Expression Of Zinc Finger Protein 488 In Nasopharyngeal Carcinoma And Its Mechanism

Part1 Effects of ZNF488 on the biological of nasophaygeal carcinoma HONE1 cell line Objective:To investigate the effects of zinc finger protein 488(ZNF488) on the biological behavior of nasopharygeal carcinoma (NPC) HONE1 cell line.Methods:Retrovirus vector pMSCV or pMSCV-ZNF 488 was transfected into 293FT cells to produce retrovirus, then the Nasopharyngeal carcinoma cell line HONE1 was infected to construct ZNF488-expressing stable cell line and vector control group. The cells were screened by puromycin. MTT assay, colony formation assay and flow cytometry cell cycle analysis were applied to evaluate the proliferation in both HONE1-ZNF488 cells and the control vector. Wound and healing assay and Transwell assay were performed to analyze the effect on cell migration and invasion. Western-blotting was used to dectect protein levels of EMT markers and suppressor gene PTEN.Results:After stable cell lines expressing ZNF488 and the control vector were successfully established, MTT assay and Colony formation assay showed that overexpression of ZNF488 significantly enhanced cell proliferation compared with control group. The flow cytometer anslysis result indicated ZNF488 might accumulated cells in S phase. And wound and healing and Transwell assay showed that ZNF488-expressing cells endowed the cells with much more migratory and invasive properties than the vector. Mechanistically, we found that overexpression of ZNF488 lead to the down-regulation of epithelial markers E-Cadhesion、α-Catenin and up-regulation of mesenchymal markers Fibronectin、Vimentin, as well as the downregulation of tumor suppressor gene PTEN.Conclusion:ZNF488 might promote potentiation of growth, proliferation, invasion and metastasis of HONE1 cell line, which might be associated with induced EMT and downregulation of suppressor gene PTEN.Part2 ZNF488 Induces the Epithelial-Mesenchymal Transition and Promotes Human Nasopharyngeal Carcinoma Progression through the Wnt and TGF-βSignaling pathway Postgraduate Zong Dan Supervisor Professor He XiaObjective:Nasopharyngeal carcinoma (NPC) shows a higher incidence and risk of invasion and metastasis than other head and neck malignancies. However, the molecular mechanisms underlying the invasiveness and metastatic progression remain elusive. Here, we investigated the role of the Zinc Finger Protein 488 (ZNF488) in promoting invasion, migration and malignancy of nasopharyngeal carcinomas (NPCs).Methods:The endogenous expression of ZNF488 in NPC tissues, non-cancerous tissues and NPC cell lines were detected by Reverse transcriptase-Quantitative polymerase chain reaction (RT-qPCR). Immunohistochemisty (IHC) was used to study protein expression in paraffin-embedded NPC tissues. The invasion and migration capacities were evaluated by Wound healing and Transwell assays in ZNF488-expressing and control cells. Small interfering RNA (siRNA) technique was employed to knock-down ZNF488 protein expression. Immunofluorescence (IF) and immunoblotting analysis were used to examine protein changes following ZNF488 overexpression. Colony formation, soft-agar and xenograft experiments were performed to study transforming ability in vitro, and tumourigenic ability in vivo. Gene-Chip analysis showed gene expression differences between ZNF488-expressing and vector cells. We also performed cluster analysis to find the pathway. Luciferase assay was performed to detect the transcription factor TCF/LEF. Western blotting was performed to verify the pathway. Statistical tests were as follows:Student’s t-test, x 2 test, Kaplan-Meier method and the log-rank test and multivariate analysis using the Cox proportional hazard model.Results:ZNF488 was overexpressed in NPC tissues compared with non-cancerous tissues, and was highly expressed in the established high-metastasis NPC clone 5-8F and S18 relative to their corresponding low-metastasis clones. Survival analyses revealed that higher expression of ZNF488 correlated with poor clinical outcomes, and it was an independent prognostic factor for overall survival, progression-free survival, distant-metastasis-free survival and locoregional recurrence-free survival. Intriguingly, ZNF488 is located predominantly at the leading edge of tumour nests; and functional studies indicate that overexpression of ZNF488 provokes invasion, migration and malignancy of NPC cells, whereas knockdown of ZNF488 alleviates invasion, migration and malignant capabilities. The overexpression of ZNF488 also promotes NPC tumour growth in vitro and in vivo. Finally, we show that ZNF488 induces Epithelial-Mesenchymal Transition (EMT) by activating both Wnt pathway and TGF-β pathway which induce EMT transcription factors TCF/LEF that is capable of triggering FOS expression.Conclusion:Together, our findings suggest that ZNF488 plays a crucial role in promoting NPC progression and could be a useful biomarker forpredicting clinical outcome.