【摘要】 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MDAMB-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。更多还原

【Abstract】 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, T47 D, MDA-MB-231, and MDA-MB-468). A plasmid expressing TMPRSS4 was transfected into MDA-MB-468 and MDA-MB-231 cells, 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). qRTPCR 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.更多还原