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  南方医科大学学报  2019, Vol. 39Issue (3): 313-319  DOI: 10.12122/j.issn.1673-4254.2019.03.09.
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引用本文 [复制中英文]

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): 313-319. DOI: 10.12122/j.issn.1673-4254.2019.03.09.
DENG Xiaoya, LUO Qinli, DONG Fei, XU Li, TANG Xiaokui. Tristetraprolin inhibits autophagy in cultured lung cancer cells via the nuclear factor-κB pathway[J]. Journal of Southern Medical University, 2019, 39(3): 313-319. DOI: 10.12122/j.issn.1673-4254.2019.03.09.

基金项目

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

作者简介

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

通信作者

通信作者:
徐莉, 高级实验师, E-mail: xuliglee@126.com
唐小葵, 教授, E-mail: txk1200@126.com

文章历史

收稿日期:2018-12-01
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     
重庆医科大学附属第一医院呼吸与危重症医学科, 重庆 400016
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。
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
DENG Xiaoya , LUO Qinli , DONG Fei , XU Li , TANG Xiaokui     
Department of Respiratory and Critical Care Medicine, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
Supported by National Natural Science Foundation of China (81602430)
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显着抑制肿瘤细胞增殖, 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)。

图 1 Tristetraprolin在肺腺癌细胞H1975及H1299中低表达 Fig.1 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 2ABFH), 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 2ACDFIJ, P < 0.05), 同样, LC3 Ⅱ/LC3Ⅰ比值明显降低, 差异具有统计学意义(图 2AEFK, 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 2AF)。

图 2 Tristetraprolin在肺腺癌H1975及H1299细胞中对自噬相关基因表达影响 Fig.2 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 3BCD, 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。

图 3 Tristetraprolin抑制肺腺癌H1975细胞中NF-κB p-65, c-rel核移位 Fig.3 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 4ACD, P < 0.05), LC3Ⅱ表达也增多, 差异具有统计学意义(图 4AE, 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。

图 4 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 Fig.4 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。

