OBJECTIVE: To explore the expression of the RNA-binding protein tristetraprolin in lung adenocarcinoma cells and its molecular mechanism for inhibiting autophagy. METHODS: Quantitative real-time PCR and Western blotting were performed to detect the expression of autophagy-related genes (including Beclin1, LC3-Ⅱ/LC3-Ⅰ and SQSTM1/p62) in cultured lung adenocarcinoma cells at 24, 48 and 72 h after transient transfection with a tristetraprolin-overexpressing plasmid and the empty plasmid. The effects of transfection with the tristetraprolin-overexpressing plasmid and empty plasmids in the presence or absence of tumor necrosis factor- (TNF-) on the expressions of nuclear factor-κB (NF-κB) p65, c-rel, and p50 were examined in lung adenocarcinoma cells using immunofluorescence assay and Western blotting. The cells were also transfected with the IκBα-mut plasmid and the tristetraprolin-overexpressing plasmid, either alone or in combination, and the changes in the expressions of tristetraprolin and autophagy-related genes were detected using RT-qPCR and Western blotting. RESULTS: The expressions of tristetraprolin were significantly reduced at both the mRNA and protein levels in lung adenocarcinoma cells ( < 0.001). Overexpression of tristetraprolin in the cells significantly lowered the expressions of autophagy-related genes Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ at the mRNA and protein levels ( < 0.001), obviously lowered the expressions of NF-κB p65 and c-rel, and almost totally blocked the nuclear translocation of NF-κB p65 and c-rel ( < 0.05); the expression of p50, however, did not undergo significant changes in response to tristetraprolin overexpression ( > 0.05). The inhibitory effect of tristetraprolin overexpression on autophagy was abrogated by transfection of the cells with IκBα-mut plasmid, which blocked the NF-κB signaling pathway. Co-transfection of the cells with IκBα-mut also attenuated the inhibitory effect of tristetraprolin overexpression on Beclin1 and the LC3-Ⅱ/LC3-Ⅰ ratio at both the mRNA and protein levels ( < 0.05). CONCLUSIONS The expression of tristetraprolin is low in lung adenocarcinoma cells. Tristetraprolin overexpression causes inhibition of autophagy in lung adenocarcinoma cells possibly by blocking NF-κB p65 and c-rel nuclear translocation.
Autophagy ; Cell Line ; Humans ; Lung Neoplasms ; NF-kappa B ; Signal Transduction ; Tristetraprolin

PURPOSE: Overexpression of cyclooxygenase 2 (COX-2) is thought to promote survival of transformed cells. Transforming growth factor beta (TGF-beta) exerts anti-proliferative effects on a broad range of epithelial cells. In the current study, we investigated whether TGF-beta can regulate COX-2 expression in A549 human lung adenocarcinoma cells, which are TGF-beta-responsive and overexpress COX-2. MATERIALS AND METHODS: Western blotting, Northern blotting, and mRNA stability assays were performed to demonstrate that COX-2 protein and mRNA expression were suppressed by TGF-beta. We also evaluated the effects of tristetraprolin (TTP) on COX-2 mRNA using RNA interference. RESULTS: We demonstrated that COX-2 mRNA and protein expression were both significantly suppressed by TGF-beta. An actinomycin D chase experiment demonstrated that COX-2 mRNA was more rapidly degraded in the presence of TGF-beta, suggesting that TGF-beta-induced inhibition of COX-2 expression is achieved via decreased mRNA stability. We also found that TGF-beta rapidly and transiently induced the expression of TTP, a well-known mRNA destabilizing factor, before suppression of COX-2 mRNA expression was observed. Using RNA interference, we confirmed that increased TTP levels play a pivotal role in the destabilization of COX-2 mRNA by TGF-beta. Furthermore, we showed that Smad3 is essential to TTP-dependent down-regulation of COX-2 expression in response to TGF-beta. CONCLUSION: The results of this study show that TGF-beta down-regulated COX-2 expression via mRNA destabilization mediated by Smad3/TTP in A549 cells.
Adenocarcinoma ; Blotting, Northern ; Blotting, Western ; Cyclooxygenase 2 ; Dactinomycin ; Down-Regulation ; Epithelial Cells ; Humans ; Lung ; Lung Neoplasms* ; RNA Interference ; RNA Stability ; RNA* ; RNA, Messenger ; Transforming Growth Factor beta* ; Tristetraprolin

OBJECTIVETo assess the association of genetic polymorphisms of mitogen-activated protein kinase activated protein kinase 2 gene (MK2) and zinc finger protein 36 gene (ZFP36) with high density lipoprotein cholesterol (HDL-C) in Xinjiang Urgur population.

METHODSNine hundred thirty Uygur individuals were randomly recruited from Hetian area. Single nucleotide polymorphisms (SNP) in MK2 gene (rs44890 and rs45514798) and ZFP36 gene (rs251864 and rs3746083) were determined with Taqman-PCR. All subjects were investigated with questionnaire, physical examination and measurement of lipid levels and plasma tumor necrosis factor-alpha.

RESULTS(1)In Uygur men younger than 50 years old, SNP rs45514798 was associated with HDL-C [dominant model P=0.054, OR(95%CI)0.261(0.082-0.833) after age, smoking, drinking, abdominal circumference, waist/hip ratio and body mass index and tumor necrosis factor were controlled]. (2) For males younger than 50 years old, the concentration of total cholesterol, HDL-C, low density lipoprotein cholesterol were different in dominant model of rs45514798(P< 0.05). Female: total cholesterol, low density lipoprotein cholesterol were different in dominant model of rs45514798(P< 0.05). (3) The distribution of genotype of ZFP36 gene did not differ significantly between the low HDL-C groups and the control group.

CONCLUSIONMK2 gene rs45514798 polymorphisms may be associated with HDL-C in Uygur men younger than 50 years old from Hetian area of Xinjiang. ZFP36 gene is not associated with HDL-C in Uygur people from Xinjiang.

Adult ; Age Factors ; Aged ; Base Sequence ; China ; ethnology ; Cholesterol, HDL ; blood ; Female ; Genetic Association Studies ; Genotype ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Middle Aged ; Phenotype ; Polymorphism, Single Nucleotide ; Protein-Serine-Threonine Kinases ; genetics ; Sex Factors ; Tristetraprolin ; genetics