YAP对卵巢癌干细胞增殖的影响

Effect of YAP gene on proliferation of ovarian cancer stem cells

目的:探讨YAP基因在卵巢癌干细胞中的表达和作用。方法:取对数期人卵巢细胞株OVCAR-3,胰酶消化后制备单细胞悬液,利用流式细胞术鉴定卵巢癌干细胞。将YAP 5个磷酸化位点突变(YAP-5SA)及C末端缺失(YAP-△C)载体构建在慢病毒载体上,并且将YAP-5SA,YAP-△C载体的病毒转染卵巢癌干细胞,筛选稳定转染的细胞进行MTT细胞增殖实验、平板克隆实验。取卵巢癌干细胞,YAP-5SA和YAP-△C稳定转染的卵巢癌干细胞,接种在15只裸鼠中,观察4周,每周记录1次瘤体体积。结果:分离制备的细胞接种在DMEM培养基中培养24 h后细胞数量开始增多,生长5 d时不同细胞长满瓶底,细胞成干细胞球形,流式细胞仪测定该细胞CD44阳性率为99.8%,CD117为0.76%。YAP在卵巢癌干细胞高表达且YAP-5SA促进卵巢癌干细胞增殖和平板细胞克隆形成,YAP-△C抑制卵巢癌干细胞增殖和平板细胞克隆形成。YAP-△C稳转卵巢癌干细胞促进瘤体生长,YAP-△C抑制卵巢癌干细胞瘤体生长。结论:YAP-5SA促进卵巢干细胞增殖,参与卵巢癌的发生、发展。

Objective： To investigate the role of YAP in ovarian cancer stem cells and its influence on biologic function. Methods： A single cell suspension was prepared after digestion of pancreatic enzymes with OVCAR-3 cells. And then, the ovarian cancer stem cells were identified by flow cytometry. The YAP five phosphorylation site mutation （YAP-5SA） and C terminal deletion （YAP-△C） in the construction of vector lentiviral packaging vector, and the virus core particles transfected with YAP-5SA, YAP-△C vector in ovarian cancer stem cells, then the cell proliferation and stability were examined by MTT assay and colony-forming assay. The ovarian cancer stem cells, YAP-5SA and YAP-△C transfected ovarian cancer stem cells were inoculated in 15 the nude mice, the volume of tumors were measurand for 4 weeks, once a week. Results： After the ovarian cancer stem cells inoculated in DMEM culture medium for 24 h, the number of cells were increased, and 5 days the cells presenting stem cell globules and covering the bottom of the bottle. And the cells had 99.8% CD44 and 0.76% CD117 examined by flow cytometry. YAP level was higher in ovarian cancer stem cells than ovarian cancer cells. And YAP-5SA promoted stem cell proliferation and tablet cell cloning in ovarian cancer stem cells, however, YAP-△C showed the opposite phenomenon. YAP-5SA transfected ovarian cancer stem cells promoted the growth of the tumor, however, YAP-△C transfected ovarian cancer stem cells suppressed the growth of the tumor. Conclusion： These results suggest that YAP contribute to the proliferation of ovarian stem cells and participate in the development and progression of ovarian cancer.