切换至 "中华医学电子期刊资源库"

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: 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]. 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]. 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)
1
Cheng D, Liang B, Li Y. High TMPRSS4 expression is a predictor of poor prognosis in cervical squamous cell carcinoma [J]. Cancer Epidemiol, 2013, 37(6):993-997.
2
Ohler A, Becker-Pauly C. TMPRSS4 is a type II transmembrane serine protease involved in cancer and viral infections [J]. Biol Chem, 2012, 393(9):907-914.
3
Shi G, Yang X, Dai B, et al. Clinical significance of TMPRSS4 in prostate cancer [J]. Int J Clin Exp Pathol, 2014, 7(11):8053-8058.
4
Hooper JD, Clements JA, Quigley JP, et al. Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes [J]. J Biol Chem, 2001, 276(2):857-860.
5
Min HJ, Lee Y, Zhao XF, et al. TMPRSS4 upregulates uPA gene expression through JNK signaling activation to induce cancer cell invasion [J]. Cell Signal, 2014, 26(2):398-408.
6
Choi SY, Shin HC, Kim SY, et al. Role of TMPRSS4 during cancer progression [J]. Drug News Perspect, 2008, 21(8):417-423.
7
Wang CH, Guo ZY, Chen ZT, et al. TMPRSS4 facilitates epithelial-mesenchymal transition of hepatocellular carcinoma and is a predictive marker for poor prognosis of patients after curative resection [J]. Sci Rep, 2015, 5:12366.
8
Larzabal L, Nguewa PA, Pio R, et al. Overexpression of TMPRSS4 in non-small cell lung cancer is associated with poor prognosis in patients with squamous histology [J]. Br J Cancer, 2011, 105(10):1608-1614.
9
Guan H, Liang W, Liu J, et al. Transmembrane protease serine 4 promotes thyroid cancer proliferation via CREB phosphorylation [J]. Thyroid, 2015, 25(1):85-94.
10
Polyak K,Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits [J]. Nat Rev Cancer, 2009, 9(4):265-273.
11
Jung H, Lee KP, Park SJ, et a1. TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an epithelial-mesenchymal transition [J]. Oncogene, 2008, 27(18):2635-2647.
12
Lee Y, Ko D, Min HJ,et al. TMPRSS4 induces invasion and proliferation of prostate cancer cells through induction of Slug and cyclin D1 [J]. Oncotarget, 2016, 7(31):50315-50332.
13
Kim S, Kang HY, Nam EH,et al. TMPRSS4 induces invasion and epithelial-mesenchymal transition through upregulation of integrin a5 and its signaling pathways [J]. Carcinogenesis, 2010, 31(4):597-606.
14
Jin J, Shen X, Chen L, et al. TMPRSS4 promotes invasiveness of human gastric cancer cells through activation of NF-kB/MMP-9 signaling [J]. Biomed Pharmacother, 2016, 77:30-36.
[1] 邵华, 那子悦, 荆慧, 李博, 王秋程, 程文. 术前经皮超声造影对乳腺癌腋窝前哨淋巴结转移及负荷的诊断价值[J]. 中华医学超声杂志(电子版), 2023, 20(08): 849-853.
[2] 张旭, 徐建平, 苏冬明, 王彩芬, 王大力, 张文智. 男性乳腺肿块的超声造影特征[J]. 中华医学超声杂志(电子版), 2023, 20(08): 854-859.
[3] 孙帼, 谢迎东, 徐超丽, 杨斌. 超声联合临床特征的列线图模型预测甲状腺乳头状癌淋巴结转移的价值[J]. 中华医学超声杂志(电子版), 2023, 20(07): 734-742.
[4] 马伟强, 马斌林, 吴中语, 张莹. microRNA在三阴性乳腺癌进展中发挥的作用[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 111-114.
[5] 唐旭, 韩冰, 刘威, 陈茹星. 结直肠癌根治术后隐匿性肝转移危险因素分析及预测模型构建[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 16-20.
[6] 冯冰, 邹秋果, 梁振波, 卢艳明, 曾奕, 吴淑苗. 老年非特殊型浸润性乳腺癌超声征象与分子生物学指标的临床研究[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 48-51.
[7] 栗艳松, 冯会敏, 刘明超, 刘泽鹏, 姜秋霞. STIP1在三阴性乳腺癌组织中的表达及临床意义研究[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 52-56.
[8] 袁育韬, 邢金琳, 谢克飞, 殷凯. CT征象及BRAFV600E基因突变与甲状腺乳头状癌中央区淋巴结转移的相关性[J]. 中华普外科手术学杂志(电子版), 2023, 17(06): 611-614.
[9] 江振剑, 蒋明, 黄大莉. TK1、Ki67蛋白在分化型甲状腺癌组织中的表达及预后价值研究[J]. 中华普外科手术学杂志(电子版), 2023, 17(06): 623-626.
[10] 晏晴艳, 雍晓梅, 罗洪, 杜敏. 成都地区老年转移性乳腺癌的预后及生存因素研究[J]. 中华普外科手术学杂志(电子版), 2023, 17(06): 636-638.
[11] 徐成, 王璐璐, 王少华. 洗脱液甲状腺球蛋白在甲状腺乳头状癌转移淋巴结中的应用[J]. 中华普外科手术学杂志(电子版), 2023, 17(06): 701-704.
[12] 刘飞, 王影新, 马骍, 辛灵, 程元甲, 刘倩, 王悦, 张军军. 不同介质腔内心电图定位技术在乳腺癌上臂输液港植入术中应用的随机对照研究[J]. 中华临床医师杂志(电子版), 2023, 17(07): 760-764.
[13] 岳瑞雪, 孔令欣, 郝鑫, 杨进强, 韩猛, 崔国忠, 王建军, 张志生, 孔凡庭, 张维, 何文博, 李现桥, 周新平, 徐东宏, 胡崇珠. 乳腺癌HER2蛋白表达水平预测新辅助治疗疗效的真实世界研究[J]. 中华临床医师杂志(电子版), 2023, 17(07): 765-770.
[14] 邓世栋, 刘凌志, 郭大勇, 王超, 黄忠欣, 张晖辉. 沉默SNHG1基因对膀胱癌细胞增殖、凋亡、迁移和铁死亡的影响[J]. 中华临床医师杂志(电子版), 2023, 17(07): 804-811.
[15] 孔凡彪, 杨建荣. 肝脏基础疾病与结直肠癌肝转移之间关系的研究进展[J]. 中华临床医师杂志(电子版), 2023, 17(07): 818-822.
阅读次数
全文


摘要