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Journal of Southern Medical University logoLink to Journal of Southern Medical University
. 2019 Mar 20;39(3):313–319. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2019.03.09

Tristetraprolin通过NF-ĸB通路抑制肺腺癌细胞自噬

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

Xiaoya DENG 1, Qinli LUO 1, Fei DONG 1, Li XU 1,*, Xiaokui TANG 1,*
PMCID: PMC6765686  PMID: 31068309

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.

Keywords: tristetraprolin, nuclear factor-κB, autophagy, Beclin1

肺癌是世界上癌症死亡的首要原因, 其中非小细胞肺癌约占85%[1]。在目前精准医疗的大环境下, 寻找有效的分子标志物及对其相应分子机制的研究有重要价值。tristetraprolin通过与mRNA 3'非翻译区上的AU富集元件结合, 诱导靶基因mRNA降解, 直接参与肿瘤相关基因的转录[2], 同时也可调控转录因子从而影响肿瘤细胞发生、发展[3-5]。tristetraprolin在多种肿瘤中低表达, 与患者疾病进展、生存时间呈负相关[6]。tristetraprolin显著抑制肿瘤细胞增殖, 其主要机制包括对细胞周期调控关键基因的转录调控, 如p21[7], cyclin D1[8], E2F1[9], LATS2[10]等, 引起细胞周期阻滞; 通过调控凋亡相关基因或通过其他分子机制诱导细胞凋亡[11-12]; 通过调控自噬相关基因诱导或抑制细胞自噬[13-16]。

目前对tristetraprolin与自噬相关性研究较少, 对二者相互关系的研究存在争议。tristetraprolin可诱导自噬, 促进肿瘤细胞增殖, 同时, tristetraprolin也可通过下调自噬相关基因, 抑制肿瘤细胞生长; 前期研究发现, tristetraprolin可能通过抑制自噬从而抑制肺癌细胞增殖, 导致细胞死亡[17], 但未对其可能的分子机制或信号通路深入研究。本文在前期研究基础上进一步探索其可能通过NF-κB发挥作用的具体分子机制, 首次在肺癌中阐明tristetraprolin可能通过NF-kappaB信号通路调控自噬的发生, 进而影响肿瘤细胞的命运, 是对tristetraprolin抑癌机制的重要探索。

1. 材料和方法

1.1. 实验材料

人胚肺细胞系MRC-5、肺腺癌细胞系NCI-H1975及H1299 (中国科学院上海细胞库提供); 总RNA抽提试剂盒、第一链cDNA合成试剂盒、PCR试剂盒及PCR引物(Takara); tristetraprolin过表达质粒由美国圣路易斯医学院刘建国教授赠送; jetPrime DNA transfection reagent及人TNF-α生长因子(Polyplus-transfection); 质粒提取试剂盒(Omega Bio-tek); DAPI显色液、BCA蛋白定量试剂盒、RIPA裂解液、细胞核蛋白提取试剂盒(碧云天); 大肠杆菌DH5α感受态细胞(Takara); 兔抗人tristetraprolin抗体(Sigma-Aldrich)、兔抗人NF-κB p65抗体、兔抗人NF-κB p50抗体、兔抗人NF-κB c-rel抗体、兔抗人LC3Ⅱ/LC3Ⅰ抗体、兔抗人p62抗体(CST); 兔抗人Beclin1抗体、兔抗人GAPDH抗(Proteintech); 兔抗人LaminB抗体(万类生物); 鼠抗人tristetraprolin抗体、鼠抗人β-actin (Santa Cruz); 鼠抗人荧光二抗及兔抗人荧光二抗(中杉金桥生物); ECL化学发光液购(Millipore); Marker(Fermentas); RPMI 1640培养液(Hyclone); LB液体培养基、LB营养琼脂(索莱宝); 优等胎牛血清(PAN Biotech); PVDF膜(Millipore), 其余试剂均为分析纯。

1.2. 实验方法

1.2.1. 细胞培养

人胚肺细胞系MRC-5、肺腺癌细胞系NCI-H1975细胞及H1299细胞均生长于含10%胎牛血清及1%青霉素/链霉素的RPMI 1640培养基, 培养于37℃、5%CO2体积分数的恒温无菌培养箱, 0.25%的胰蛋白酶传代, 取对数期细胞进行试验。

