HIF-1? Induces Cancer Stem Cells Properties Of Lung Adenocarcinoma Cells Through N1ICD

Chapter 1 IntroductionA small subpopulation of tumor cells carries stem cell-like properties and behaviors of self-renewal,multi-potential differentiation,tumor initiation and propagation,which was termed as cancer stem cells(CSCs).How do the CSCs come from and be maintained are still unclear.Previous studies have found that hypoxia and HIFs can induce tumor cells to obtain stem cells properties.However,the molecular mechanism is still not clear.Notch signaling is a highly conservative evolutionary pathway between the neighboring cells.Some Studies have showed that Notch signaling pathway plays an important role in maintaining properties of lung CSCs,but exact part of the Notch which functions in the CSCs is unknown.We hypothesized that hypoxia induce tumor cells to acquire properties of CSCs depending on HIF-1?/N1 ICD pathway.We constructed a lung adenocarcinoma cell line with stable expression of HIF-1? or N1 ICD mediated by lentiviral vector.q PCR was used to investigate the expression level of stem cell markers after hypoxia in lung adenocarcinoma cells and the role of Notch1 signaling pathway in the process.We further explored the expression of the stem cell markers in lung adenocarcinoma cell line with stable expression of HIF-1? or N1 ICD by q PCR.The influence of HIF-1?/ N1 ICD on properties of CSCs in lung adenocarcinoma was explored by experiments including drugs sensitivity test,tumorsphere formation assays and tumor formation in nude mice.Chapter 2 Construction of a lung adenocarcinoma cell line with stable expression of HIF-1?Objective: To construct a lung adenocarcinoma cell line with stable expression of HIF-1?.Methods: The HIF-1? fragment was amplified by PCR,then it was used to construct p HBLV-HIF-1?-RFP-Puro plasmid.The recombinant plasmid was identified by PCR and gene sequencing.LV-HIF-1? was packaged by transfection 293 T cell with p HBLV-HIF-1?-RFP-Puro and Auxiliary plasmids which were purified without endotoxin extraction.Lentivirus empty plasmid(LV-RFP-Puro)was also constructed.Dilution counting method was used to determine the titer of LVHIF-1? and LV-RFP-Puro.Puromycin was used to screen stable expression cell line after of A549 cells being infected with LV-HIF-1? or LV-RFP-Puro.Transfection efficiency was observed by fluorescence microscope and Western blotting.Cells proliferation and apoptosis were detected by MTT and flow cytometry.Results: The HIF-1? fragment and recombinant plasmid were verified by PCR and gene sequencing.High-titer of LV-HIF-1? and LV-RFP-Puro was packaged and purified successfully.After being infected with LV-HIF-1? or LV-RFP-Puro,70-80% A549 cells were detected red fluorescence by fluorescence microscope.The expression of HIF-1? in A549-HIF-1? group was significant higher than of control through Western blotting.The Notch1 signaling was activated in A549-HIF-1? group.Overexpression of HIF-1? promotes cell proliferation and induces cell apoptosis.Conclusions: A lung adenocarcinoma cell line with stable expression of HIF-1? was constructed successfully ? Overexpression of HIF-1? can promote cell proliferation and induce cell apoptosis.Chapter 3 Construction of a lung adenocarcinoma cell line with stable expression of N1ICDObjective: To construct a lung adenocarcinoma cell line with stable expression of Notch1 intracellular domain(N1ICD).Methods: Primers were designed according to the NCBI Reference Sequence of Notch1.The N1 ICD fragment was amplified by PCR,then it was used to construct p HBLV-N1ICD-Zs Green-Puro plasmid.The recombinant plasmid was identified by PCR and gene sequencing.The recombinant plasmid was used to transfect 293 T cell with the auxiliary plasmids.Lentivirus empty plasmid(LV-Zs Green-Puro)was also constructed.Dilution counting method was used to determine the titer of LV-N1 ICD and LV-Zs Green-Puro.Puromycin was used to screen stable transfection cell line after infection of A549 cell with LV-N1 ICD or LV-Zs Green-Puro.Transfection efficiency was observed by fluorescence microscope and Western blotting.Cell proliferation and apoptosis were detected by MTT and flow cytometry.Results: p HBLV-N1ICD-Zs Green-Puro plasmid was verified by PCR and gene sequencing.High-titer of LV-N1 ICD and LV-Zs Green-Puro were acquired by packaging and purification.About 70~80% cells was detected green fluorescence by fluorescence microscope after drug screening.The expression of N1 ICD in A549-N1 ICD group was significant higher than in A549-Zs Green-Puro through Western blotting.The Notch1 signaling was activated in A549-N1 ICD group.Overexpression of N1 ICD promotes cell proliferation.Conclusions: A lung adenocarcinoma cell line with stable expression of N1 ICD was constructed successfully.Overexpression of N1 ICD can promote cell proliferation.Chapter 4 HIF-1? induces cancer stem cells properties of lung adenocarcinoma cells through N1ICDObjective: To explore the role of HIF-1? and N1 ICD in CSCs properties of lung adenocarcinoma cells induced by hypoxia.Methods: q PCR was used to investigate the effect of hypoxia on the expression of stem cell markers in lung adenocarcinoma cells and the role of Notch1 signaling pathway in the process.q PCR was also used to investigate the influences of HIF-1? and Notch1 signaling pathway on the expression of stem cell markers in lung adenocarcinoma cells.To explore the effects of HIF-1? and Notch1 signaling pathways on chemo-sensitivity of lung adenocarcinoma cells,the cell viability was detected by CCK8 after DDP or Docetaxel administration and IC50 was calculated respectively.The influence of HIF-1? and Notch1 signaling pathway on self-renewal ability of lung adenocarcinoma cells was checked with tumorsphere formation assays.The effects of HIF-1? and Notch1 signaling pathway on the ability of forming tumor of lung adenocarcinoma cells was studied by transplantation experiment.Results: The expression of CD133,Oct4,c-Myc,ABCG2,ALDH1 and Sox2 was induced by hypoxia treatment in A549 cells,while it was inhibited by interfering Notch1 pathway.Overexpression of HIF-1? or N1 ICD can induce higher expression of multiple stem cell marks and the ability of HIF-1? inducing expression of stem cell marks was suppressed by Notch1 inhibition.Overexpression of HIF-1? or N1 ICD resulted in resistance to DDP and Docetaxel in A549 cells.After Notch1 was inhibited,A549-HIF-1? cells recovered sensitivity to DDP and Docetaxel.The numbers of tumorsphere in A549-HIF-1? and A549-N1 ICD group were more than of control group.HIF-1? inducing self-renewal was suppressed by Notch1 inhibition.The volume and weight of A549-HIF-1? or A549-N1 ICD group was significantly greater than control group.GSI administration can suppress tumor growth in A549-HIF-1? cells.Conclusions: HIF-1? induces CSCs properties of lung adenocarcinoma cells through N1 ICD.