| Objective:Ovarian cancer is one of the most common malignant tumor of the female reproductive system,and has one of the highest mortality rates among gynecological malignancies,performing a threat to women's lives.To improve the therapeutic effect of ovarian cancer considerable efforts have been made,but 5-year survival rate remains at only 30%,and the death of most patients was distant metastasis.Despite all this,the pathogenesis of distant metastasis is still poorly understood.Therefore,investigating the mechanism and developing a effective indicator of metastasis and invasion of ovarian cancer are required.Hypoxia is the basic characteristic of the vast majority of solid tumors and it can contribute to accelerate malignant progression,tumor cell proliferation and metastasis.In vitro researches have shown that hypoxia enhances invasion of the extracelluar matrix.Clinical studies have also demonstrated that tumor hypoxia is correlated with metastatic potential.Hypoxia-inducible factor 1?(HIF-1?),a pivotal role in regulating the intracellular oxygen metabolism,is up-regulated under hypoxic conditions which is associated with tumor cell proliferation,invasion and metastasis.The aim of the present study is to elucidate the effect of hypoxia-induced HIF-la on the invasive potential and the expression of MMP-13 in ovarian cancer cell in response to hypoxia.Methods:1.Hypoxia incubator was used to mimic tumor hypoxia microenvironment.Ovarian cancer A2780 cells were cultured in the normoxic environment and hypoxic environment,respectively.2.The mRNA and protein expression levels of HIF-1? and MMP13 at normoxic environment and hypoxic environment were detected by semi-quantitative RT-PCR and western blot,respectively.3.Small interfering RNA(siRNA)was utilized to transfect ovarian cancer A2780 cell.We evaluate the transfection efficiency by fluorescence microscopy.The expression level of HIF-1? after siRNA transfection was verified by semi-quantitative RT-PCR.4.After being treated with siRNA,the mRNA and protein expression levels of HIF-1?and MMP13 were assayed by semi-quantitative RT-PCR and western blot.5.The effect of hypoxia and siRNA on migration and invasion were tested by counting the cell number through artificial matrix membrane in Transwell chamber.Results:1.Ovarian cancer cell line A2780 was subjected to hypoxia for various time periods,hypoxia increased HIF-1? mRNA expression in a time-dependent manner;under hypoxia,protein level of HIF-1? increased obviously,which was significantly increased after exposure to hypoxia for 24h.2.We observed that fluorescence was widely distributed at the microscope field after siRNA transfection,and the transfection efficiency was above 85%.The result showed that siRNA has been transferred into the ovarian cancer cells,and the experiment acquired well transfection efficiency.3.HIF-1? expression at mRNA and protein levels were significantly inhibited after siRNA transfection compared with untreated control and negative control.The inhibition rate at mRNA level reached 80%and at protein level reached 60%.4.Under hypoxia,mRNA and protein levels of MMP13 were up-regulated,the expression of MM13 and HIF-1? were positively correlated.With siRNA inhibiting the expression of HIF-1?,the mRNA and protein levels of MMP13 were down-regulated.5.The cell migration and invasion in normoxia group were lower than hypoxia group.The migration and the number of invading cells decreased after the treatment of siRNA under hypoxia.Conclusion:Hypoxia can increase protein level of HIF-1? in ovarian cancer A2780 cell,which promote migration and invasion possibly through up-regulating MMP13.It might be an effective strategy targeting HIF-1?-MMP13 to inhibit invasion and metastasis of ovarian cancer.Objective:Dendritic cells are considered as the most powerful antigen presenting cells.Dendritic cells participate in the initiation,maintenance and regulation of the immune response.Among professional antigen-presenting cells,DCs are the most effective in activating naive T cells,which express high levels of major histocompatibility complex class ? and ?,costimulatory molecules and adhesion molecules.The main DC-based immunotherapy is loading DCs with tumor antigens and immune cofactor ex vivo improves DCs' ability of presenting,which induces specific cytotoxic T lymphocytes and killing malignant tumor cells.At present,the DC vaccine application in tumor therapy has become one of the hot spot in tumor immunotherapy.Although the DC-based vaccine has developed for decades,clinical trials have produced contrasted results and no definitive conclusion can be drawn about the real efficacy of this approach.Basic questions,concerning ftinctional deficiency of DCs in tumor microenvironment,are currently unresolved.Finding which factor in tumor microenvironment can impair the maturation of DCs and how to effect on it with help to improve tumor immunotherapy.Methods:1.Cervical cancer cell culture and collection of conditioned medium;Cancer cells were maintained in RPlVn-1640 medium for 24 h.Cuhural supernatants were collected for conditioned medium.2.Bone marrow derived dendritic cell culture: Bone marrow was isolated from femurs and tibias of mice and red blood cells were eliminated.Bone marrow cells were maintained in RPMI-1640 supplemented with GM-CSF and IL-4 for 6 days.LPS was added for stimulation for another 48 h,then non-adherent and loosely adherent cells were harvested.3.Flow cytometry analysis of cell surface molecules: Cells were incubated for 30 min wkh antibodies spedfic for CD80,CD86 and MHC?,washed,and then resuspended with PBS for analysis using FACcan flow cytometer.4.DNA microarray: Gene expression profiling(n=3/group)analyzed by Agilent mouse whole-genome chip,were performed in Shanghai KangCheng bio-techcorporation.Differentially expressed genes were determined via the fold change and p values5.RT-PCR and western blot were used to verify the microarray results.Results:1.To study the influence of tumor microenvironment on DC maturation,normal media and tumor conditional-instructed DCs were activated with LPS as a stimulus.As judged by microscopy,DCs acquired typical morphology in both group.2.In the cancer-instructed cells,the number of cells expressing maturation markers CD80,MHC ? and costimulatory molecules CD86 was significantly decreased comparing with the control group.3.Agilent whole genome microarray assay was used for comparison of gene expression profiles between the control and cancer group.When the fold change is set >1.5 and the p value at <0.05,we found that the expressions of 125 genes were significantly altered in cancer-instructed DC,compared to control cells.4.The accuracy of gene expression level changes from the microarray analysis was validated by RT-PCR and Wesern blot.Tumor cidture supernatant treatment significantly decreased TRAF6 mRNA and protein expression levels of DCs compared to the control cells.Conclusion:DC fails to maturate is an important part of the overall mability of the immune system to sufficiently respond to tumor challenge.One potential mechanism involved in DC dysmaturity in tumor microenvironment is suppression of TRAF6.However,the story of DC maturation in cancer is far from complete.Many questions remain to be answered. |
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