Study On Invasion And Metastasis Of ZIP4 In HNSCC

Head and neck carcinoma is the 6th most common type of cancer in the incidence of human malignant tumors,the large majority are squamous cell carcinomas(HNSCC).Due to the local invasion as well as metastasis,the prognosis is very poor.To further clearly understood the mechanisms of local invasion and metastatic have significant implications for finding new therapy target and improving the prognosis of HNSCC.Some researches found that ZN2+ play a significant role in the local invasion as well as metastasis,because of the functions of changing the tight junctions and enhancing epithelial barriers.ZRT-and IRT-like protein(ZIP)family is are a new class of Metal ion transporter,its function is releasing zinc to the cytosol,and work with zinc transporter to maintain the homeostasis of ZN2+ in the plasm.ZIP4 is a member of ZRT-and IRT-like protein family,encoded by SLC39A4,plays an important function in maintaining an intracellular zinc level by the uptake of nourishment zinc from intestinal epithelium cells and release of zinc from the vesicular compartments.ZIP4 is up-regulated in lymphoma?malignant melanoma and metastatic colon cancer,besides,it is correlated with malignancy?clinical stage and so on.Thus,we want to explore the function of ZIP4 and the molecular mechanism by which ZIP4 regulates HNSCC developmemt,and provide experimental evidence for finding therapy target for HNSCC treatment.Methods Part I Validation of ZIP4 expression signature in HNSCC tissues,major experiment:(1)Western blot analysis was used to assess ZIP4 expression in HNSCC tissues and relationships between the expression and the clinical stage.(2)Real-time PCR analysis was used to assess the relative expression levels of SLC39A4 genes.(3)Western blot analysis was used to assess the relationships between ZIP4 expression and the patients' prognosis.Part II In the study involving Fadu cells,as fellows:(1)Cells were treated with varying concentrations of Zinc ion and Zinc ion chelating agent TPEN,and Western Blot analysis was conducted to investigate the alteration in protein expression(2)Transwell assay and wound-healing assay were used to detect the change of tumor invasion ability and migration ability.(3)To established ZIP4 overexpression tumour cell lines,and research the change of cells in morphology and phenotype.(4) Western Blot analysis was conducted to study the alteration in EMT protein expression at different ZIP4 expression status.(5) We used transwell assay and wound-healing assay to evaluate the capacity change of invasion ability and migration ability.(6)Immunofluorescence staining was utilized to dectect the pseudopod change of Fadu tumor cells.Part III: Fadu and Fadu-ZIP4 cells were infected with Luciferase lentiviruses to establish foot metastasis model,and verify by fluorescence intensity,Tumor volume and metastasis were measured by bioluminescence imaging.Further investigation is conducted to known about the invasion and metastasis of HNSCC in vivo.Results IHC results are shown that ZIP4 expression are higher in cells at anterior edge of the invasion and aggressive to adipose tissue than well-differentiated tumors.Overexpression of ZIP4 in HNSCC cells with spindle-shaped and hyperchromatic nuclei,are completely different with tumor cells in keratin pearls,has typical features of mesenchymal cells.q RT-PCR examination revealed that ZIP4 expression at gne levels was higher in tumors tissues compared with adjacent normal tissues,and metastasis has the highest expression.WB detection find that Patients with lowexpression tumors have an excellent prognosis.Fadu was treated with different concentrations of ZN2+,at concentrations as low as 50 u M,the expression of E-cadherin is up-regulated,and N-cadherin and ZIP4 were down-regulated,treated with a relative high concentration,there are opposite results.when treated with Zn2+ chelating agents,these changes can be reversed.The low concentration of ZN2+resulted in impaired migration in both scratchwound experiments and in migration assays is opposite to high ZN2+.After the established of ZIP4 high expression cells,WB assay find the migration and invasion ability was correlated with the expression of ZIP4.Increased pseudopodia formation was associated with increasing ZIP4 expression,and enhancing invasiveness.HNSCC foot metastasis animal model results indicated:ZIP4 high-expression was higher than control group(P<0.05).Femur dissection was conducted after the animals were killed by cervical dislocation,the results shown that carcinoma metastases to in each distal femur in the ZIP4 high-expression groups.The metastatic capacity is significantly higher than control.Conclusion: 1? In HNSCC patient,the expression of ZIP4 is more significant than normal.2? The expression level of ZIP4 in HNSCC patients is associated with outcomes.3? There was a significant effect of the concentration of ZN2+ in migration and invasion ability 4? The overexpression of ZIP4 enhances migration and invasion ability of HNSCC 5? UP-regultaion of ZIP4 markably enhanced tumor metastasis in vivo.