沉默Kif2a基因抑制舌癌侵袭转移及其机制研究

Cytoskeletal reorganizations play an important role in tumor cell invasion and metastasis. Microtubule depolymerase Kif2a is a member of the Kinesin-13 family, which may be involved in changing cytoskeleton of the cells by regulating the microtubules dynamics. Our previous studies showed that the expression of Kif2a was higher in lymph node metastasis than that in primary foci in tongue squamous cells carcinoma, and its expression was positively correlated with lymphatic metastasis. Moreover, the results of gene expression profile showed the gene-silence of Kif2a mediated by siRNA led to downregulation of the Rho family, which were the main components of Rho/ROCK signal pathway. Therefore we speculate that Kif2a might regulate cell invasion and metastasis via Rho/ROCK signal pathway besides depolymerizing microtubules. In this study, RNAi was used in both in vitro and in vivo experiments in order to observe the effects of Kif2a on the invasion and metastasis of Tca8113 and SAS cells. In addition, the mechanism of Kif2a regulating the invasion and metastasis of tumor cells were explored by analyzing the relationship between Kif2a and Rho/ROCK signal pathway. Therefore this research will provide a new target and theoretical basis for anti-cancer therapy.

细胞骨架在肿瘤细胞侵袭及转移过程中发挥重要的作用，微管是细胞骨架的重要组成部分，微管解聚蛋白（Kif2a）是一种驱动蛋白，能够通过解聚微管而影响细胞骨架。我们前期研究发现，Kif2a在舌鳞癌淋巴结转移灶中的表达显著强于原发灶，并与淋巴结有无转移成正相关； 此外，Kif2a基因沉默后，Rho/ROCK信号转导通路中Rho家族主要基因表达下调。由此推测，Kif2a除了自身解聚微管影响细胞骨架外，还可能通过Rho/ROCK信号转导通路调控细胞的侵袭及转移。本研究拟以Tca8113、SAS细胞及裸鼠移植瘤为研究对象，采用RNAi技术，从体内、体外两个层面探讨Kif2a对细胞侵袭转移能力的影响，同时通过研究Kif2a与Rho/ROCK信号转导通路的关系，探讨Kif2a调控细胞侵袭转移的分子机制。该研究将为以细胞骨架为靶点的抗肿瘤治疗提供新型靶分子及理论依据。

细胞骨架在肿瘤细胞侵袭及转移过程中发挥重要作用，微管是细胞骨架的重要组成部分，微管解聚蛋白（Kif2a）是一种驱动蛋白，能通过解聚微管而影响细胞骨架。我们前期研究发现，Kif2a在舌鳞癌淋巴结转移灶中表达显著强于原发灶，并与淋巴结有无转移成正相关； 此外，Kif2a基因沉默后，Rho/ROCK信号转导通路中Rho家族主要基因表达下调。由此推测，Kif2a除了自身解聚微管影响细胞骨架外，还可能通过Rho/ROCK信号转导通路调控细胞侵袭及转移。本研究旨在探讨Kif2a对Tca8113细胞增殖及侵袭转移能力的调控及其分子机制。我们的实验结果显示，Kif2a基因沉默后Tca8113细胞增殖、侵袭及粘附能力明显降低，凋亡率增加。Kif2a基因沉默能显著增加化疗药物5-Fu对细胞增殖的抑制效应。Kif2a干扰组裸鼠移植瘤生长显著减慢。Kif2a干扰组细胞内RhoA、ROCK1、Rac1、Cdc42及PI3K、Akt、Bcl-2的基因和蛋白表达水平均显著下降，而Bax的表达显著升高。通过加入Rho/ROCK信号通路特异性抑制剂LY-27632和激动剂溶血磷脂酸LPA，进一步观察Kif2a基因沉默对细胞迁移能力的影响。与si-NC及si-Kif2a+LPA组比较，si-Kif2a及si-NC+LY-27632组细胞迁移及粘附能力显著下降。在加入胰岛素生长因子IGF-1 30分钟后, si-NC组细胞出现显著Akt磷酸化，而si-Kif2a组Akt磷酸化水平在各时间点都显著减少。裸鼠转移瘤实验结果显示，Lenti-NC及Lenti-Kif2a两组裸鼠双肺重量有显著性差异，Lenti-NC组转移瘤结节明显多于Lenti-Kif2a组；RhoA，ROCK1，Rac1及Cdc42蛋白在Lenti-Kif2a组的表达显著低于si-NC组。由此我们得出以下结论：Kif2a基因沉默能抑制Tca8113细胞的增殖、侵袭能力并提高Tca8113细胞对化疗药物5-Fu的敏感性。Kif2a基因沉默可能是通过调控Rho/ROCK1信号通路抑制Tca8113细胞的侵袭转移能力并通过PI3K/Akt信号通路诱导细胞凋亡。该研究结果将为以细胞骨架为靶点的抗肿瘤治疗提供新型靶分子及理论依据。