| Backgroud In recent years,cytokine-mediated treatment has become an important anti-tumor treatment strategy.Cytokines are a class of immunomodulatory proteins that activate or inhibit the activity of the immune system.Their biological functions are often related to their own properties,concentration used,and microenvironment and so on.A number of cytokines,such as IL-2,TNF,IFN-? and the like,have been extensively studied and are being tried for the treatment of certain types of tumors.IL-18 has been shown to play an important role in tumorigenesis and the progression of tumors.However,the exact role that IL-18 plays in the development of tumors is still controversial.Objective To investigate the effects of interleukin-18 over-expression on the proliferation of human colo cancer HCT-116 cells in vitro and in vivo.Methods 1.A recombinant lentivirus vector containing human IL-18 gene fragment was constructed.2.The HCT-116 cell line stably expressing human IL-18(HCT-116/IL-18)was obtained by recombinant lentivirus transfection.3.The lentivirus transfection efficiency was observed under a fluorescence microscope,and the expression of IL-18 protein in cells was detected by Western blotting.4.The proliferation of HCT-116/IL-18 cells and mock HCT-116 cells was determined by CCK-8 method.5.The expressions of IL-18,Cyclin D1,proliferating cell nuclear antigen(PCNA)and DNA damage repair enzyme(PARP)were detected by Western blotting.6.mock HCT-116 and HCT-116/IL-18 cells were inoculated into left and right axillas of Balb/c nude mice,respect tively.Then the tumorigenicity and the growth of transplanted tumor were observed.7.The expressions of IL-18 and PCNA in xenograft tissues were detected by immunohistochemistry analysis.Results 1.IL-18 gene over-expression in HCT-116 cells can control the proliferation of HCT-116 cells(P<0.05 or P<0.01).2.PARP expression was increased significantly and PCNA,Cyclin D1 expression were decreased in HCT-116-IL-18 cells as compared to that of mock HCT-116 cells(P<0.01).3.The tumorigenicity of HCT-116-IL-18 cell was significantly decreased in nude mice with a tumor-formation rate of 43%,the rate of tumor formation in the mock HCT-116 group was 100%;Compared with control group,HCT-116/IL-18 cell line had a longer tumorigenesis time,slower growth and smaller tumor volume;4.PCNA protein expression was down-regulated in HCT-116-IL-18 xenograft tissue shown by immunohistochemistry analysis(P<0.01).Conclusion IL-18 over-expression inhibited the growth and proliferation of HCT-116 cells both in vitro and in vivo,and themechanismmight be related with IL-18 regulating cell cycle and poiy ADP-ribose polymerase.Background Oxaliplatin is a non-nephrotoxic third-generation platinum complex with strong antitumor activity and less hematological toxicity.It is widely used as a malignant tumor chemotherapy drug in combination with other drugs.Chemotherapy for patients with gastric cancer,colorectal cancer and so on.Thanks to the numerous studies that have been carried out recently in the field of cytosolic DNA sensing,STING(Stimulator of Interferon Genes)is now recognized as a key mediator of innate immune signaling.A substantial body of evidence derived from in vivo mouse models demonstrates that STING-regulated pathways underlie the pathogenesis of many diseases including infectious diseases and cancers.Therefore,STING may become a new target of anti-tumor therapy.Objective To study the changes of oxaliplatin sensitivity and the possible molecular mechanism of colorectal cancer HCT-116 cells transfected with IL-18 gene.Methods 1.The sensitivity of mock HCT-116 group cells(control group)and HCT-116/IL-18 cells(experimental group)to different concentrations of OXA was determined by CCK-8 method.2.The two groups cell were cultured in DMEM containing 10 ?g/ml of OXA,and the protein expression of interferon-stimulator genes(STING),TBK-1,IRF3,IL-18 and PARP was determined by Western Blot.3.The two groups cell were treatment with 10?g/ml OXA medium,and the cell cycle and apoptosis were detected by flow cytometry.4.Plasmids down-regulated STING expression were transiently transfected into HCT-116 cells,and the difference of cell viability was determined by CCK-8 method of mSTING/HCT-116 and mock HCT-116 group cells as treated with OXA.Results 1.Under the same concentration of OXA,the mortality of HCT-116/IL-18 cells decreased compared with mock HCT-116 group cells.2.Compared with mock HCT-116 group cells,the expression of STING,TBK1,IRF3,and the expression of IL-18 and PARP was increased in HCT-116/IL-18 cells.The expression of STING,TBK1,IRF3,IL-18 and PARP were up-regulated after both groups of cells were given the same dosage of oxaliplatin.3.Overexpression of IL-18 gene had no significant effect on the cell cycle of HCT-116 cells.After killing for 48 h by 10 ?g/ml OXA,the cells in the S phase of the experimental group increased significantly,the number of cells in the G2 phase decreased,and the cells percentage in the G1 phas did not change significantly.4.Apoptosis in the experimental group was significantly lower than that in the control group After culturing in 10 ?g/ml OXA medium for 48 h.5.The sensitive to oxaliplatin was reduced afte the express of STING down-regulatedConclusion Overexpression of IL-18 can reduce the sensitivity of CRC HCT-116 cells to OXA,and its mechanism may be that over-expression of IL-18 inhibits the expression of STING gene. |
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