Regulation Of TNF-? On NF-?B/PXR Signaling Pathway And Related Effects Of DeSUMOylation Modification In Lung Adenocarcinoma

Objective: 1.To explore the expression differences,correlations and relationship with clinicopathological characteristics between TNF-?,NF-?B,PXR and SENP1 in lung adenocarcinoma tissues.2.To explore the regulatory effect of TNF-? on NF-?B/PXR singnaling pathway in lung adenocarcinoma cell line and the role of SENP1 in the activation of NF-?B/PXR pathway induced by TNF-? in lung adenocarcinoma.Methods: 1.76 cases of lung invasive adenocarcinoma and paracancer tissues as well as 20 cases of lung adenocarcinoma in situ and paracancer tissues were collected.Immunohistochemistry staining was used to detect the expression levels of TNF-?,NF-?B,PXR and SENP1.2.Human normal lung(bronchial)epithelial cell line BEAS-2B and lung adenocarcinoma cell line A549 were cultured.The m RNA expression levels of NF-?B,PXR,SENP1 and PXR target gene MDR-1 were detected by real-time PCR before and after TNF-? treatment.3.Immunocytochemistry was performed to identify the protein expression levels of NF-?B,PXR and SENP1 in A549 cell line.4.Western blot was used to detect the protein expression levels of NF-?B,p-NF-?B,PXR and SENP1 in BEAS-2B and A549 cells after TNF-? treatment.5.Statistical analysis software IBM SPSS 20.0,Graph Pad Prism and image analysis software Image-pro plus?Image J were used for statistical analysis.?2 test,t test and multiple t test were performed for statistical comparison.Spearman test was used for correlation analysis.P <0.05 indicates that the difference is statistically significant.Results: 1.The expressions of TNF-?,NF-?B,PXR and SENP1 in lung invasive adenocarcinoma and lung adenocarcinoma in situ were significantly higher than paracancer tissues,the difference was statistically significant(P<0.001);the expression of SENP1 in lung invasive adenocarcinoma tissue was higher than lung adenocarcinoma in situ,the difference was statistically significant(P<0.01),and there was no differential expression of TNF-?,NF-?B and PXR between lung invasive adenocarcinoma and lung adenocarcinoma in situ(P>0.05).2.Correlations between NF-?B,PXR and SENP1 expression were determined in lung adenocarcinoma tissues.TNF-? was positively correlated with NF-?B and SENP1(P<0.01),while negatively correlated with PXR(P<0.01).NF-?B was negatively correlated with PXR(P<0.01).The expression of TNF-? had no correlations with age,gender,tumor size and lymph node metastasis(P>0.05),but was correlated to the TNM stage(P<0.05).3.The baseline expression levels of NF-?B m RNA were similar between A549 cells and control and the difference was not statistically significant(P=0.745).Increased expression of NF-?B m RNA after TNF-? treatment was found in A549 cells and normal control BEAS-2B cells(P<0.05,P<0.001).Moreover the expression levels were significantly higher in A549 cells compared to BEAS-2B cells and the difference was statistically significant(P<0.05).4.The baseline m RNA expression levels of PXR?MDR-1 and SENP1 were higher in A549 cells than BEAS-2B cells and the difference was statistically significant(P<0.01 for PXR,P<0.05 for MDR-1,P<0.001 for SENP1).The m RNA expression levels of PXR?MDR-1?SENP1 were decreased in A549 cells after TNF-? treatment(P<0.01 for MDR-1,P<0.001 for PXR,SENP1).However the m RNA expression levels were increased in BEAS-2B cells after TNF-? treatment(P<0.01 for PXR ?P<0.01 for MDR-1,P<0.001 for SENP1).The m RNA expression levels were higher in A549 cells than BEAS-2B cells and the difference was statistically significant(P<0.001).5.The results of immunocytochemistry showed that after TNF-? treatment,the protiein expression levels of NF-?B in A549 cells were increased and the expression levels of PXR and SENP1 were decreased.The difference was statistically significant(P=0.003 for NF-?B,PXR,P=0.009 for SENP1).6.Western blot results showed that after TNF-? treatment,the protein expression levels of NF-?B and p-NF-?B were increased in BEAS-2B and A549 cells,and the difference was statistically significant(P<0.001).The protein expressions of PXR and SENP1 were increased in BEAS-2B cells.The protein expressions of PXR and SENP1 were decreased in A549 cells,the differences was statistically significant(P<0.001).Changes in protein expression levels are consistent with changes in m RNA expression levels.Conclusions: 1.TNF-? may involve in tumorigenesis of lung adenocarcinoma via activating NF-?B expression and repressing transcriptional activity of PXR.2.De-SUMOylation may play an important role in the regulation of TNF-? on NF-?B/PXR pathway in lung adenocarcinoma.3.The expression levels of SENP1 were higher in invasive adenocarcinoma and adenocarcinoma in situ when compared to normal controls.Moreover,the expression levels of SENP1 were higher in invasive carcinoma in comparison to adenocarcinoma in situ.The results suggested SENP1 was involved in tumor initiation and development in lung adenocarcinoma.4.Positive correlations between TNF-? and SENP1 were observed in lung adenocarcinoma tissues.On the contrary,TNF-? can cause decreased expression levels of SENP1 in A549 cells,suggesting that more complicated regulation mechanisms for de-SUMOylation modification are present in solid tumors of lung adenocarcinoma.