Department of Urology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Hubei, Republic of China.
Urol Oncol. 2012 Mar-Apr;30(2):167-76. doi: 10.1016/j.urolonc.2010.02.009. Epub 2010 Sep 22.
To study the underlying alteration in the expression of epithelial markers involved in epithelial-mesenchymal transition (EMT), and elucidate the potential mechanism(s) for Tβ4-induced EMT-like phenotypic changes in bladder cancer cells.
All tissue samples in this study were obtained from clinical patients of the Union Hospital of Tongji Medical College, and were confirmed by surgery and pathology. Of these, normal bladder tissues (control), primary urothelial carcinoma of different grades (Stage pTa, Stage pT3), bladder paracancerous tissues, accompanied with 2 bladder cancer cell lines (BIU-87 and T24), were divided into 6 groups. Quantitative RT-PCR, Western blotting, and immunohistochemical study of adhesion molecules Tβ4, ILK, E-cadherin, and β-catenin involved in EMT were carried out. A lentiviral gene transferring vector containing the RNA polymerase III-dependent U6 promoter to express short hairpin RNA (shRNA) directed against Tβ4 was also applied. In the present study, all agents were evaluated using commercial kits.
A strong correlation between the expression levels of Tβ4, ILK, E-cadherin, and β-catenin was found in the bladder transitional cell carcinoma (TCC) patients. In the BIU-87 and T24 bladder cancer cells overexpressing Tβ4, which were accompanied by a loss of E-cadherin as well as a cytosolic accumulation of β-catenin, up-regulation of ILK was also revealed. The inhibition of the Tβ4 expression with lentiviral shRNA vector could raise EMT-like phenotypic changes, significantly depressed motility, and subsequent invasiveness of bladder cancer cells.
Our results imply that the Tβ4 is likely to play a crucial role in EMT progression, and that inhibition of the Tβ4 expression or interactions with other genes should be novel therapeutic targets for bladder cancers with high invasive and metastatic potential.
研究上皮细胞标志物在 EMT 中表达的潜在改变，并阐明 Tβ4 诱导膀胱癌细胞发生 EMT 样表型改变的潜在机制。
本研究中的所有组织样本均来自华中科技大学同济医学院附属协和医院的临床患者，并通过手术和病理证实。其中，正常膀胱组织（对照组）、不同分级的原发性尿路上皮癌（pTa 期、pT3 期）、膀胱旁组织、伴有 2 种膀胱癌细胞系（BIU-87 和 T24），分为 6 组。采用定量 RT-PCR、Western blot 和免疫组织化学方法研究 EMT 相关黏附分子 Tβ4、ILK、E-钙黏蛋白和β-连环蛋白。还应用了一种含 RNA 聚合酶 III 依赖性 U6 启动子的慢病毒基因转移载体来表达针对 Tβ4 的短发夹 RNA（shRNA）。在本研究中，所有试剂均采用商业试剂盒进行评估。
在膀胱癌患者中发现 Tβ4、ILK、E-钙黏蛋白和β-连环蛋白的表达水平之间存在很强的相关性。在过表达 Tβ4 的 BIU-87 和 T24 膀胱癌细胞中，E-钙黏蛋白表达下降，β-连环蛋白在细胞质中积累，同时也发现 ILK 的上调。用慢病毒 shRNA 载体抑制 Tβ4 的表达可导致 EMT 样表型改变，显著抑制膀胱癌细胞的迁移和随后的侵袭能力。
我们的结果表明，Tβ4 可能在 EMT 进展中发挥关键作用，抑制 Tβ4 的表达或与其他基因的相互作用可能是具有高侵袭性和转移性潜能的膀胱癌的新治疗靶点。