1.2.2. 基因过表达

将tristetraprolin过表达及IKBα-mut质粒转化后进行测序, 测序成功后进行单克隆扩增, 按照质粒提取试剂盒说明书提取DNA并测浓度、将对数生长期细胞按照质粒转染试剂说明书转染tristetraprolin及IκBα-mut过表达质粒, 同时转入相应空载作为对照组, 转染后5 h进行换液, 每隔24 h提取蛋白、RNA验证相关目的基因表达及进行下一步实验, 人TNF-α生长因子于提取蛋白及RNA前2 h加入细胞培养瓶。

1.2.3. Real-time PCR检测RNA水平

按照Takara总RNA提取试剂盒提取纯化RNA并测RNA浓度, 用Takara逆转录试剂盒将RNA转为cDNA, 经过染色后进行荧光实时定量PCR反应。PCR反应条件为: 95℃预变性30 s, PCR反应(95℃5 s, 60℃30 s, 40个循环)。tristetraprolin上游引物: 5'-GGAGTGTCTTCCGAGG TTCTTG-3', 下游引物: 5'-AACGGCTTTGGCTACTT GCTT-3'; Beclin1上游引物: 5'-TACCACAGCCCAGG CGAAAC-3', 下游引物: 5'-CCAGTGACCTTCAGTC TTCGGC-3; P62上游引物: 5'-TTCCAGCACAGAGG AGAAGAGC-3', 下游引物: 5'-GATTCTGGCATCTGT AGGGACTG-3';GAPDH上游引物: 5'-TGAAGGTC GGAGTCAACGGAT-3', 下游引物: 5'-CCTGGAAGA TGGTGATGGGAT-3'。目的基因表达量以GAPDH作为参照, 采用相对定量法, 以2-△△Ct表示实验组目的基因mRNA的相对表达量。每组设置3个副孔, 实验重复3次。

1.2.4. Western blot检测蛋白水平

将各组细胞按照RIPA裂解液及细胞核提取试剂盒说明书于冰上裂解后提取各细胞总蛋白、核蛋白及细胞浆蛋白。用BCA法测定蛋白浓度, 蛋白样品与5x蛋白上样缓冲液按照4: 1混合, 100℃加热10 min, 取一部分进行试验, 其余放置- 80℃冰箱保存。分别取30 μg总蛋白、60 μg核蛋白及细胞浆蛋白样品上样, 后于10%及12%SDS-PAGE胶中进行电泳分离, 电泳后将蛋白转移至PVDF膜上, 快速封闭液封闭13 min, 于TBST中洗2 min, 加入相应特异性一抗, 于4℃房间孵育过夜(约12 h), 于TBST洗3次, 10 min/次, 加入相对应二抗, 于37℃中孵育1 h, 于TBST中洗30 min后进行ECL发光显影。采用Quantity one 4.6.2软件对条带进行分析, 总蛋白以GAPDH为内参, 胞浆蛋白以β-actin为内参, 核蛋白以LaminB为内参, 以目的蛋白与内参蛋白之比表示蛋白的相对表达水平, 实验重复3次。

1.2.5. 细胞免疫荧光检测核移位

将对数期生长细胞接种于铺好无菌载玻片的6孔板中, 继续培养40 h, 按照说明书加入转染试剂及tristetraprolin过表达质粒, 5 h后换液, 继续培养48 h。4%多聚甲醛固定10 min, PBS洗3次, 5 min/次, 加入0.1%TritonX-100破膜10 min, PBS洗3次, 5 min/次。山羊血清封闭, 37℃恒温箱中敷育1 h, 吸去多余血清, 加入anti-NF-κB p65、p50、c-rel及anti-tristetraprolin抗体4℃孵育过夜, PBS洗3次, 分别用FTTC及PE荧光二抗避光孵育1 h, PBS洗3次, DAPI染细胞核5 min, PBS洗3次, 用抗荧光淬灭剂封片后于显微共聚焦镜下拍照。每组样品为2, 实验重复3次。

