| Head and neck squamous cell carcinoma (HNSCC) is a common malignancy,characterized with insidious initiation, aggressive invasion, predisposed recurrence and metastasis, and poor prognosis. Despite that technique advances in therapy in recent years have improved quality of life, 5-year survival rate of HNSCC has remained nearly unchanged for past 20 to 30 years. Mortality of the disease remains high because of the development of distant metastases, the emergence of therapy-resistance and locoregional recurrences. The therapy-resiatance and recurrence in HNSCC is a troublesome and an unsolved problem. It is the crucial to get a deeper insight into the biology and the mechanism behind the therapeutic resistance of this disease to find more effective modalities for overcoming this specific situation.Recent studies have demonstrated that a subpopulation of tumor cells residing in static cell cycle, known as cancer stem cells (CSCs), deserve the ability to undergo self-renewal and di?erentiation potency (properties of normal stem cells) and are responsible for the tumorigenesis and tumor growth. Scholars have demonstrated the existance of CSCs in many solid tumours, including HNSCC, which offer a new scope and breakthrough for exploration of therapy-resiatance in HNSCC and improvement in treatment.Hypoxia induces a wide range of changes in cellular biology and molecular biology, which may affect the progress of proliferation, apoptosis and damage repair. The hypoxic microenvironment in tumor is considered to play a critical role in induction of therapeutic resistance in many solid tumors (including HNSCC) and is correlated with post-treatment recurrences, and poor outcomes and prognosis. The central regulator of oxygen and energy supply in tumor is hypoxia-inducible factor, an oxygen-dependent nucleus transcription protein. Hypoxia-inducible factor have become an important target gene in the investigation of the hypoxic microenvironment in tumor. It plays a critical role in the process of the adaptation to hypoxia by inducing related gene expression and participating in the regulation of many downstream genes.As a subpopulation of tumor cells, CSCs are maintained in the same environment with full-differentiation tumor cells. The living microenvironment of tumor is hypoxic, which implys that CSCs are also surviving in hypoxic microenvironment. It is of great interest to know whether hypoxic microenvironment can influence the proliferation and differentiation of CSCs, and how the hypoxic microenvironment-mediated therapy-resistance is related to CSCs. Based on the theory of hypoxic microenvironment and CSCs, our investigation is to explore the influence of hypoxic microenvironment on CSCs'proliferation and differentiation, with further insight into the relationship between microenvironment-mediated chemotherapy-resistance and CSCs.Part one Impact of the hypoxic microenvironment on the phenotype of cancer stem cells in hep-2 cells Objective:By testing the effects of hypoxic microenvironment on CD133 phenotype,proliferation, apoptosis, cell cycle and related gene expression of hep-2 cells, we explored the impact of the hypoxic microenvironment on the phenotype of cancer stem cells in hep-2 cells, and further discussed the mechanism of by which the cells are influenced. Methods:1 hep-2 cells were cultured in hypoxic and normoxic environment for different times (6h, 12h, 24h, 36h and 48h), then the CD133 phenotype of the cells'was measured with flow cytometry.2 hep-2 cells were cultured in hypoxic and normoxic environment, after which the growth curve was measured with MTT method. 3 hep-2 cells were cultured in hypoxic and normoxic environment, followed by measured of cell cycle and apoptosis with flow cytometry.4 clone formation of hep-2 cells cultured in hypoxic and normoxic environment were measured with soft agar clonal formation experiment.5 hep-2 cells were cultured in hypoxic and normoxic environment, and mRNA expression of HIF-1αwas measured with RT-PCR.6 hep-2 cells were cultured in hypoxic and normoxic environment, then protein expression of HIF-1αwas measured with Western blot. Results: 1 CD133 was expressed in the hep-2 cells cultured under both hypoxia and normoxia.The rate of CD133+ cells under normoxia is 0.82±0.13%, whereas it counted 1.41±0.11%, 5.16±0.23%, 2.43±0.15%, 2.07±0.33% and1.05±0.23% under hypoxia for different times (6h, 12h, 24h, 36h, 48h).2 measured with MTT, the proliferation activity of hep-2 cells in hypoxia is higher than that in normoxia.3 measured with flow cytometry, the apotosis rate of hep-2 cells in hypoxia is lower than that of in normoxia, and hep-2 cells in hypoxia expressed apparente block in G0/G1.4 measured with soft agar clonal formation experiment, the number of clones'formed in hypoxia is more than that in normoxia.5 measured with RT-PCR, the mRNA expression of HIF-1αin hep-2 cells showed no difference between hypoxia and normoxia groups.6 measured with Western blot, the protein expression of HIF-1αin hep-2 cells in hypoxia is higher than that in normoxia.Conclutions:The hypoxic microenvironment can induce and reinforce the features of cancer stem cells in hep-2 cells, which might be correlated with the changes of HIF-1αexpression levels.Part two Construction of RNA