YAP调控肝癌细胞增殖活性及对索拉非尼敏感性的影响

YAP regulates the proliferation and modifies the sensitivity to sorafenib in hepatoceUular carcinoma cells

目的检测YAP在肝癌细胞中的表达水平，并探讨其对肝癌细胞增殖活性以及对索拉非尼敏感性的影响。方法采用Western blot方法检测人肝癌细胞株SMMC－7721、SK－Hep－1、HepG－2、Huh7和正常人肝细胞株L－O2中YAP的蛋白表达水平。以YAP的小干扰RNA或过表达质粒分别转染SK－Hep－1和Huh7细胞，采用细胞计数试剂盒8（CCK－8）法检测细胞的增殖能力，流式细胞术检测细胞周期。将SK－Hep－1和SK－Hep－1 si－YAP细胞接种于裸鼠皮下，并以索拉非尼灌胃，观察移植瘤与对照组的差异。结果YAP蛋白在肝癌细胞株中表达升高。下调SK－Hep－1细胞的YAP表达后，细胞存活率为（78.5±0.3）%，与对照组[（92.3±0.2）%]比较明显下降，差异有统计学意义（P＝0.025）；G0/G1期细胞占（65.4±3.3）%，与对照组[（55.7±3.4）%]比较明显升高，差异有统计学意义（P＝0.039）。上调Hun7细胞的YAP表达后，细胞存活率为（81.2±1.3）%，与对照组[（62.5±1.1）%]比较明显升高，差异有统计学意义（P＝0.013）; G0/G1期细胞占（38.2±3.8）% ，与对照组[（48.8±2.9）%]比较明显下降，差异有统计学意义（P＝0.019）。si－YAP＋索拉非尼组SK－Hep－1细胞的存活率为（31.13±1.79）%，低于索拉非尼组[（48.87±0.58）%]，差异有统计学意义（P〈0.0001）。PC3.1－YAP＋索拉非尼组的细胞存活率为（69.98±2.94）%，高于索拉非尼组[（53.53±1.93）%]，差异有统计学意义（P〈0.0001）。裸鼠移植瘤模型实验显示，SK－Hep－1组、SK－Hep－1＋索拉非尼组、SK－Hep－1 si－YAP组和SK－Hep－1 si－YAP＋索拉非尼组瘤重分别为（0.96±0.08）g、（0.62±0.08）g、（0.70±0.06）g和（0.27±0.02）g。SK－Hep－1＋索拉非尼组和SK－Hep－1 si－YAP组瘤重均低于SK－Hep－1组（P值分别为0.012和0.031），SK－Hep－1 si－YAP＋索拉非尼组瘤重低于SK－Hep－1 si－YAP组（P＝0.001）。结论YAP在肝癌细胞系中表达上调，其表达变化影响细胞增殖能力和细胞周期进程，并影响肝癌对索拉非尼的敏感性。YAP是肝癌治疗的一个潜在分子靶点。

ObjectiveTo detect the expression level of YES-associated protein 1 （YAP） in hepatocellular carcinoma （HCC） cell lines and investigate its effects on the proliferation activity and the sensitivity to sorafenib in HCC cells.MethodsWestern blot was used to detect the protein expression levels of YAP in SMMC-7721, SK-Hep-1, HepG-2, Huh7 and the normal liver cell line L-O2. YAP specific small interfering RNA （si-YAP） or YAP expression plasmid were transfected in SK-Hep-1 or Huh7 cells, respectively. Cell counting kit-8 （CCK-8） test was used to detect the cell proliferation activity and the cell cycle test was conducted by flow cytometry. SK-Hep-1 and SK-Hep-1 si-YAP cells were subcutaneously injected into the nude mice which were sequentially treated by intragastric administration of sorafenib, and the tumor growth in vivo were observed and compared.ResultsThe expression of YAP was upregulated in HCC cell lines. Deletion of YAP expression significantly decreased the survival rate of SK-Hep-1 cells [（78.5±0.3）% vs （92.3±0.2）%, P=0.025]. Knockdown of YAP significantly increased the percentage of G0/G1-phase cells [ （65.4±3.3） % vs （55.7±3.4） %, P=0.039]. On the contrary, upregulation of the YAP expression in Huh7 cells significantly increased the cell survival rate [（81.2±1.3）% vs （62.5±1.1）%, P=0.013] and reduced the percentage of G0/G1-phase cells [（38.2±3.8）% vs （48.8±2.9）%, P=0.019]. The survival rate of SK-Hep-1 cells treated by si-YAP combined with sorafenib was （31.13±1.79）%, significantly lower than （48.87±0.58） % of SK-Hep-1 cells treated by sorafenib alone （P=0.001）, while overexpression of YAP attenuated the inhibitory effect of sorafenib on the survival of Huh7 cells [（69.98±2.94） % vs （53.53±1.93）%, P=0.001]. The tumor weights of SK-Hep-1 group, sorafenib alone group, SK-Hep-1 si-YAP group and SK-Hep-1 si-YAP combined with sorafenib group were （0.96±0.08） g, （0.62±0.08） g, （0.70±0.06） g and （0.27±0.02） g, respectively. The tumor weights of sorafenib alone group and SK-Hep-1 si-YAP group were significantly lower than that of SK-Hep-1 group （P=0.012 and P=0.031, respectively）. The tumor weight of SK-Hep-1 si-YAP combined with sorafenib group was significantly lower than that of SK-Hep-1 si-YAP group （P=0.001）.ConclusionsThe expression of YAP is upregulated in HCC cell lines, which regulates the proliferation, cell cycle, and sensitivity to sorafenib of HCC cells. YAP is a potential molecular target for HCC treatment.