Tristetraprolin Inhibit Autophagy In Lung Cancer Though NF-κB Pathway

Objective : To investigate the expression of RNA binding protein tristetraprolin in lung adenocarcinoma cells and its molecular mechanism on autophagy.Methods:Quantitative real-time PCR and WB were used to detect the expression of TTP in human lung adenocarcinoma cell lines H1975,H1299 and normal human embryonic lung fibroblasts cells(MRC-5).WB and qRT-PCR performed to detect the expression of autophagy-related genes,including Beclin1,LC3-Ⅱ/LC3-Ⅰ and SQSTM1/p62 in cells transiently transfected with TTP expression constructions after 24,48 and 72 h.Cells were transfected with TTP and empty construction followed by treatment of act D(10μg/m L)to stop the transcription.qRT-PCR was used to detect the expression level of Beclin1 mRNA at indicated time points.Nuclear and cytoplasmic protein was extracted to examine the effect of TTP on NF-κB p65,c-rel,p50 in lung adenocarcinoma cells which were treated with empty vectors,TTP over-expression vectors,empty vectors+TNF-αand TTP over-expression vectors +TNF-α.Immunofluorescence and Confocal microscopy was performed to detect the Nuclear translocation ofNF-κB in Lung cancer cells with or without TTP overexpression.RT-qPCR and WB were used to detect the expression of TTP and the autophagy-related gene in the lung adenocarcinoma cells which were transiently transfected with TTP empty vectors(PCMV),IκBα-mut empty vectors(PCR3.1),TTP over-expression vectors,IκBα-mut vectors and IκBα-mut+TTP over-expression vectors.Results : 1.The TTP mRNA expression and protein level in lung adenocarcinoma cells was low(P<0.001)compared with normal lung epithelial cells MRC-5.2.The expression of autophagy-related genes Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰwere decreased at the RNA and protein levels in lung adenocarcinoma cells transiently transfected with TTP overexpression plasmid compared with transfected with empty vector(P<0.001).3.After actinomycin D terminated transcription,there was no significant difference in the expression level of autophagy-related gene Beclin1 mRNA between the empty group and the TTP group(P>0.05).4.The expression of nuclear p65 and c-rel protein were decreased in lung adenocarcinoma cells transiently transfected with TTP overexpression plasmid compared with empty vector and empty vector+TNF-α(P<0.05).Confocal microscopy revealed NF-κB p65,c-rel nuclear translocation was almost completely blocked after overexpression of TTP.However,the expression of p50 was no difference in cells transfected with empty vectorand TTP overexpression vector(P>0.05).5.The expression of Beclin1,and the ration of LC3-Ⅱ/LC3-Ⅰin co-transfected with IκBα-mut and TTP overexpression vector were increased compared with only transfected with TTP overexpression vector at RNA and protein levels in lung adenocarcinoma cells(P<0.05).Conclusion:The expression of TTP is low in lung adenocarcinoma cells.TTP might downregulate autophagy through NF-κB signaling by blocking p65 and c-rel nuclear translocation in lung adenocarcinoma cells.