interference expressing plasmid of human HIF-1αand establishment of knockdown cell line in laryngeal squamous cell carcinoma hep-2 cells Objective: To construct RNAi plasmid targeting HIF-1αand to establish HIF-1αknockdown hep-2 cells.Methods:1 the RNA interference fragments targeting HIF-1αwere designed and synthesized, anneal them into vector plasmid and construct recombinant plasmid pGPU6/Hygro-HIF-RNAi.2 the recombinant plasmids were transfected into human laryngeal carcinoma hep-2 cells with liposome, selected with antibiotics for stable transfection.3 RT-PCR and Western blot were used to investigate the changes of HIF-1αexpression.Results:1 recombinant plasmid pGPU6/Hygro-HIF-RNAi and HIF-1αknockdown cell line in human laryngeal carcinoma hep-2 cells was successfully established.2 measured with RT-PCR and Western blot, the genetic transcription and protein expression of HIF-1αwere inhibited significantly.Conclusion:Recombinant plasmid pGPU6/Hygro-HIF-RNAi was proved to be effectively transfected into hep-2 cells and downregulate the expression of HIF-1α, which lays the foundation for further exploration of the gene. Part three Impact of HIF-1αknockdown on the phenotype of cancer stem cells in hep-2 cellsObjective:By testing the changes of CD133 phenotype, proliferation, apoptosis, cell cycle in HIF-1αknockdown hep-2 cells, to explore the impact of HIF-1αon the phenotype of cancer stem cells in hep-2 cellsMethods:1 hep-2 cells and HIF-1αknockdown hep-2 cells were cultured in hypoxic environment for different times (12h and 24h), after which the CD133 phenotype of the cells'was measured with flow cytometry. 2 hep-2 cells and HIF-1αknockdown hep-2 cells were cultured in hypoxic environment, followsd by the proliferation was measured with MTT method.3 hep-2 cells and HIF-1αknockdown hep-2 cells were cultured in hypoxic environment, then the cell cycle and apoptosis was measured with flow cytometry.4 clonal formation of hep-2 cells and HIF-1αknockdown hep-2 cells cultured in hypoxic environment were measured with soft agar clonal formation experiment.Results:1 Both hep-2 cells and HIF-1αknockout hep-2 cells cultured under hypoxic environment for 12 and 24 hours expressed CD133, the expression rate in hep-2 cells is 5.16±0.23% and 2.43±0.15%,whereas the expression rate in HIF-1αknockdown hep-2 cells is 1.86±0.15% and 1.21±0.13%.2 Measured with MTT, the proliferation activity of hep-2 cells in hypoxia decreased after knockdown of HIF-1α.3 Measured with flow cytometry, after knockdown of HIF-1αthe apotosis rate of hep-2 cells in hypoxia increased, meanwhile the G0/G1 block. alleviated. 4 Measured with soft agar clonal formation experiment, the number of clones'formed of hep-2 cells in hypoxia is more than that of HIF-1αknockdown hep-2 cells.Conclutions:Knockdown of HIF-1αcan weaken the features of cancer stem cells induced by hypoxic microenvironment in hep-2 cells, HIF-1αplays an important role in the enrichment of CSC induced by hypoxic microenvironment and might participate in the proliferation regulation of CSC. Part four The role of HIF-1αin regulating proliferation and DDP resistance of cancer stem cells under hypoxic microenvironmentObjective:To explore the role of HIF-1αin regulating proliferation and DDP resistance of CD133+ cells under hypoxic microenvironment, and further address the mechanism behind the phenomenon.Methods:1 CD133+ cells were sorted from hep-2 cells and HIF-1αknockdown hep-2 cells with flow cytometry.2 clonal formation of CD133+ cells cultured in hypoxic environment were measured with limiting dilution assay clonal formation test.3 CD133+ cells cultured in hypoxic environment were treated with DDP, after which the proliferation inhibition was measured with MTT method.4 CD133+ cells were cultured in hypoxic environment, followed by the expressions of related genes were measured with flow cytometry.Results:1 CD133+ cells were successfully sorted from hep-2 cells and HIF-1αknockdown hep-2 cells with flow cytometry.2 measured with limiting dilution assay clonal formation test, the clonal formation of CD133+ cells from hep-2 cells was higher than that of HIF-1αknockdown hep-2 cells, the clonal formation rate was 85.35±3.34% and 50.43±5.12%.3 measured with MTT, the proliferation inhibition of CD133+ cells in hypoxia treated with DDP increased after knockdown of HIF-1α.4 the expressions of related genes(Oct-4, suvivin and p53) in CD133+ cells cultured in hypoxic environment were measured with flow cytometry, the fluorescence index of Oct-4, suvivin and p53 in CD133+ cells from hep-2 was 3.64±0.21, 0.88±0.16 and 1.39±0.23, while the fluorescence index of Oct-4, suvivin and p53 in CD133+ cells from HIF-1αknockdown hep-2 was 2.23±0.15, 1.25±0.11 and 1.07±0.17.Conclutions:CD133 + cells of hep-2 in hypoxia possess higher proliferation and differentiation activity, and resist to DDP treatment. HIF-1αknockdown can decrease the proliferation and differentiation activity and the resistance to DDP treatment, accompanied with changes of Oct-4, p53 and Survivin. HIF-1αplays a role in regulating CSC in terms of proliferation, differentiation and DDP resistance under hypoxic microenvironment, probably by regulating Oct-4, p53 and Survivin expression.
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