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中华临床医师杂志(电子版) ›› 2018, Vol. 12 ›› Issue (05) : 279 -287. doi: 10.3877/cma.j.issn.1674-0785.2018.05.005

所属专题: 乳腺疾病 文献

临床研究

TMPRSS4通过上皮间质转化促进乳腺癌细胞的增殖、侵袭和转移
李晓梅1,(), 叶红1, 秦书明1, 林敏1, 侯刚1   
  1. 1. 271000 山东泰安,泰安市中心医院病理科
  • 收稿日期:2017-12-26 出版日期:2018-03-01
  • 通信作者: 李晓梅
  • 基金资助:
    泰安市科技发展计划(2016NS1200)

TMPRSS4 promotes breast cancer cell migration and invasion by regulating epithelial-mesenchymal transition

Xiaomei Li1,(), Hong Ye1, Shuming Qin1, Min Lin1, Gang Hou1   

  1. 1. Department of Pathology, Taian Central Hospital, Taian 271000, China
  • Received:2017-12-26 Published:2018-03-01
  • Corresponding author: Xiaomei Li
  • About author:
    Corresponding author: Li Xiaomei, Email:
引用本文:

李晓梅, 叶红, 秦书明, 林敏, 侯刚. TMPRSS4通过上皮间质转化促进乳腺癌细胞的增殖、侵袭和转移[J/OL]. 中华临床医师杂志(电子版), 2018, 12(05): 279-287.

Xiaomei Li, Hong Ye, Shuming Qin, Min Lin, Gang Hou. TMPRSS4 promotes breast cancer cell migration and invasion by regulating epithelial-mesenchymal transition[J/OL]. Chinese Journal of Clinicians(Electronic Edition), 2018, 12(05): 279-287.

目的

通过检测Ⅱ型跨膜丝氨酸蛋白酶4(TMPRSS4)在乳腺癌细胞中的表达情况,分析其在乳腺癌细胞增殖、侵袭和转移过程中的作用及其与上皮间质转化(EMT)的关系。

方法

采用实时荧光定量PCR(qRT-PCR)和Western blot法检测5种不同乳腺癌细胞系中TMPRSS4 mRNA和蛋白水平的表达情况。将过表达质粒转染至乳腺癌细胞中,采用qRT-PCR方法检测转染效率,并通过MTS和EdU细胞增殖实验、Transwell和Matrigel细胞迁移和侵袭实验,研究过表达TMPRSS4对乳腺癌细胞增殖、侵袭和迁移的作用。采用qRT-PCR和Western blot法检测过表达TMPRSS4后细胞EMT相关基因E-cadherin、Vimentin、Claudin-1、Slug、ZEB1的表达变化。

结果

TMPRSS4在乳腺癌MDA-MB-468和MDA-MB-231细胞系中高表达。MTS实验显示TMPRSS4过表达组乳腺癌细胞MDA-MB-468和MDA-MB-231的增殖能力与阴性对照组相比明显增加,差异具有统计学意义(P=0.039和0.038),EdU实验进一步证实过表达TMPRSS4促进乳腺癌细胞的增殖,MDA-MB-468细胞和MDA-MB-231细胞的增殖率与阴性对照组比较差异具有统计学意义(P=0.001和0.008)。Transwell实验显示,TMPRSS4过表达组MDA-MB-468细胞和MDA-MB-231细胞的迁移能力与阴性对照组比较明显提高,差异具有统计学意义(p均=0.001)。Matrigel实验显示,TMPRSS4过表达组MDA-MB-468细胞和MDA-MB-231细胞的侵袭浸润能力与阴性对照组比较明显提高,差异具有统计学意义(P=0.012和0.000)。与阴性对照组比较,过表达TMPRSS4后,EMT相关基因包括上皮性标记E-cadherin和Claudin-1的表达均显著下降,而间质标记Vimentin和Slug的表达均明显提高,差异具有统计学意义(P分别=0.024,0.003,0.002和0.012)。

结论

TMPRSS4参与了乳腺癌EMT的发生过程,并通过EMT促进乳腺癌细胞的增殖、侵袭和转移。

Objective

To investigate the expression of TMPRSS4 in breast cancer (BC) cells and to analyze the relationship between TMPRSS4 expression and epithelial-mesenchymal transition (EMT) as well as the significance of TMPRSS4 expression in the development and progression of BC.

