YAP调控人胶质瘤细胞生长的体外研究

于福华; 贾志凡; 浦佩玉; 王广秀; 张安玲; 杨卫东

doi:10.3969/j.issn.1000-8179.20140237

YAP调控人胶质瘤细胞生长的体外研究

Yes-associated protein modulation of human glioma cell growth in vitro

摘要

摘要:
目的研究YAP（Yes-associated protein）调控人脑胶质瘤细胞系LN229细胞生长的作用。
方法采用YAP干扰RNA（siRNA）敲低LN229细胞中YAP的表达，Western Blot法鉴定肿瘤细胞中YAP是否已被敲低；MTT比色法测定YAP敲低后对肿瘤细胞增殖的影响；Transwell实验检测胶质瘤细胞侵袭能力的变化；流式细胞术和Annexin Ⅴ标记法分别检测胶质瘤细胞周期及凋亡的变化。Western Blot法检测相关蛋白。
结果经Western Blot法检测证实转染YAP siRNA后LN229细胞中YAP被有效敲低；敲低LN229细胞中YAP表达，可以抑制胶质瘤细胞增殖，阻滞细胞周期于G₀/G₁期，并可抑制细胞侵袭能力，促使细胞凋亡数显著增加。促增殖蛋白Ki-67、促侵袭蛋白MMP-9、促周期进展蛋白Cyclin D1以及凋亡相关蛋白Bcl-2表达明显下调。
结论敲低人胶质瘤细胞中YAP活性可以抑制肿瘤细胞增殖和侵袭，促进凋亡，为进一步探究Hippo-YAP信号通路在胶质瘤中的分子病理机制中的作用提供依据。

Abstract:
Objective This study aimed to explore the effect of Yes-associated protein (YAP) on the growth of the human glioma cell line LN229.
Methods YAPsiRNA was transfected into LN229 cells to knock down the YAP expression. The downregulation of the YAP expression was identified through Western Blot analysis. Colorimetric assay using methyl-thiazolyl-tetrazolium was applied to evaluate cell proliferation ability. Cell invasive activity was examined using Transwell assay. Flow cytometry and AnnexinV were used to detect cell cycle and apoptosis, respectively. The relevant molecules regulating proliferation, invasion, cell cycle progression, and apoptosis were examined through Western Blot analysis.
Results The YAP expression was downregulated after YAPsiRNA was transfected into LN229 glioma cells. Reduced YAP expression could arrest the cell cycle at G₀/G₁ phase, inhibit cell proliferation and invasion, and promote apoptosis. The expression of the proliferating cell nuclear antigen (Ki-67), matrix metallopeptidase-9 (MMP-9), cyclin D1, and Bcl-2 were downregulated.
Conclusions The downregulation of YAP in LN229 cells suppresses cell proliferation and invasion, as well as promotes cell apoptosis. This study provides a novel evidence for further study on Hippo-YAP signal pathway in molecular pathology of glioma.

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