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中华细胞与干细胞杂志(电子版) ›› 2020, Vol. 10 ›› Issue (01) : 20 -25. doi: 10.3877/cmj.j.issn.2095-1221.2020.01.004

所属专题： 文献；

论著

肿瘤坏死因子-α促进乳腺癌转移的分子机制研究
徐瑞1, 邱彤璐1, 梅杰2, 沈书凝1, 朱一超3,†()   
  1. 1. 211166　南京医科大学生理学系
    2. 211166　南京医科大学生理学系；214023　无锡，南京医科大学附属无锡人民医院肿瘤科
    3. 211166　南京医科大学生理学系；211166　南京医科大学生殖医学国家重点实验室
  • 收稿日期:2019-05-08 出版日期:2020-02-01
  • 通信作者: 朱一超

The mechanism of tumor necrosis factor-α in promoting breast cancer metastasis

Rui Xu1, Tonglu Qiu1, Jie Mei2, Shuning Shen1, Yichao Zhu3,†()   

  1. 1. Department of Physiology, Nanjing Medical University, Nanjing 211166, China
    2. Department of Physiology, Nanjing Medical University, Nanjing 211166, China;Department of Oncology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
    3. Department of Physiology, Nanjing Medical University, Nanjing 211166, China;State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 211166, China
  • Received:2019-05-08 Published:2020-02-01
  • Corresponding author: Yichao Zhu
  • About author:
    Corresponding author:ZhuYichao, Email:
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徐瑞, 邱彤璐, 梅杰, 沈书凝, 朱一超. 肿瘤坏死因子-α促进乳腺癌转移的分子机制研究[J]. 中华细胞与干细胞杂志(电子版), 2020, 10(01): 20-25.

Rui Xu, Tonglu Qiu, Jie Mei, Shuning Shen, Yichao Zhu. The mechanism of tumor necrosis factor-α in promoting breast cancer metastasis[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(01): 20-25.

目的

探究肿瘤坏死因子-α （TNF-α）促进乳腺癌转移的分子机制。

方法

采集病理学确诊为乳腺癌的患者血液标本53例，首先把MDA-MB-231乳腺癌细胞分为对照组、TNF-α诱导组，再把乳腺癌细胞分为空载对照组（转染空载体）、Arf6-T27N组（转染Arf6-T27N）、空载转染+TNF-α组（转染空载体后加500 ng/ml TNF-α）、Arf6-T27N+TNF-α组（转染Arf6-T27N后加500 ng/ml TNF-α）。采用酶联免疫检测法（ELISA）对乳腺癌患者血浆中TNF-α含量进行检测，采用划痕实验检测细胞的迁移能力，采用小G蛋白活化实验（GLISA）筛选并检测对照组和TNF-α诱导组的小G蛋白及其诱导效应。两组间比较采用t检验，多组间比较采用单因素方差分析，两两比较采用LSD检验；采用χ2检验或Fisher确切概率法分析TNF-α表达高低与临床病理参数的关系。

结果

与未发生淋巴结转移的乳腺癌患者比较，已发生淋巴结转移的患者血浆中TNF-α的含量（391.24±307.35比709.58±277.51）升高，差异具有统计学意义（P < 0.001）；与对照组比较，TNF-α诱导组乳腺癌细胞相对愈合面积（1.00±0.04比2.34±0.25）增大，细胞小G蛋白Arf6活性（1.00±0.02比3.11±0.14）升高，差异有统计学意义（P均< 0.001）；与空载转染+TNF-α组比较，Arf6-T27N+TNF-α组乳腺癌细胞的迁移能力（2.33±0.14比1.83±0.15）降低，差异具有统计学意义（P < 0.001）。

结论

本研究阐明TNF-α通过激活Arf6促进乳腺癌细胞迁移，提示TNF-α/Arf6可作为控制乳腺癌转移的新靶点。

关键词: TNF-α, Arf6, 乳腺癌, 淋巴结转移
Objective

Tumor necrosis factor-α (TNF-α) , as one of the inflammatory factors, plays an important role in the oncogenesis and development of tumors. This study aimed to explore the mechanism of TNF-α in promoting breast cancer metastasis.