Methods

qRT-PCR and Western blot analysis were used to determine the expressions levels of TMPRSS4 mRNA and protein in five BC cell lines (MCF-7, ADM, T47D, MDA-MB-231, and MDA-MB-468). A plasmid expressing TMPRSS4 was transfected into MDA-MB-468 and MDA-MB-231cells, and transfection efficiency was evaluated. The MTS assay, EdU assay, and Transwell and Matrigel invasion assays were used to assess the proliferation, invasion, and metastasis of BC cells. Following transfection with the TMPRSS4 expressing plasmid, the mRNA and protein expression of key biomarkers of EMT (E-cadherin, Vimentin, Claudin-1, Slug, and ZEB1) was detected by qRT-PCR and Western blot analysis, respectively.

Results

qRT-PCR and Western blot analysis showed that TMPRSS4 was differentially expressed in five BC cell lines, with MDA-MB-468 cells having the highest expression. MTS assay showed that the cell proliferation rate was significantly increased in TMPRSS4 overexpressing MDA-MB-468 cells (P=0.039) and MDA-MB-231 cells (P=0.038) than in non-transfected cells. EdU assay further confirmed that TMPRSS4 overexpression promoted BC cell proliferation. The proliferation of TMPRSS4 overexpressing MDA-MB-468 cells and MDA-MB-231 cells was significantly higher than that of the negative control group (P=0.001 and P=0.008, respectively). Transwell and Matrigel assays showed that the migration and infiltration capacities of TMPRSS4 overexpressing MDA-MB-468 cells and MDA-MB-231 cells were significantly increased compared with those of the negative control group (P=0.001, 0.001, 0.012, and 0.000, respectively). qRT-PCR and Western blot analysis revealed that the expression of E-cadherin and Claudin-1 was significantly decreased, while the expression of Vimentin and Slug was increased after the overexpression of TMPRSS4 (P=0.024, 0.003, 0.002, and 0.012, respectively).

Conclusion

TMPRSS4 participates in the occurrence of EMT in BC and promotes the proliferation, invasion, and metastasis of BC cells through regulating EMT.

表1 基因名称及引物序列
表2 5种乳腺癌细胞系中TMPRSS4 mRNA和蛋白质的表达水平(±s
图1 TMPRSS4在5种乳腺癌细胞系中的表达 图1a qRT-PCR显示TMPRSS4在MDA-MB-468、MDA-MB-231中高表达;图1b Western blot实验显示TMPRSS4在MDA-MB-468、MDA-MB-231中高表达
图2 pCMV-myc-TMPRSS4过表达质粒测序图
图3 qRT-PCR检测所得乳腺癌细胞转染效率MDA-MB-468和MDA-MB-231两个细胞系均显示转染过表达质粒pCMV-myc-TMPRSS4后,细胞内TMPRSS4 mRNA含量较阴性对照组(Vector)明显升高(*P<0.05)
图4 MTS实验所示过表达TMPRSS4对乳腺癌细胞增殖的影响 图4a MDA-MB-468细胞转染过表达质粒pCMV-myc-TMPRSS4 96 h后,与阴性对照组(Vector)比较细胞增殖率显著提高(*P<0.05);图4b MDA-MB-231细胞转染过表达质粒pCMV-myc-TMPRSS4 96 h后,与阴性对照组比较细胞增殖率显著提高(*P<0.05)
图5 EdU实验所示过表达TMPRSS4对乳腺癌细胞增殖的影响 图5a,图5c 蓝色荧光(DAPI)代表细胞总数,红色荧光(EdU)代表增殖的细胞数,混合荧光(Merged)代表增值细胞数占细胞总数的比例;图5b,图5d MDA-MB-468细胞和MDA-MB-231细胞的增殖率与阴性对照组(Vector)比较显著提高(**P<0.01)
图6 过表达TMPRSS4对乳腺癌细胞迁移和侵袭浸润的影响 图6a,图6c MDA-MB-468细胞和MDA-MB-231细胞过表达TMPRSS4组穿过滤膜(Migration)和基质胶(Invasion)的细胞数显著多于阴性对照组(Vector);图6b,图6d MDA-MB-468细胞和MDA-MB-231细胞的迁移和侵袭能力较阴性对照组明显升高(*P<0.05,**P<0.01,***P<0.001)
图7 过表达TMPRSS4对乳腺癌细胞EMT相关基因的影响 图7a,图7b qRT-PCR检测,MDA-MB-468和MDA-MB-231细胞过表达TMPRSS4后,与阴性对照组(Vector)比较,EMT相关基因上皮性标记E-cadherin和Claudin-1的表达均明显下降,间质标记Vimentin和Slug的表达均明显提高(*P<0.05,**P<0.01);图7c-图7f Western blot检测,MDA-MB-468和MDA-MB-231细胞过表达TMPRSS4后,与阴性对照组比较,EMT相关基因蛋白的表达结果与qRT-PCR检测结果一致(*P<0.05,**P<0.01,***P<0.001)
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