YAP effect on C6 glioma stem cell properities

doi:10.3969/j.issn.2095-4344.0523

Abstract

Abstract:

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 studies 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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neoplastic Stem Cells, Glioma, Tissue Engineering

CLC Number:

R394.2

Ma Yi-ming, Xia Xiang-guo. YAP effect on C6 glioma stem cell properities[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3335-3340.

Figures/Tables 2

2.1 C6胶质瘤干细胞CD133和Nestin免疫荧光鉴定 C6胶质瘤干细胞球贴壁后使用CD133、Nestin免疫荧光染色，在激光共聚焦显微镜下可见C6胶质瘤干细胞CD133与Nestin强阳性表达，证实培养的肿瘤球是C6胶质瘤干细胞(C6 GSCs)，见图1。 2.2 C6胶质瘤干细胞相对C6胶质瘤细胞YAP表达上调 C6胶质瘤细胞和C6胶质瘤干细胞正常培养，蛋白提取后使用Western blot检测YAP蛋白表达差异，结果发现C6胶质瘤干细胞相对C6胶质瘤细胞YAP蛋白表达明显上调(图2A)，蛋白相对内参灰度值比值差异有显著性意义(P < 0.05，图2B)。提取mRNA使用qRT-PCR检测YAP mRNA表达差异，显示C6胶质瘤干细胞相对C6胶质瘤细胞YAP mRNA表达明显上调(图2C)，差异有显著性意义(P < 0.05)。使用免疫荧光检测YAP蛋白表达及分布差异，发现C6胶质瘤干细胞内YAP表达明显升高，且核内YAP蛋白量增多(图2D)。 2.3 YAP过表达C6胶质瘤细胞的干细胞特性增强 C6胶质瘤细胞正常培养，使用YAP过表达慢病毒构建YAP过表达细胞系，使用Western blot和qRT-PCR检测过表达细胞YAP表达情况，结果显示YAP蛋白(图3A)和YAP mRNA(图3B)均显著过表达，与转染前比较差异有显著性意义(P < 0.05)。检测过表达细胞系干细胞标志物CD44、Nanog和Oct-4表达情况，发现YAP过表达后干细胞标志物均明显上调(图3C)；过表达细胞系进行肿瘤干细胞肿瘤球形成实验，结果显示YAP过表达后干细胞肿瘤球体积增大、数量增多(图3D)，与转染前比较差异有显著性意义(P < 0.05)。 2.4 YAP敲低C6胶质瘤干细胞的干细胞特性减弱 C6胶质瘤干细胞正常培养，用两种不同位点YAP shRNA慢病毒构建YAP敲低细胞系，采用Western blot和qRT-PCR检测敲低细胞YAP表达情况，结果显示YAP蛋白(图4A)和YAP mRNA(图4B)均显著降低，与转染前比较差异有显著性意义(P < 0.05)。检测干细胞标志物CD44、Nanog和Oct-4蛋白表达情况，发现YAP敲低后干细胞标志物显著下调(图4C)；肿瘤干细胞肿瘤球形成实验结果显示YAP敲低后干细胞肿瘤球体积减小、数量减少(图4D)，与转染前比较差异有显著性意义(P < 0.05)。"

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