YAP蛋白对C6胶质瘤干细胞特性的影响

YAP effect on C6 glioma stem cell properities

背景:YAP蛋白能够促进与干细胞特性相关蛋白基因的活化,因此推测YAP在维持C6胶质瘤干细胞特性中发挥重要作用,且暂无此方面研究报道。目的:探讨YAP蛋白在胶质瘤干细胞干性维持中发挥的作用。方法:使用无血清培养基培养C6胶质瘤细胞获取C6胶质瘤干细胞,细胞免疫荧光染色检测干细胞标志物CD133和Nestin蛋白表达;Western blot、qRT-PCR和细胞免疫荧光染色观察胶质瘤干细胞和胶质瘤细胞YAP表达差异。进一步使用慢病毒转染将C6胶质瘤细胞YAP蛋白过表达,使用shRNA慢病毒转染将C6胶质瘤干细胞YAP蛋白敲低,Western blot检测CD44、Nanog和Oct-4蛋白表达,干细胞克隆球形成实验鉴定细胞成球能力。结果与结论:(1)C6胶质瘤干细胞同时表达CD133和Nestin;(2)C6胶质瘤干细胞与C6胶质瘤细胞相比,YAP蛋白和mRNA表达升高;(3)YAP过表达后C6胶质瘤细胞的干细胞标志物CD44、Nanog和Oct-4表达明显上调,肿瘤干细胞克隆球体积明显增大、数量明显增多;(4)YAP敲低后C6胶质瘤干细胞的干细胞标志物CD44、Nanog和Oct-4表达明显下调,肿瘤干细胞克隆球体积明显减小、数量明显减少;(5)结果表明,C6胶质瘤细胞中存在肿瘤干细胞,YAP蛋白在胶质瘤干细胞中高表达并且与胶质瘤干细胞特性的维持密切相关。

BACKGROUND： YAP protein can promote the activation of stem cell-associated proteins. Therefore, it is speculated that YAP plays an important role in maintaining the stem cell characteristics of C6 glioma cells, but no tudies have been reported in this regard.OBJECTIVE： To explore the function of YAP protein in the maintenance of C6 glioma stemness.METHODS： C6 glioma cells were cultured in serum-free medium to obtain C6 glioma stem cells, and immunofluorescence staining was used to detect CD133 and Nestin. Western blot, qRT-PCR and immunofluorescence staining were used to observe the differences of YAP expression between glioma stem cells and glioma cells. Lentiviral transfection was further used to transfect C6 glioma cells and glioma stem cells to make the cells overexpress or knock down the YAP protein. The protein levels of YAP, CD44, Nanog and Oct-4 were tested by western blot assay. The stem cell formation ability of tumor cells was determined in the cloning sphere test. RESULTS AND CONCLUSION： C6 glioma stem cells expressed CD133 and Nestin. The protein and mRNA levels of YAP in C6 glioma stem cells were higher than those in C6 glioma cells. Overexpression of YAP in C6 glioma cells significantly up-regulated the expression of CD44, Nanog and Oct-4, enlarged and increased the tumor spheres of tumor stem cells. Conversely, knockdown of YAP in C6 glioma stem cells obviously decreased the stem cell properties. In conclusion, tumor stem cells exist in C6 glioma cells, and YAP overexpression in the glioma stem cells is highly associated with the maintenance of stem cell properties.