1.3. 统计学分析

所有资料采用SPSS 17.0和GraphPad Prism 6软件统计并进行处理, 数据以均数±标准差表示, 多样本均数比较用单因素方差分析, 两独立样本间比较采用t检验, P < 0.05为差异有统计学意义。

2. 结果

2.1. tristetraprolin在肺腺癌细胞中低表达

采用Western blot及RT-qPCR检测人胚肺细胞系MRC-5、肺腺癌细胞系NCI-H1975细胞及H1299细胞表达水平。结果显示, tristetraprolin蛋白水平在肺腺癌H1975及H1299中的表达低(图 1A), 几乎检测不出其表达量, 同样在mRNA水平, 其表达量也极低(图 1B)。

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Tristetraprolin在肺腺癌细胞H1975及H1299中低表达

Low expression of tristetraprolin in lung adenocarcinoma cell lines H1975 and H1299. A: Protein expression of tristetraprolin in lung adenocarcinoma cells detected by Western blotting; B: mRNA expression level of tristetraprolin in lung adenocarcinoma cells detected by RT-qPCR. ***P < 0.001 vs MRC- 5 cells (a normal lung epithelial cell line).

2.2. 过表达tristetraprolin抑制自噬相关基因表达

将肺腺癌细胞系H1975细胞及H1299细胞瞬时转染tristetraprolin后检测自噬相关基因Beclin1、LC3Ⅱ/ LC3Ⅰ及P62在mRNA及蛋白水平表达变化。结果显示, 在肺腺癌H1975细胞中, 转染48 h后tristetraprolin mRNA及蛋白表达水平最高(图 2A、B、F、H), 同时发现Beclin1表达量随tristetraprolin增高而降低, 与空载组相比较, 其在mRNA及蛋白水平表达均明显降低, 差异具有统计学意义(图 2A、C、D、F、I、J, P < 0.05), 同样, LC3 Ⅱ/LC3Ⅰ比值明显降低, 差异具有统计学意义(图 2A、E、F、K, P < 0.01)。p62在H975及H1299肺腺癌细胞中表达变化不稳定, 差异没有统计学意义(图 2A、F)。

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Tristetraprolin在肺腺癌H1975及H1299细胞中对自噬相关基因表达影响

Effect of tristetraprolin overexpression on the expression of autophagy-related genes in lung adenocarcinoma H1975 and H1299 cells. The relative expressions of tristetraprolin and autophagy-related mRNA in H1975 and H1299 cells (B, C, H, I) were analyzed by RT-qPCR. Western blotting was used to detect the expressions of autophagy-related proteins (A, D, E, F, J, k). The samples were detected every 24 h until 3 days after transfection. EV: H1299 and H1975 cells transfected with the empty vector; tristetraprolin: H1299 and H1975 cells transfected with tristetraprolin-overexpressing plasmid. *P < 0.05, **P < 0.01, ***P < 0.001 vs EV.

2.3. 过表达tristetraprolin可能抑制NF-κB核移位

将肺腺癌系H1975细胞瞬时转染tristetraprolin过表达质粒, 转染40 h后用人TNF-α处理细胞2 h, 采用Western blot和细胞免疫荧光检测细胞核中NF-κB p65, p50, c-rel蛋白表达水平。在细胞核中可见少量细胞浆蛋白β-actin表达, 细胞浆中无细胞核蛋白LaminB表达, 细胞浆及细胞核蛋白分离成功(图 3B)。过表达tristetraprolin后细胞核中NF-κB p65, c-rel表达量与空载组相比减少, 差异具有统计学意义(图 3B、C、D, P < 0.001), 但p50在细胞核及细胞浆中无明显变化(图 3B)。同样, 细胞免疫荧光显示, 过表达tristetraprolin组较空载组细胞核中NF-κB p65, c-rel表达减少(图 3A), 而p50无明显变化。

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Tristetraprolin抑制肺腺癌H1975细胞中NF-κB p-65, c-rel核移位

