Molecular Mechanism Of Resolvin D1 In The Invasion And Metastasis Of Non-keratinizing Nasopharyngeal Carcinoma

Part I Epithelial to mesenchymal transition in non-keratinizing nasopharyngeal carcinoma and its association with high mobility group box 1Objective: To detect the different expression of EMT markers in non-keratinizing nasopharyngeal carcinoma and nasopharyngitis.The expression and localization of HMGB1 in the two types of tissues were detected,and the role of HMGB1 in EMT in non-keratinizing nasopharyngeal carcinoma was explored.Methods: Forty-two cases of non-keratinizing nasopharyngeal carcinoma and 30 cases of nasopharyngitis were collected.The correlation between HMGB1 expression and clinicopathological features,as well as the correlation between HMGB1 expression and EMT marker expression was analyzed.The expression and localization of Ecadherin,vimentin and HMGB1 in tissues were detected by immunohistochemistry and immunofluorescence.Western-blot was used to detect the protein expression of ZO-1,N-cadherin,E-cadherin,vimentin and HMGB1 in the tissues of non-keratinizing nasopharyngeal carcinoma and nasopharyngitis.Results: The expression of HMGB1 was not significantly correlated with the age and gender of patients,but was highly correlated with TNM staging.HMGB1 expression was negatively correlated with E-cadherin expression,while positively correlated with vimentin expression.With immunohistochemistry and immunofluorescence,the expression of E-cadherin in non-keratinizing nasopharyngeal carcinoma was lower than that in nasopharyngitis(P<0.05),while the expression of vimentin was higher in nasopharyngeal carcinoma.HMGB1 was highly expressed in non-keratinizing nasopharyngeal carcinoma and was mainly expressed in cytoplasm,with statistically significant difference(P<0.01).Epithelial cell markers ZO-1 and Ecadherin were decreased in non-keratinizing nasopharyngeal carcinoma tissues(P<0.05),mesenchymal cell markers N-cadherin and vimentin were increased in nasopharyngeal carcinoma tissues(P<0.05)and HMGB1 expression was higher in nasopharyngeal carcinoma by western blot assay(P<0.05).Conclusion: Compared with chronic nasopharyngitis,the expression of epithelial cell markers in non-keratinizing nasopharyngeal carcinoma tissues was decreased,while the expression of mesenchymal cell markers was increased.HMGB1 expression increased in nasopharyngeal non-keratinizing carcinoma tissues,and the same time intracytoplasmic expression increased.There was a correlation between EMT and HMGB1 expression in nasopharyngeal carcinoma.Part ? Role of high mobility group box 1 in invasion,metastasis and epithelial-mesenchymal transition of nasopharyngeal carcinoma cell linesObjective: To detect the EMT markers in CNE1 and CNE2 cell lines after incubating with rhHMGB1 and explore the role of HMGB1 in the invasion and metastasis of nasopharyngeal carcinoma cells.Methods: Western blot assay was used to detect the expression of ZO-1,Ncadherin,E-cadherin and vimentin in CNE1 and CNE2 nasopharyngeal carcinoma cell lines with rhHMGB1 or si HMGB1.Rh HMGB1 incubated the cells for 24 and 48 hours and observed the morphological changes of the cells.The effects of rhHMGB1 and si HMGB1 on cell invasion and metastasis were analyzed by scratch assay and transwell chamber.Results: RhHMGB1 could induce EMT in nasopharyngeal carcinoma cells.In CNE1 cells,500 ng/ml rhHMGB1 can cause significantly decreased the expression of ZO-1 and E-cadherin(P<0.05),increased the expression of N-cadherin(P<0.05),while there was not significant difference in vimentin expression(P>0.05).In CNE2 cell lines,the expressions of these markers were significant difference.The cell morphology changed after incubation with rhHMGB1,and the cells extended pseudopod obviously.In the scratch assay,the cell migration ability of the rhHMGB1 treatment group was enhanced,and after 72 hours,the scratch part was completely covered.After adding glue in Transwell chamber,the number of cells which passed through the membrane in the rhHMGB1 incubating group was significantly increased compared with that of HMGB1 gene silencing group.And the cell migration and invasion ability were enhanced.Conclusion: Exogenous rhHMGB1 can induce EMT in nasopharyngeal carcinoma cell lines in a dose-dependent manner.It can enhance the invasion and migration of nasopharyngeal cells in this way.Part ? RvD1 attenuates epithelial-mesenchymal transition of nasopharyngeal carcinoma cells by inhibiting high mobility group box 1Objective: to analyze the mechanism of RvD1 on the invasion and metastasis of nasopharyngeal carcinoma by detecting the EMT markers in the nasopharyngeal carcinoma cells which incubated with rhHMGB1.Methods: Immunofluorescence and western blot assay were used to detect the locations and semi-quantitative of EMT markers in cells with rhHMGB1 intervention,HMGB1 silencing and RvD1+rhHMGB1 intervention groups.Western-blot was used to detect the expression of HMGB1 in cells after RvD1 intervention,and the expression of ALX/FPR2 and GPR32 in cells after rhHMGB1 intervention,to explore the relationship between RvD1 and HMGB1.Gene transfection was performed to silence and overexpression of RvD1 receptor ALX/FPR2 and GPR32,and the pathway of RvD1 inhibiting HMGB1 induced EMT was analyzed.Results: After incubating with 100 n MRvD1,the expression of HMGB1 in CNE2 cells was decreased by western-blot assay(P=0.0137).The expression of ZO-1 and Ecadherin in RvD1+rhHMGB1 group and HMGB1 silent group was enhanced compared with rhHNGB1 and control groups,while the expression of N-cadherin and vimentin was decreased.After RvD1 receptor silencing,the intervention effect on HMGB1-induced EMT was weakened,while after receptor overexpression,the intervention effect on HMGB1-induced EMT was enhanced,and the change caused by GPR32 was more obvious.Conclusion: RvD1 inhibits HMGB1 induced EMT mainly by binding to GPR32 receptor.