Impacts of hypoxia on the features and chemoresistance of cancer stem cells in Hep-2 cells and underlying mechanism

Abstract

To investigate the effects of hypoxia on the features and chemoresistance of cancer stem cells in Hep-2 cells and underlying mechanism. The shRNA interference recombinant plasmid targeting HIF-1α was synthesized and transfected into Hep-2 cells. The HIF-1α knockdown Hep-2 cells were established after clonal selection and the expression of HIF-1α was measured. The cellular features including proliferation, clonal formation, cell cycle, apoptosis and CD133 phenotype were measured in Hep-2 cells cultured under hypoxic condition in vitro. CD133+ cells were sorted from Hep-2 cells with flow cytometry. Clonal formation test and cisplatin treatment were carried out, and the expressions of related genes (Oct-4, suvivin and p53) in CD133+ cells were measured. HIF-1α knockdown Hep-2 cells was successfully established, as evidenced by the reduced mRNA and protein expressions of HIF-1α. The Hep-2 cells cultured under hypoxic microenvironment showed higher proliferation and clonal formation activity, cell cycle arrest in G0/G1, lower apoptosis, up-regulated CD133, however the effects of hypoxia reduced in HIF-1α knockdown Hep-2 cells. CD133+ cells were successfully sorted from Hep-2 cells, and the CD133+ cells showed increased clonal formation activity and cisplatin treatment resistance in hypoxia. Also the effects of hypoxia on CD133+ cells decreased with HIF-1α knockdown, showing down-regulated Oct-4 and survivin and up-regulated p53. Hypoixa can induce the features of cancer stem cells in Hep-2 cells and increase proliferation, differentiation and chemoresistant ability of CD133+ cells, which might be correlated with the changes in expressions of HIF-1α and related genes regulated by HIF-1α.