FAK选择性剪接体Δ26FAK与乳腺癌转移的关系及其机制研究

Numerous evidence indicates that breat cancer-associated splicing variants play an important role in breast cancer initiation and progression. Focal adhesion kinase(FAK) is an intra-cellular non-receptor tyrosine kinase that frequently correlate with breast cancer metastasis and poor prognosis. Our previous results indicate that a splicing variant of FAK(Δ26 FAK) may play important role in breat cancer cell migration. Yet the precise mechanism needs to be further studied. And the mechanism leading to the generation of Δ26 FAK is still unclear. Our hypothesis is that alterations of the protein structure lead to constitutive activation and aberrant intermolecular interaction since a deletion of 26exon. Then this property correlates with the breast cancer metastasis. To demonstrate our hypothesis, we use various biochemical and biological assays, such as protein immunorecipitation, immunoblotting, IP, EMSA, cell migration assays, immunofluorescence. This alternative splicing study is opening new avenue of research in breast metastasis. The alternatively spliced protein isoforms and the alternative splicing process itself are potential targets for breast cancer therapy.

乳腺癌相关基因的选择性剪接体在乳腺癌发生发展中起着重要作用。黏着斑激酶(FAK)是一种与乳腺癌转移及预后密切相关的非受体蛋白酪氨酸激酶。我们前期研究提示它的选择性剪接体Δ26 FAK与乳腺癌的转移有关，但具体作用机制及它的形成原因还有待进一步探讨。我们推测Δ26 FAK可能因为第26个外显子缺失引起蛋白质分子构象改变，导致它的持续激活及与下游蛋白相互作用异常而促进乳腺癌转移；Δ26 FAK的形成或与基因突变及反式作用因子SF2/ASF的表达水平有关。我们将通过RT-PCR、Western Blot、IP、EMSA、免疫荧光、Transwell等从分子、细胞、组织以及动物水平等多方面探讨Δ26 FAK在乳腺癌转移中的作用及Δ26 FAK的形成原因。本研究从选择性剪接这个视点为揭示乳腺癌转移机制奠定基础，以选择性剪接蛋白为药物靶点或纠正剪接调控机制为治疗乳腺癌转移提供新途径。

乳腺癌相关基因的选择性剪接体在乳腺癌发生发展中起着重要的作用。我们新发现的黏着斑激酶(FAK)的异常选择性剪接体Δ 26 FAK 与乳腺癌发生发展有关。通过RT-PCR及DNA测序等方法对102例乳腺癌标本及配对的52例癌旁组织进行检测发现了Δ 26 FAK，在MCF-10A细胞中过表达Δ 26 FAK，然后通过免疫共沉淀以及Western Blot等方法检测激酶活性，免疫荧光等检测它在细胞内的定位，Transwell检测细胞迁移能力，并用流式细胞技术，TUNEL等方法检测细胞凋亡水平。结果提示Δ 26 FAK与野生型FAK具有相似的生物学功能，但是比野生型FAK具有更强的抑制细胞凋亡的能力。因为野生型FAK会被caspase降解而Δ 26 FAK则会持续存在并激活，从而发挥抑制细胞凋亡的作用。