参考文献
[1]
Torre LA, Bray F, Siegel RL, et al. Global cancer statistics[J]. CA Cancer J Clin, 2012, 65(2): 87-108.
[2]
Guo J, Wang H, Jiang SY, et al. The cross-talk between tristetraprolin and cytokines in cancer[J]. Anticanc Agents Med Chem, 2017, 17(11): 1477-86.
[3]
Gu L, Ning H, Qian XE, et al. Suppression of IL-12 production by tristetraprolin through blocking NF-kappa B nuclear translocation[J]. J Immunol, 2013, 191(7): 3922-30. DOI: 10.4049/jimmunol.1300126.
[4]
Schichl YM, Resch U, Martin R. Tristetraprolin impairs NF-kappaB/p65 nuclear translocation[J]. J Biol Chem, 2009, 284(43): 29571-81. DOI: 10.1074/jbc.M109.031237.
[5]
Liang J, Lei TH, Song YT, et al. RNA-destabilizing factor tristetraprolin negatively regulates NF-kappa B signaling[J]. Biol Chem, 2009, 284(43): 29383-90. DOI: 10.1074/jbc.M109.024745.
[6]
Park JM, Lee TH, Kang TH. Roles of tristetraprolin in tumorigenesis[J]. Int J Mol Sci, 2018, 19(11): 3384-95. DOI: 10.3390/ijms19113384.
[7]
Al-Haj A, L, Blackshear PJ, et al. Regulation of p21/CIP1/WAF-1 mediated cell-cyclearrest by RNase L and tristetraprolin, and involvement of AU-rich elements[J]. Nucleic Acids Res, 2012, 40(16): 7739-52. DOI: 10.1093/nar/gks545.
[8]
Marderosian M, Sharma A, Funk AP, et al. Ttristetraprolin regulates cyclin D1 and c-Myc mRNA stability in response to rapamycin in an Akt-dependent manner via p38 MAPK signaling[J]. Oncogene, 2006, 25(47): 6277-90. DOI: 10.1038/sj.onc.1209645.
[9]
Lee HH, Lee SR, Leem SH. Tristetraprolin regulates prostate cancer cell growth throughsuppression of E2F1[J]. Microbiol Biotechnol, 2014, 24(2): 287-94.
[10]
Lee HH, Vo MT, Kim HJ, et al. Stability of the LATS2 tumor suppressor gene is regulated by tristetraprolin[J]. J Biol Chem, 2010, 285(23): 17329-37. DOI: 10.1074/jbc.M109.094235.
[11]
Wang H, Ding NN, Guo J, et al. Dysregulation of TTP and HuR plays an important role in cancers[J]. Tumour Biol, 2016, 37(11): 14451-61. DOI: 10.1007/s13277-016-5397-z.
[12]
Hai DD, Koch A, Allister A, et al. Treatment with MAPKAP2 (Mk2) inhibitor and DNA methylation inhibitor, 5-aza dC, synergistically triggers apoptosis in hepatocellular carcinoma (HCC) via tristetraprolin (TTP)[J]. Cell Signal, 2016, 28(12): 1872-80. DOI: 10.1016/j.cellsig.2016.09.002.
[13]
Bourcier C, Griseri P, Grépin R, et al. Constitutive ERK activity induces downregulation of tristetraprolin, a major protein controlling interleukin8/CXCL8 mRNA stability in melanoma cells[J]. Am J Physiol Cell Physiol, 2011, 301(3): C609-18. DOI: 10.1152/ajpcell.00506.2010.
[14]
Suswam EA, Shacka JJ, Walker K, et al. Mutant tristetraprolin:a potent inhibitor of malignant glioma cell growth[J]. Neuro Oncol, 2013, 113(2): 195-205. DOI: 10.1007/s11060-013-1112-8.
[15]
Tilija PN, Park PH. Adiponectin inhibits inflammatory cytokines production by Beclin-1 phosphorylation and Bcl-2 mRNA destabilization:role for autophagy induction[J]. Br J Pharmaco, 2018, 175(7): 1066-84. DOI: 10.1111/bph.v175.7.
[16]
María VG, Albana G, Johanna M. Expression of the mRNA stability regulator Tristetraprolin is required for lactation maintenance in the mouse mammary gland[J]. Oncotarget, 2018, 9(9): 8278-89.
[17]
Dong F, Li C, Xu L. The RNA binding protein tristetraprolin downregulates autophagy in lung adenocarcinoma cells[J]. Exp Cell Res, 2018, 367(1): 89-96. DOI: 10.1016/j.yexcr.2018.03.028.
[18]
Hitti E, Bakheet T, Al-Souhibani N, et al. Systematic analysis of AURich element expression in cancer reveals common functional clusters regulated by key RNA-Binding proteins[J]. Cancer Res, 2016, 76(14): 4068-80. DOI: 10.1158/0008-5472.CAN-15-3110.
[19]
Mohammad F, Antonio LA, John LC. CREB targets define the gene expression signature of malignancies having reduced levels of the tumor suppressor tristetraprolin[J]. PLoS One, 2014, 9(12): e115517-26. DOI: 10.1371/journal.pone.0115517.
[20]
Rebecca VW, Amaravadi RK. Emerging strategies to effectively target autophagy in cancer[J]. Oncogene, 2016, 35(1): 1-11. DOI: 10.1038/onc.2015.99.
[21]
Liu GB, Pei F, Yang FQ, et al. Role of autophagy and apoptosis in non-small-cell lung cancer[J]. Int J Mol Sci, 2017, 18(2): E367-75. DOI: 10.3390/ijms18020367.
[22]
Karsli UG, Jy G, Price S, et al. Autophagy is required for glucose homeostasis and lung tumor maintenance[J]. Cancer Discov, 2014, 4(8): 914-27. DOI: 10.1158/2159-8290.CD-14-0363.
[23]
Lee SJ, Kim HP, Jin Y, et al. Beclin1 deficiency is associat-ed with increased hypoxia-induced angiogenesis[J]. Autophagy, 2011, 7(8): 829-39. DOI: 10.4161/auto.7.8.15598.
[24]
Wang XF, Du ZY, Li LY, et al. Beclin1 and p62 expression in nonsmall cell lung cancer:relation with malignant behaviors and clinical outcome[J]. Int J Clin Exp Pathol, 2015, 8(9): 10644-52.
[25]
Wu SF, Su J, Qian H, et al. SLC27A4 regulate ATG4B activity and control reactions to chemotherapeutics-induced autophagy in human lung cancer cells[J]. Tumour Biol, 2016, 37(5): 6943-52. DOI: 10.1007/s13277-015-4587-4.
[26]
Xu L, Ning H, Gu L, et al. Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-kappa B pathway[J]. Oncotarget, 2015, 6(39): 41679-91.
[27]
Salminen A, Hyttinen JM, Kauppinen A, et al. Context-dependent regulation of autophagy by IKK-NF-κB signaling:impact on the aging process[J]. Int J Cell Biol, 2012, 20(18): 849541-53.
[28]
Pankiv S, Clausen TH, Lamark T, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[J]. J Biol Chem, 2007, 282(33): 24131-45. DOI: 10.1074/jbc.M702824200.
[29]
Seibenhener ML, Geetha T, Wooten MW. Sequestosome 1/p62-more than just a scaffold[J]. FEBS Lett, 2007, 581(2): 175-9. DOI: 10.1016/j.febslet.2006.12.027.