| Background: Laryngeal cancer is the most common malignant tumor in the head and neck,and the mortality rate is extremely high.But the molecular mechanisms that influence the development and development of laryngeal cancer have not been systematically elucidated at present.NK4,as a hepatocyte growth factor(HGF)antagonist,not only It acts as an antagonist of inhibiting HGF/c-Met-induced tumor growth,metastasis and invasion,and can also inhibit HGF/c-Met pathwayindependent angiogenesis factor(VEGF,b FGF)-induced tumor angiogenesis,which eventually leads to tumor cell apoptosis.At present,no research has confirmed the role of NK4 gene in laryngeal cancer and whether it plays a specific biological function in the malignant progression of laryngeal cancer and by which mechanism.The occurrence of laryngeal cancer is closely related to the dysregulation of various signal transduction pathways.Wnt / ?-catenin signaling pathway is one of the most common deregulated signaling pathways in laryngeal cancer,and abnormal activation of Wnt / ?-catenin signaling is closely associated with initiation,development and infiltration.It has been confirmed that it is related to metastasis and recurrence of laryngeal cancer.Objective: To investigate the effects and related mechanisms of the NK4 gene on laryngeal cancer AMC-HN-8 cell proliferation,migration,apoptosis,cell cycle and tumorigenesis in nude mice.Methods: Laryngeal carcinoma AMC-HN-8 cells were transfected with attenuated Salmonella carrying NK4 gene,and the expression of NK4 in AMC-HN-8cells was detected by quantitative real-time polymerase chain reaction(q RT-PCR).The effects of NK4 gene on the proliferation,migration,invasion,apoptosis and cell cycle of laryngeal carcinoma AMC-HN-8 cells were detected by MTT assay,cell scratch assay,Transwell assay and flow cytometry.NK4 was evaluated by a nude mouse tumorigenesis model.Effect of genes on AMC-HN-8 cell proliferation in vivo;tumor tissue was observed by HE staining,Western blotting and immunohistochemistry were used to detect the expressions of DKK1,Wnt1 and ?-Catenin in transplanted tumor tissues and control tissues.Results: The morphology and number of cells changed after transfection;q RTPCR results showed significantly higher expression of NK4 in the transfection group than in the control group(P <0.01),with expression over time of transfection.It was shown to be increased;MTT results showed that NK4 was highly expressed.It can inhibit the proliferative capacity of AMC-HN-8 cells(P <0.01).With the addition of30% expression supernatant,the inhibitory activity did not increase with increasing dose.The scratch test results showed that with the increase of transfection time,the healing rate of cell scratches was lower than that of the control group,and the cell migration ability decreased(P<0.01).Transwell invasion assay detected that NK4 inhibited the invasion of laryngeal cancer cells;Annexin V/PI double staining assay showed that NK4 gene induced increased apoptosis of AMC-HN-8 cells(P<0.01).After 48 hours of transfection,the proportion of G1 and G2/M phases in the transfection group was lower than that of the control group,and the proportion of cells in S phase was higher than that of the control group,which induced cell cycle arrest in S phase(P<0.01).Nude mice subcutaneous tumorigenic experiments showed that the tumorigenic ability of AMC-HN-8 cells was attenuated after high expression of NK4(P<0.05).WB detection showed that the expression of DKK1 protein was increased after the high expression of NK4,and the protein expression of Wnt1 and ?-Catenin was decreased.The results of HE staining showed that the squamous cell carcinoma cells in the control group were distributed in nests,with large and dark nuclei,and the split images were easy to see;in the treatment group,most of the cancer tissues were necrotic,with only a few remaining cancer cells,and some cancer cells had degenerated.Immunohistochemical detection showed that the cancer cells in the control group had low expression of NK4,while the treatment group had high expression of NK4,and some cancer cells had no expression.Compared with the control group,the expression of DKK1 was increased in the treatment group,and the expression of ?-Catenin protein was decreased compared with the control group.Conclusion: The NK4 gene carried by attenuated Salmonella can inhibit the proliferation,migration and invasion of laryngeal carcinoma AMC-HN-8 cells,promote apoptosis,and inhibit the tumorigenicity of AMC-HN-8 cells in nude mice.The anti-laryngeal cancer mechanism of NK4 may be related to its participation in the regulation of DKK1/Wnt/?-catenin signaling pathway. |
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