| Objective: To explore the relationship between Octamer4(Oct4) andABCG2in liver CSCs self-renewal ability and chemotherapy resistance,we enriched CD90+CD133+HCC cancer stem cells.Methods: Liver cancer stem cells were isolated from establishedhepatocellular carcinoma (HCC) cell lines (HepG2line and Hep3B line)via flow cytometry. To enrich liver cancer stem cells we used sphereformation by serum-free medium. Cells were divided into two groups,including liver cancer cells group and liver cancer stem cells group. Cellproliferation capacity was detected using colony formation assays and cellactivity was detected using MTT assays after doxorubicin treatment.Expression of CD90, CD133, Oct4and ABCG2mRNA and protein levelswere detected by PCR and western blot, respectively.Results: We found that the single cell proliferation was stronger inliver cancer stem cells, single-cell colony formation assay showed thatcultured after14days colony formation rate was8/27(30%) in Hep3Bcells group,12/23(52%) in Hep3B cancer stem cells group (p<0.05);7/38(18%) in HepG2cells group,9/27(35%) in HepG2cancer stem cells group (p<0.05). After treated by doxorubicin for48hours, MTT assaysshowed that liver caner stem cells were more drug-resistant whencompared to parental cancer cells. The cell activity of the HepG2cell groupwas (38.17±6.92)%, the HepG2cancer stem cells group was (69.88±5.43)%(p<0.05); the Hep3B cells group was (50.16±4.89)%and theHep3B cancer stem cells group was (78.53±7.86)%(p<0.05). RT-PCRand real-time PCR analysis showed that CD90, CD133, Oct4and ABCG2genes were significantly over-expressed on mRNA level in liver cancerstem cells compared to liver cancer cells (p<0.05). Western blot analysisshowed that Oct4and ABCG2were increased in liver CSCs group, therewas significant difference between liver CSCs and parental liver cancercells (p<0.05).Conclusion: Oct4and ABCG2are highly expressed in enrichedCD90+CD133+liver CSCs and are closely associated with chemotherapydrug resistance. |
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