南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (03): 313-.doi: 10.12122/j.issn.1673-4254.2019.03.09

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Tristetraprolin通过NF-?B 通路抑制肺腺癌细胞自噬

邓小垭，罗勤利，董飞，徐莉，唐小葵   

  • 出版日期:2019-03-20 发布日期:2019-03-20

Tristetraprolin inhibits autophagy in cultured lung cancer cells via the nuclear factor-κB pathway

  • Online:2019-03-20 Published:2019-03-20

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摘要： 目的研究RNA结合蛋白tristetraprolin在肺腺癌中的表达及抑制自噬作用的分子机制。方法瞬时转染tristetraprolin过 表达质粒，分别在转染tristetraprolin 24、48 及72 h 后采用实时荧光定量PCR（RT-qPCR）和Western blot 检测肺腺癌细胞中 tristetraprolin表达及自噬相关分子Beclin1、LC3II/LCI及p62的表达变化。瞬时转染tristetraprolin过表达质粒及加入TNF-α，将 肺腺癌细胞分为空载组、tristetraprolin 组、空载+TNF-α组及tristetraprolin+TNF-α组，应用免疫荧光和Western blot检测NF-ĸB p65、c-rel、p50分子在细胞核中表达情况。共转染tristetraprolin过表达质粒及IκBα-mut质粒，将肺腺癌细胞分为tristetraprolin 空载组、IκBα-mut 空载组、tristetraprolin 组、IκBα-mut 组及tristetraprolin+IκBα-mut 组，采用RT-qPCR 和Western blot 检测 tristetraprolin表达及自噬相关基因表达变化。结果Tristetraprolin在肺腺癌细胞中RNA及蛋白水平表达低（P<0.001）。过表达 tristetraprolin 后，自噬相关基因Beclin1、LC3-Ⅱ/LC3-Ⅰ在RNA及蛋白水平表达均较空载组降低（P<0.001）。同时，过表达 tristetraprolin后，细胞核内的p65及c-rel蛋白表达较空载组及空载+TNF-α组减少（P<0.05），但p50表达无明显变化（P>0.05）。 过表达tristetraprolin后，p65及c-rel核移位较空载组减少。共转染IκBα突变质粒及tristetraprolin过表达质粒后，NF-κB信号通 路被阻断，自噬相关基因Beclin1、LC3-Ⅱ/LC3-Ⅰ在RNA及蛋白水平表达较tristetraprolin过表达组升高（P<0.05），NF-ĸB信号 通路被阻断后tristetraprolin对自噬的抑制作用减弱。结论Tristetraprolin在肺腺癌细胞中低表达，过表达tristetraprolin可能通 过抑制NF-ĸB p65及c-rel核移位而抑制肺腺癌细胞自噬。

Abstract: 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-α (TNF-α) 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 (P<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 (P<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 (P<0.05); the expression of p50, however, did not undergo significant changes in response to tristetraprolin overexpression (P>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 (P<0.05). Conclusion 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.