Tristetraprolin blocks NF-кB nuclear translocation. A: H1975 cells were transiently transfected with tristetraprolin or control vector, and 40 h later, the transfected cells were treated with TNF-α (10 ng/mL) for 2 h, followed by immunostaining with anti-tristetraprolin Ab (red) and anti-NF-кB p65 Ab (green), or anti-NF-кB c-Rel Ab (green), or anti-NF-кB p50 (green) Ab. DAPI labeling was used to identify the cell nuclei (blue); B-D: Western blot analysis of the endogenous p65, c-rel, and p50 protein in cytoplasmic and nuclear extracts of tristetraprolin-overexpressing H1975 cells stimulated with TNF-α (10 ng/mL) for 2 h. β-actin was used as the cytoplasmic protein loading control. Lamin B was used as the nuclear protein loading control. EV: H1975 cells transfected with empty plasmid; tristetraprolin: H1975 cells transfected with tristetraprolin-overexpressing plasmid. *P < 0.05, **P < 0.01 vs EV.

2.4. Tristetraprolin通过NF-κB通路抑制肺腺癌细胞自噬作用

为进一步验证tristetraprolin通过NF-κB信号通路影响肺腺癌细胞自噬, 在H1975肺腺癌细胞中瞬时转染IκBα-mut质粒抑制NF-κB信号通路, 然后再转染tristetraprolin过表达质粒, 观察抑制NF-κB通路后, tristetraprolin+ IκBα-mut组和tristetraprolin组中自噬相关基因表达差异。结果显示, tristetraprolin+IκBα-mut组中, 自噬相关基因Beclin1在mRNA及蛋白水平较单过表达tristetraprolin组明显增加(图 4A、C、D, P < 0.05), LC3Ⅱ表达也增多, 差异具有统计学意义(图 4A、E, P < 0.05)。结果显示, 抑制NF-κB信号通路后, 肺腺癌细胞中tristetraprolin对自噬基因Beclin1及LC3Ⅱ抑制作用减弱。

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抑制NF-κB信号通路后, tristetraprolin对肺腺癌细胞自噬相关基因表达影响

Tristetraprolin overexpression inhibits autophagy in lung H1975 cancer cells through NF-κB signal pathway. H1975 cells were co-transfected with tristetraprolin and IκBα-mut plasmid for 48 h, and the mRNA and protein expressions of Beclin1 were measured by RT-qPCR and Western blotting. A, D, E: The expression of tristetraprolin and autophagy-related protein in lung H1975 cancer cells by Western blotting; B, C: The mRNA expression level of tristetraprolin and Beclin1 in lung H1975 cancer cells by RT-qPCR. PCMV: H1975 cells transfected with tristetraprolin-empty vector; PCR3.1: H1975 cells transfected with IκBα-mut-empty vector; tristetraprolin: H1975 cells transfected with tristetraprolin-overexpressing plasmid. IκBα-mut: H1975 cells transfected with IκBα-mut plasmid. *P < 0.05, **P < 0.01, ***P < 0.001 vs tristetraprolin.

3. 讨论

本研究在前期实验基础上进一步探究在肺腺癌细胞中tristetraprolin与自噬关系及具体分子机制, 结果显示, tristetraprolin在肺腺癌中低表达, tristetraprolin抑制肺腺癌自噬相关基因Beclin1及LCⅡ/LCⅠ的表达, 同时tristetraprolin抑制肺腺癌中NF-κB p65, c-rel核移位, 阻断NF-κB信号通路后, tristetraprolin对自噬基因Beclin1及LC3Ⅱ的抑制作用减弱, 提示在肺腺癌细胞中, tristetraprolin可能通过NF-κB信号通路抑制自噬。tristetraprolin在多种肿瘤组织中低表达, 与恶性肿瘤的发生、发展密切相关[18]。肺癌患者中, tristetraprolin低表达患者预后较差, 生存率更低, 同时与肿瘤的基因突变频率相关[19]。同样, 本研究发现与正常肺上皮细胞MRC-5比较, 肺腺癌H1975及H1299中tristetraprolin表达降低, 这表明可能仅仅tristetraprolin的低表达就是肺腺癌发生的关键驱动力, 对其作用的具体机制的研究具有重要意义。

