Stathmin在乳腺癌转移中的作用研究

The microtubule-destabilizing protein stathmin is closely associated with breast cancer development. The phosphorylation status of stathmin plays an important role in a lot of malignancies metastasis. Moreover, the regulation role of stathmin phosphorylation in breast cancer metastasis is poorly understood. In our previous study, the expression levels of stathmin in invasive breast cancer cells were significantly higher than in noninvasive breast cancer cells. The mechanisms of stathmin regulation in breast cancer metastasis are not clear and will be explored for further study. In this study, we will construct the cell models and mouse models with high stathmin expression in order to discover the relationship between stathmin and breast cancer metastasis. Meanwhile, we have collected breast cancer cases for clinical research to explore the correlation of stathmin expression and phosphorylation status in breast cancer metastasis. Then we will respectively mutate four serine sites of stathmin to alanine (Ser16, Ser25, Ser38 and Ser63) by point-mutated method to construct the recombinant plasmids with mutant stathmin. The recombinant plasmids will be transfected into breast cancer cells in order to the regulation role of stathmin phosphorylation in breast cancer metastasis through cell adhesion, motility and invasion assays. The further study will be performed in mouse models. Altogether, these studies may discover the regulation role of stathmin in breast cancer metastasis and provide an attractive target for breast cancer biological therapy.

微管解聚蛋白stathmin与乳腺癌的发生发展密切相关。已有研究报道，stathmin的磷酸化修饰在恶性肿瘤的转移中发挥重要作用。我们前期的研究表明，stathmin在侵袭性乳腺癌细胞中的表达水平明显高于非侵袭性乳腺癌细胞，但目前stathmin在乳腺癌转移中的作用机理尚不清楚。本课题拟通过构建stathmin表达增加的乳腺癌细胞模型和动物模型，评价stathmin与乳腺癌转移发生的相关性；同时开展临床研究，收集乳腺癌组织，探讨stathmin表达水平及磷酸化修饰在乳腺癌转移中的相互关系；通过点突变的方式分别将stathmin蛋白4个丝氨酸位点突变为丙氨酸，构建4种磷酸化位点突变的重组载体，转染乳腺癌细胞，通过检测细胞粘附、运动及侵袭的变化在细胞水平探讨stathmin磷酸化修饰对乳腺癌转移的调控作用，进一步在动物体内验证这种调控作用，希望为乳腺癌生物治疗发掘新靶点奠定实验基础。

微管解聚蛋白Stathmin在许多恶性肿瘤包括乳腺癌中都呈高表达状态，并且它可以通过调控微管的解聚与聚合影响肿瘤细胞的运动，从而参与恶性肿瘤的侵袭及转移。但是目前Stathmin通过何种方式调控微管参与乳腺癌转移的作用机制还不清楚。为了探讨Stathmin及其磷酸化在乳腺癌转移中的作用机理，我们在本课题的实施过程中（1）初步证实了在MDA-MB-231、SKBR3、MCF-7三种细胞中，细胞侵袭力差异和Stathmin表达水平相关，高侵袭性乳腺癌细胞中Stathmin表达水平明显高于低侵袭性乳腺癌细胞；并且Stathmin表达水平上调促进乳腺癌细胞粘附、运动、侵袭和成瘤能力，Stathmin表达水平下调抑制细胞增殖、粘附、运动、侵袭和成瘤能力；（2）发现了不同乳腺癌细胞四个丝氨酸位点的磷酸化水平不同。在MDA-MB-231、SKBR3、MCF-7三种乳腺癌细胞中，Ser16和Ser63表达相同，不同主要在Ser25和Ser38，MDA-MB-231细胞两个位点都为阳性，SKBR3细胞Ser25为阴性，Ser38为阳性，而MCF-7的Ser25为阳性，Ser38为阴性；（3）乳腺癌组织的Stathmin表达量和磷酸化状态与肿瘤组织学分级和转移相关。Stathmin及磷酸化表达在乳腺癌肿瘤组织中阳性率明显高于癌旁组织； Stathmin及磷酸化位点Ser16、Ser25、Ser38和Ser63在肿瘤组织分级高的乳腺癌组织中阳性率明显高于分级低的乳腺癌组织，存在差异并有统计学意义（P<0.05）；同时Stathmin表达和Ser16磷酸化在伴转移的乳腺癌组织中的阳性率高于不伴转移的乳腺癌组织，差异有统计学意义（P<0.05）；（4）通过细胞模型和动物模型进一步证实了Stathmin磷酸化状态与细胞的侵袭相关，与Stathmin高表达细胞相比，Stathmin磷酸化位点突变抑制细胞粘附、运动和侵袭。本课题通过研究初步阐明Stathmin在乳腺癌转移中的调控机理，为发掘乳腺癌治疗新靶点奠定理论基础。