Ehm2可变剪接体在乳腺癌中表达的临床意义及分子机制

Ehm2, a member of FERM family proteins, is overexpressed in breast cancers and prostate cancers. Ehm2 plays an important role in the metastasis of breast cancer. Ehm2 gene can produce two mRNA transcript variants by alternative splicing. Ehm2 transcript variant1 lacks several exons and includes an alternate 3' terminal exon, compared to variant 2, which contains exons 1-26. In our previous study we found that the two Ehm2 transcript variants were both expressed in breast cancer cell lines but had different expression patterns, which played an opposite role in cell adhesion and cell migration. However, express pattern of two Ehm2 alternative splice variants and their functional roles in breast cancer are very unclear. In this study we will examine the expression profiles of two alternative splice variants of Ehm2 in breast cancer species, and assess the correlation of Ehm2 splice variants expression with histologic type, grade, lymph node involvement, tumor metastasis stage, and prognostic relevance. Then we will interfere with the amount of two Ehm2 splice variants by transfecting breast cancer cells with expression vectors of Ehm2 splice variants and lentiviral shRNA vectors targeting to Ehm2 splice variants, analyze cell functions and molecular mechanisms for the effects of Ehm2 splice variants on cell functions. Then we will study the effects of two Ehm2 splice variants on tumor growth and tumor metastasis by transplanting tumor in nude mice. According to the in vitro results, further experiments are will be carried out in clinical specimens and tumor bearing nude mice to verify the mechanism of different Ehm2 splice variants in the invasion and metastasis of breast cancer. This study aims to clarify the function of Ehm2 splice variants in breast cancer and their regulation mechanism, to provide a new way for the study of breast cancer metastasis and to explore new ways for the treatment of breast cancer.

高转移细胞中表达蛋白Ehm2在乳腺癌的转移中起重要作用。前期研究表明，在乳腺癌细胞系中Ehm2存在两种mRNA可变剪接体，在细胞黏附迁移中起相反作用，但其表达模式及功能仍缺乏深入研究。本研究拟通过检测乳腺癌标本中Ehm2两个可变剪接体的表达并结合病人资料分析Ehm2可变剪接体与肿瘤病理级别及病人预后的关系；构建Ehm2可变剪接体表达载体及靶向Ehm2可变剪接体的shRNA载体，干预细胞内Ehm2不同可变剪接体的含量，在细胞水平观测Ehm2可变剪接体对肿瘤细胞功能的影响，并在分子水平探讨其作用机制；通过裸鼠荷瘤实验研究Ehm2不同可变剪接体对肿瘤生长和转移的影响；根据体外机制研究结果，在临床标本和活体动物水平进一步验证Ehm2不同可变剪接体调控乳腺癌侵袭转移的作用机制。本研究力图阐明乳腺癌中Ehm2不同可变剪接体的功能及调控机制，为乳腺癌转移机制的研究提供新思路，为乳腺癌治疗挖掘新途径。

乳腺癌是女性排名第一的常见恶性肿瘤，转移是乳腺癌治疗失败的首要原因，探明乳腺癌转移的机制有助于发现新的作用靶点，指导转移性乳腺癌的诊断和个性化治疗。高转移细胞中表达的蛋白Ehm2（Expressed in high-metastatic cells）通过可变剪接产生两种可变剪接体——Ehm2/1和Ehm2/2，这两种可变剪接体在肿瘤发生发展中发挥何种作用尚不清楚。通过本项目研究，我们首先检测了乳腺癌临床样本中Ehm2两个可变剪接体的表达情况，并分析其与病人预后的相关性；在乳腺癌细胞中过表达/敲减Ehm2，分析这两个可变剪接体对乳腺癌细胞生长、侵袭和迁移能力的影响；在机制研究方面，我们详细探讨了Ehm2/1影响钙黏蛋白E-cadherin蛋白水平的分子机制。结果表明，在正常乳腺组织中，Ehm2主要表达可变剪接体1，呈明显的极性分布，主要分布在上皮细胞的顶膜和侧膜，在乳腺癌中表达显著下调；可变剪接体2分布无极性，在乳腺癌组织中表达增加。Ehm2可变剪接体1高表达乳腺癌患者预后较好，可变剪接体2高表达与患者不良预后相关。过表达Ehm2可变剪接体1可显著降低细胞的恶性表型，而过表达Ehm2可变剪接体2促进细胞的恶性表型。过表达可变剪接体1能提高E-cadherin的蛋白水平，不影响其mRNA水平，而Ehm2/2作用相反。进一步的结果表明，Ehm2/1能提高E-cadherin的半衰期，增强E-cadherin蛋白稳定性，抑制其泛素化水平及蛋白酶体降解。蛋白质相互作用结果显示，Ehm2/1与E-cadherin存在相互作用。此外，我们还筛选鉴定了E-cadherin的特异E3泛素连接酶为Smurf1，过表达Ehm2/1可以抑制Smurf1对E-cadherin的泛素化修饰。这些结果表明，Ehm2/1过表达通过降低E-cadherin泛素化降解发挥抑癌作用。