Methods

Plasma TNF-α levels of 53 breast cancer patients were detected by enzyme-linked immunosorbent assay (ELISA) . The migration ability of MDA-MB-231 breast cancer cells treated with vehicle, TNF-α, transfected with vector, Arf6-T27N, vectorplus TNF-α, Arf6-T27N plus TNF-α was detected by wound healing assay. TNF-α-induced activization of small G proteins of MDA-MB-231 cells treated with vehicle or TNF-α was screened and detected by small G protein activation assay (GLISA) . The comparison of means between the two groups was performed using t-test. The comparison of means between multiple groups was performed using a one-way analysis of variance, and the LSD test was used in the post hoc comparisons. The relationships between the levels of TNF-α and clinicopathological parameters were analyzed by χ2 test or Fisher exact probability method.

Results

Compared with breast cancer patients without lymph node metastasis, the plasma levels of TNF-α in patients with lymph node metastasis were significantly increased (709.58±277.51 vs 391.24±307.35, P< 0.001) .Wound healing assay showed that TNF-α could significantly promote the migration of MDA-MB-231 cells compared to the control cells (2.34±0.25 vs 1.00±0.04, P< 0.001) . TNF-α notably activatedthe small G protein Arf6 compared to the control group (3.11±0.14 vs 1.00±0.02, P< 0.001) , and overexpressed Arf6 (Arf6-T27N) significantly inhibited TNF-α-induced migration of breast cancer cells compared to theTNF-α-treatedcells (1.83±0.15 vs 2.33±0.14, P< 0.001) .

Conclusion

This study demonstrates that TNF-α promotes breast cancer cell migration by activating Arf6, suggesting that TNF-α/Arf6 might be used as a new target for controlling breast cancer metastasis.

Key words: TNF-α, Arf6, Breast cancer, Lymph node metastasis
表1 血浆TNF-α表达量与乳腺癌病理参数间的关系
临床病理参数 例数 TNF-α表达量 χ2 P值
低表达 高表达
年龄       0.016 0.901
≤60岁 29 14 15    
> 60岁 24 12 12
大小       0.212 0.646
≤2 cm 33 17 16    
>2 cm 20 9 11
淋巴结转移     9.294 0.002
N0 34 22 12    
N1-3 19 4 15
远端转移     - 0.491*
M0 51 26 25    
M1 2 0 2
临床分期     1.072 0.503
1-2 44 23 21    
3-4 9 3 6
ER表达状态     3.592 0.058
阴性 12 3 9    
阳性 41 23 18
PR表达状态     0.774 0.379
阴性 13 5 8    
阳性 40 21 19
HER2表达状态     0.040 0.842
阴性 17 8 9    
阳性 36 18 18
表2 不同淋巴结转移状态的乳腺癌患者血浆TNF-α表达量
淋巴结转移 例数 TNF-α表达量
未发生 34 391.24±307.35
已发生 19 709.58±277.51
t值   3.740
P值   < 0.001
图1 正置明视场显微镜下观察两组乳腺癌细胞迁移能力（×10）
表3 TNF-α对常见小G蛋白活化程度的影响（±s，n = 3）
小G蛋白 相对活化程度 t值 P值
对照组 诱导组
Rac1 1.00±0.04 1.03±0.06 0.737 0.502
Rac2 1.03±0.07 0.93±0.13 0.122 0.909
Rac3 1.00±0.02 0.98±0.66 0.505 0.640
RhoA 1.00±0.03 0.98±0.13 0.044 0.967
Cdc42 0.98±0.06 1.00±0.12 0.216 0.840
Arf1 1.00±0.04 1.00±0.05 0.100 0.925
Arf6 1.00±0.02 3.11±0.14 25.720 < 0.001
RalA 0.97±0.09 1.00±0.07 0.441 0.682
图2 正置明视场显微镜下观察不同分组乳腺癌细胞的迁移能力（×10）
表4 不同TNF-α浓度对乳腺癌细胞中Arf6活化程度的影响（±s，n = 3）
分组 Arf6相对活化程度
对照组 1.00±0.02
100 ng/ml诱导组 1.13±0.11
500 ng/ml诱导组 3.17±0.23ab
1000 ng/ml诱导组 3.12±0.10a
F值 230.276
P值 < 0.001
表5 不同分组乳腺癌细胞的Arf6相对活化程度及迁移能力（±s，n = 3）
分组 Arf6相对活化程度 相对愈合面积
空载对照组 1.00±0.02 1.00±0.03
Arf6-T27N组 1.00±0.05 1.03±0.11
空载转染+TNF-α组 2.95±0.23a 2.33±0.14a
Arf6-T27N+TNF-α组 1.92±0.13b 1.83±0.15b
F值 138.330 97.516
P值 < 0.001 < 0.001
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