Dong Ruofan, Pu Hong, Wang Yuan, Yu Jinjin, Lian Kuixian, Mao Caiping
Department of Reproductive Center, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China; Department of Obstetrics and Gynecology, The Affiliated Hospital of Jiangnan University and the Fourth People's Hospital of Wuxi, Wuxi, Jiangsu Province, China.
APMIS. 2015 May;123(5):394-400. doi: 10.1111/apm.12361. Epub 2015 Feb 27.
We previously reported frequent loss of TESTIN in human endometrial carcinoma, which significantly suppressed tumor proliferation and invasion. Herein, we further explored the mechanisms underlying TESTIN loss and its roles in the epithelial-mesenchymal transition (EMT, a key step for tumor spreading). Methylation-specific PCR was performed to investigate the promoter status of TESTIN in a panel of endometrial cancer and normal endometrium tissues. The expression of TESTIN mRNA was determined by real-time PCR. Up- and down-regulation of TESTIN were achieved by transient transfection with pcDNA3.1-TESTIN and shRNA-TESTIN plasmids, respectively. The EMT alterations were observed under the optical microscope and EMT-related markers were detected by real-time PCR and western blot. Compared to the control (3.6%), TESTIN was hypermethylated in 43.7% endometrial cancer tissues (p < 0.001). Moreover, TESTIN hypermethylation was significantly correlated with advanced tumor stage, deep myometrial invasion and lymphatic node metastasis. In vitro, the demethylating agent dramatically restored the expression of TESTIN. In addition, up-regulation of TESTIN significantly suppressed the EMT procedure; whereas down-regulation of TESTIN enhanced EMT. In conclusion, we demonstrated that loss of TESTIN was mainly caused by hypermethylation, which might be a potent prognostic marker. Furthermore, we proved that TESTIN significantly suppressed the EMT procedure, proposing restoration of TESTIN to be a novel therapeutic strategy for endometrial carcinoma.
我们之前报道过，在人类子宫内膜癌中TESTIN频繁缺失，这显著抑制了肿瘤的增殖和侵袭。在此，我们进一步探究了TESTIN缺失的潜在机制及其在上皮-间质转化（EMT，肿瘤扩散的关键步骤）中的作用。采用甲基化特异性PCR检测了一组子宫内膜癌组织和正常子宫内膜组织中TESTIN的启动子状态。通过实时PCR测定TESTIN mRNA的表达。分别用pcDNA3.1-TESTIN和shRNA-TESTIN质粒进行瞬时转染，实现TESTIN的上调和下调。在光学显微镜下观察EMT变化，并通过实时PCR和蛋白质印迹法检测EMT相关标志物。与对照组（3.6%）相比，43.7%的子宫内膜癌组织中TESTIN发生了高甲基化（p<0.001）。此外，TESTIN高甲基化与肿瘤晚期、肌层深层浸润和淋巴结转移显著相关。在体外，去甲基化剂显著恢复了TESTIN的表达。此外，TESTIN的上调显著抑制了EMT过程；而TESTIN的下调则增强了EMT。总之，我们证明TESTIN的缺失主要是由高甲基化引起的，这可能是一个有效的预后标志物。此外，我们证明TESTIN显著抑制了EMT过程，提出恢复TESTIN有望成为子宫内膜癌的一种新的治疗策略。
