Researchs On The Interaction And Mechanism Between The Regulation Of Redox Capability And Characteristics Of Cancer Stem Cells In Hepatocellular Carcinoma Cell Lines

ObjectiveThe purpose of this paper is to study the mechanism of liver cancer cells regulating oxidative stress and the role of cancer stem cells in the process of anti-oxidative stress,to explore the molecular targets both involved in the regulation of tumor stemness characteristics and anti-oxidative stress process,and intervene those targets artificially.Then the changes of antioxidant stress'ability and stemness characteristics were further compared with before in liver cancer cells.Therefore,from the point of view that cancer stem cells with the stronger anti-oxidative stress capacity,new strategies and ideas for the clinical treatment,basic research and new drug research and development of tumors are provided.MethodFirst,explore the preliminary mechanism of liver cancer resistance to ROS through comparisons of the resistance to exogenous ROS?H₂O₂?and the expression differences of the moleculars involved in ROS regulation between the two Hepatocellular carcinoma?HCC?cell lines Huh7 and SMMC-7721.Second,isolate anti ROS subsets in Huh7 cells and compare anti ROS capacity and the expression of related molecules regulating ROS metabolism with normal Huh7 cells to screen out key molecules??-catenin?in the regulatory pathway.Third,the?-catenin blocker XAV939 is used to block the Wnt/?-catenin signaling pathway,and the anti-ROS capacity and stemness characteristics of Huh7 cells were observed and analysed.Four,interference ROS production gene Nox1 with lentiviral to reduce intracellular ROS generation,and compare the changes of anti ROS ability,cancer stem characteristics and related moleculars between experimental group Huh7-Nox1 and control group Huh7-GFP?green fluorescence?and then analyse and research the relationship between ROS regulation ability and the cancer stem characteristics.ResultsAfter H₂O₂ treatment,compared with SMMC-7721 cells,Huh7 cells had less apoptotic necrosis,stronger activity,lower intracellular ROS level,and relatively high expression of stem molecule CD133,Bmi-1,DNA repair gene Rad51 and ROS scavenger enzyme gene SOD2.There was a subgroup?Huh7-ROS?of stronger ability to resist ROS in Hepatocellular carcinoma cell line,which expressed high bmi-1,CD133,Bcl-2,SOD1 and had less apoptosis or necrosis,more vitality and lower intracellular ROS level than the ordinary cells after being treated with H₂O₂.Compared with Huh7 cells untreated with XAV939,the cells in Huh7-XAV939 group treated with XAV939 had less cell apoptosis and necrosis,more vitality,lower intracellular ROS levels after treatment with H₂O₂.While without H₂O₂ that is when the concentration of H₂O₂ was 0umol/ml,the ordinary Huh7 cell group and the Huh7-XAV939 group had no difference in apoptosis and necrosis and cell viability.Moreover,compared with the control cell group with the green fluorescent?Huh7-GFP?,the experimental cell group?Huh7-siNox1?of which the ROS production gene Nox1 was intervened with lentivirus not only had higher activity after H₂O₂ treatment,but also became more tumorigenic and had much stronger colony formation capacity in vivo.ConclusionThe ability of hepatoma cells to resist ROS depends on the interaction of tumor stem molecules,DNA repair genes and ROS scavenging enzyme genes;there is the presence of anti ROS cell subset in HCC cells and this subgroup highly express tumor stem molecules,DNA repair genes,ROS scavenging enzyme genes and the gene?-catenin which is a tumor stem cell signaling molecule.The gene?-catenin participating in the regulation of cancer stem characteristics is also involved in regulation of ROS resistance.XAV939,a blocker of?-catenin has the potential to be an adjuvant chemotherapy drug,for that XAV939 itself don't induce necrosis and apoptosis in HCC,but it can inhibit cancer cell's ability to resist to ROS,then it may enhance the effect of chemotherapy drug.There is a presence of anti ROS subset in HCC,and these cells which have anti-ROS ability and high expression of ROS scavenging enzyme and DNA repair enzyme may be cancer stem cells.To reduce endogenous ROS production with lentivirus Nox1-siRNA can enhance anti-ROS ability and features of CSCs in cell line Huh7,so Nox1 gene has the potential to be a reference index to clinical outcome and a clinical gene therapy target.