自噬与肿瘤的关系受到广泛关注, 自噬相关分子表达及自噬相关通路与肺癌的预后治疗相关, 但自噬对肺癌患者的预后治疗否有益存在争议[20-22]。目前tristetraprolin与自噬的关系研究较少, Bourcier等[13]发现tristetraprolin过表达能抑制黑色素瘤细胞的自噬。相反, 有研究发现tristetraprolin过表达能诱导细胞自噬[14-15]。María[16]发现tristetraprolin抑制细胞发生自噬性死亡, 其具体机制不明。本研究发现肺腺癌细胞过表达tristetraprolin后, 细胞中Beclin1在mRNA及蛋白表达水平降低, LC3 Ⅱ/LC3 Ⅰ比值降低, 同时, 当tristetraprolin表达减少时, Beclin1的表达增加。这说明在肺腺癌中, tristetraprolin可能通过调控Beclin1影响细胞自噬水平。Beclin1是自噬启动的关键基因, 在胃癌、肺癌及宫颈癌等肿瘤中表达降低, 同时在肺癌中发现Beclin1升高导致的自噬活性增强对肿瘤发展及耐药相关, 说明在肺癌的不同时期, Beclin1介导的自噬活性调控对肿瘤的发生发展具有不同的作用[23-25], 本研究发现在肺腺癌中tristetraprolin可能通过抑制Beclin1表达从而发挥抑癌作用, 而tristetraprolin对自噬基因Beclin1表达的直接调控机制还需进一步的证实。

NF-kappaB是重要的转录因子, tristetraprolin能抑制NF-kapaB p65亚基核移位从而抑制炎症相关因子及c-jun表达[4-6, 26]。同样, 我们发现肺腺癌中过表达tristetraprolin组和空载组比较, 细胞核中NF-κB p65, c-rel在蛋白水平表达降低, 但p50在细胞核及细胞质中表达无明显差异。细胞免疫荧光显示在tristetraprolin过表达组, 细胞核内NF-κB p65, c-rel减少, p50无明显差异, 这表明在肺腺癌细胞中tristetraprolin能抑制NF-κB p65, c-rel核移位, 对p50无明显影响。NF-κB对自噬的调控因细胞背景不同而不同, 具有细胞特异性[27], 为研究tristetraprolin是否通过NF-κB信号通路调控肺腺癌细胞自噬, 我们在H1975肺腺癌细胞中转染IκBα-mut质粒抑制NF-κB信号通路, 结果显示, 抑制NF-κB信号通路后, tristetraprolin对Beclin1及LC3Ⅱ抑制作用消除。这说明tristetraprolin可能通过NF-κB信号通路调控自噬相关基因, 抑制肺腺癌细胞自噬, 从而调控肿瘤的发生发展。

p62也是自噬标志物, 自噬诱导时, p62表达降低, 当自噬抑制时, p62表达升高[28]。本研究发现, 过表达tristetraprolin抑制自噬后p62在蛋白表达水平不受影响。这可能是由于p62作为自噬底物降解的标记物时, 其表达变化具有滞后性, 同时p62也参与了其他多种细胞活动, 这些途径的变化也会导致p62水平发生变化[29]。同时也说明tristetraprolin可能不直接调控p62的表达, 需对其相关机制做进一步研究。

综上所述, 本研究揭示了tristetraprolin可能通过NF-κB信号通路抑制肺腺癌细胞自噬, 进一步抑制肺腺癌发生发展, 深化了tristetraprolin的抑癌机制研究, 同时为tristetraprolin可能作为肺腺癌治疗的靶点提供理论依据。

Biography

邓小垭, 在读硕士研究生, E-mail: 921224096@qq.com

Funding Statement

国家自然科学基金(81602430);重庆市科委基础与前沿一般项目(cstc2016jcyjA0329)

Supported by National Natural Science Foundation of China (81602430)

Contributor Information

邓 小垭 (Xiaoya DENG), Email: 921224096@qq.com.

徐 莉 (Li XU), Email: xuliglee@126.com.

唐 小葵 (Xiaokui TANG), Email: txk1200@126.com.

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