The Role And Mechanism Of SATB2/NOX4 And IL-6/JAK-STAT3 Signaling Pathways In Oral Squamous Cell Carcinoma

Objective:1.To test expression level of SATB2 of 58 OSCC semples,analysis the relationship between the level of SATB2 pathologic and clinical features of OSCC.2.To detect biological function of SATB2 in HN4 cell line.3.To explore the molecular mechanism of SATB2/NOX4 and IL-6/JAK-STAT3 signaling pathway in OSCC cells' proliferation.Methods:1.Immunohistochemistry was used to detect SATB2 expression in 58 tissue samples from OSCC patients,the correlation between SATB2 expression pattern and clinicopathological parameters,prognosis in OSCC patients was analyzed;2.The expression of SATB2 in OSCC tissues and para-carcinoma tissues are tested by Western blot and RT-PCR Lenti-CMV-SATB2-IRES-eGFP-PGK-Puro encoding full-length human SATB2 cDNA was transfected into HN4 cell lines to obtain experiment group cells which express high level of SATB2.CCK-8 assays,Would healing,transwell,invasion were performed to mesasure the biological properties of HN4 cell line and SATB2 over-expression cell line;A xenograft tumor model was established to further determine if the SATB2 over-expression cell line had effect on tumor growth in vivo.3.Western blot were conducted to assess the alteration of signal pathway protein IL-6,phosphorylation of the STAT3 and expression pattern of E-cadherin and Vimentine in HN4 cell line and SATB2 over-expression cell line,which plays an important role in EMT;Western blot,RT-PCR and CHIP were conducted to study the potential mechanism of SATB2 in HN4 cells' carcinogenesis.Result:1.High expression of SATB2 in OSCC specimens was significant associated with tumor size,cervical lymph node metastasis,clinical staging and poor prognosis.2.To verify the efficiency of lentiviral vector Lenti-CMV-SATB2-IRES-eGFP-PGK-Puro transfected into HN4 cell line.In vitro,the experimental results showed that overexpression of SATB2 significantly enhances HN4 cells' proliferation,migration and invasion ability;In vivo,overexpression of SATB2 promoted the tumorigenicity of HN4 cells.3.Western blot analysis demonstrated that overexpression of SATB2 markedly activated IL-6/JAK-STAT3 signaling pathway while protein expression of E-cadherin and Vimentin are partially altered;Bioinformatics analysis showed that SATB2 protein can regulate NADPH oxidase type 4(NADPH Oxidase 4,NOX4)gene expression,Using small interfering RNA knockdowned NOX4,cells' proliferation were decreased both in vitro and in vivo,cell cycle was arrested in G1 phase;All this results suggested that SATB2 might mediate cells' proliferation not only by regulating IL-6/JAK-STAT3 signaling pathways but also by regulating NOX4.Conclusion:SATB2 is abnormally high-regulated in a large proportion of OSCC tissues and it may serve as a potential prognostic biomarker for OSCC;Moreover,overexpression of SATB2 significantly promotes HN4 cells'proliferation,migration and metastasis;Furthermore,SATB2 can be used as a potential target for the treatment of Oral squamous cell carcinoma and may provide novel perspective for developing new regimen.