5.Results: Flow cytometry was used to test the expression of ABCG2 in the PLC/PRF-5 cell line.The purity of ABCG₂⁺ cells in the PLC/PRF-5 cell line was 5.58～17.10%. The ABCG₂⁺ cells were isolated examined by flow cytometry. Its purity is more than 80%. Cell growth curve indicated that proliferative capacity of the ABCG₂⁺ cells was better than the ABCG2- cells . Clony formation assay was carried out in 6-well Plate.Plates were maintained at 37℃in humidified incubator.After 14 days,the number of the colony formation was assessed by counting under microscope. The number of clony formation of ABCG₂⁺ cells and ABCG2- cells is (47.17±10.50) and (23.33±7.31). ABCG₂⁺ cells generated a tumor with 1×10⁴ cells compared with at least 5×10⁵ cells needed for ABCG2- cells.At the same injection dose (1×10⁶ cells),the tumor generated by the ABCG₂⁺ cells was larger than that of the ABCG2- cells. Total 20 differentially expressed miRNAs were found,including 13 up-regulation and 7 down-regulation miRNAs.Conclusions: The ABCG₂⁺ cells had the properties of cancer stem-like cells, which possesd the ability of self-renew and proliferation, furthermore, they had stronger ability of forming clonies and tumorigenic in xenotransplantation .