Moreover, statistical analysis (Fig.?4b, d) suggested the increased activities of NF-B and Akt pathways in HPV positive HNSCC tissues when compared to HPV negative tissues. Open in a separate window Fig.?4 Relative activation of NF-B and Akt pathways and SLPI downregulation in HPV positive HNSCC. the regulatory mechanisms underlying E6-induced HNSCC progression. Then, exogenous secretory leukocyte protease inhibitor (SLPI) was added into the cell culture to investigate whether it could maintain its tumor suppressor effect on E6-expressing HNSCC cells. Results HPV E6 oncogene could promote the proliferation, cell cycle period, apoptosis resistance, migration and invasion of HNSCC cells by activating NF-B and Akt pathways. Immunohistochemical analysis conducted on HNSCC tissues illustrated that SLPI was further downregulated in HPV positive HNSCC compared to HNSCC without HPV infection. Exogenous SLPI significantly inhibited HPV E6-mediated malignant phenotypes in HNSCC cells by inhibiting the activation of NF-B and Akt and signaling pathways. Conclusions This study demonstrated that E6 oncogene led to the malignant transformation of HNSCC cells by regulating multiple pathways. SLPI could reverse the effect of E6 oncogene on HNSCC, implying that the functional inhibition of E6 by SLPI may be exploited as an attractive therapeutic strategy. luciferase (Beyotime, China), which was used to normalize data for transfection efficiency. After 24?h of transfection, the cells were treated with exogenous SLPI (40?g/mL) or the same volume of ddH2O. The cells were then Mouse monoclonal to IFN-gamma cultivated for 12? h and cell lysates were analyzed using a dual luciferase reporter assay kit (RG027,Beyotime, China) on Modulus? (Turner Biosystems, Sunnyvale, CA, USA). Statistical analysis DDR-TRK-1 Statistical analysis was performed with SPSS 21.0 software in this study. All numerical data was expressed as mean??SD from triplicate DDR-TRK-1 experiments and comparisons between two or more groups were performed by Students two-tailed test or one-way ANOVA. values less than 0.05 were considered statistically DDR-TRK-1 significant. Results Establishment of HPV E6-expressing HNSCC cells To analyze the functional role of E6 oncogene in HNSCC progression, the establishment of HPV E6-expressing HNSCC cells was needed. Firstly, HN4 and HN30 cells were infected with a lentiviral vector carrying HPV E6 gene. Then, the tumor cells stably expressing HPV E6 were selected with puromycin (10?g/mL). After the construction of E6 stably expressing HNSCC cells, we determined the overexpression of E6 at mRNA and protein levels. As suggested by Fig.?1a, HN4 cells with a stable transfection of E6 presented approximately 15-fold E6 mRNA overexpression when compared to E6 negative cells, while the lenti-E6 infection resulted in about 20-fold overexpression of E6 oncogene in HN30 cells. Immunofluorescence assay demonstrated that E6 protein was expressed in HNSCC cells after lentivirus transfection (Fig.?1b). Western blot results also illustrated that E6 oncogene was overexpressed in HN4 and HN30 cells (Fig.?1c). The above data revealed that we successfully established HPV E6-expressing HNSCC cells. Open in a separate window Fig.?1 Overexpression of E6 oncogene in HNSCC cells with a stable lentivirus transfection. a mRNA level of E6 oncogene was elevated in HNSCC cells with lentivirus transfection, DDR-TRK-1 as demonstrated by qPCR technique. b Immunofluorescence assay illustrated the elevated protein level of E6 oncogene in HNSCC cells after lentivirus transfection. c Western blot results demonstrated the overexpression of HPV E6 oncogene in HN4 and HN30 cells. ***P? ?0.001. ****P? ?0.0001 (scale bar: 20?m) HPV E6 oncogene influences the biological characteristics of HNSCC cells in vitro Due to previous findings that E6 oncogene may account for the malignant transformation of cancers, we aimed to investigate whether it could affect the proliferation of HNSCC cells. Firstly, MTT assay was performed to evaluate the effect of E6 oncogene on the proliferation of HNSCC cells. As a result, the growth rates of HN4 and HN30 cells with stable E6 expression were significantly higher when compared to control cells (Fig.?2a, b). Moreover, flow cytometry analysis revealed that E6.