Tristetraprolin通过NF-?B通路抑制肺腺癌细胞自噬 |
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引用本文: | 邓小垭,罗勤利,董飞,徐莉,唐小葵.Tristetraprolin通过NF-?B通路抑制肺腺癌细胞自噬[J].南方医科大学学报,2019,39(3):313. |
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作者姓名: | 邓小垭 罗勤利 董飞 徐莉 唐小葵 |
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作者单位: | 重庆医科大学附属第一医院呼吸与危重症医学科,重庆,400016;重庆医科大学附属第一医院呼吸与危重症医学科,重庆,400016;重庆医科大学附属第一医院呼吸与危重症医学科,重庆,400016;重庆医科大学附属第一医院呼吸与危重症医学科,重庆,400016;重庆医科大学附属第一医院呼吸与危重症医学科,重庆,400016 |
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基金项目: | 国家自然科学基金;国家自然科学基金 |
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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-?B
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-?B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-?B p65及c-rel核移位而抑制肺腺癌细胞自噬。
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关 键 词: | tristetraprolin 核因子-?B 自噬 Beclin1 |
Tristetraprolin inhibits autophagy in cultured lung cancer cells via the nuclear factor-κB
pathway |
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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. |
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