UM-164通过降低YAP活性抑制人胶质瘤细胞增殖的分子机制研究

UM-164 inhibits proliferation of human glioma cells by reducing the activity of YAP

目的探讨Src抑制剂UM-164对人胶质瘤细胞增殖的作用。方法采用UM-164处理人胶质瘤细胞SF767,Cell Counting Kit-8(CCK-8)检测SF767细胞活力;克隆形成实验考察SF767细胞集落形成能力;BrdU和Ki67掺入实验检测SF767细胞增殖情况;流式细胞术分析SF767细胞周期分布和凋亡;蛋白免疫印记实验检测Src、p38、YAP及Cyclin D1的蛋白表达水平;免疫荧光实验观察YAP的细胞核定位情况。结果 CCK-8实验结果显示UM-164可显著抑制SF767细胞活力,且抑制作用呈时间和剂量依赖性(P<0.05);克隆形成实验结果显示UM-164可抑制SF767细胞的集落形成能力(P<0.01);BrdU和Ki67掺入实验结果显示UM-164处理后BrdU和Ki67标记的阳性细胞显著减少(P<0.01);流式细胞术结果显示UM-164可诱导SF767细胞阻滞于G 0/G1期(P<0.01),但不能诱导SF767细胞凋亡;蛋白免疫印迹结果显示SF767细胞经UM-164处理后,p-Src、p-p38和细胞周期相关蛋白Cyclin D1的表达显著降低(P<0.01),p-YAP (S397)表达显著升高(P<0.01);免疫荧光结果显示UM-164促使YAP滞留于胞质且入核减少。结论 UM-164能够抑制人胶质瘤细胞的增殖,其机制可能与UM-164降低YAP活性有关。

Objective To investigate the effect of UM164, a Src inhibitor, on the proliferation of human glioma cells. Methods Human glioma cells SF767 were divided into blank control group and UM-164-treated group. Cell Counting kit-8(CCK-8) was employed to detect the viability of SF767 cells. Colony forming ability of SF767 cells was detected by colony formation assay. The effect of UM-164 on SF767 cells proliferation was analyzed by BrdU and Ki67 incorporation assays. Cell cycle distribution and apoptosis were analyzed by flow cytometry(FCM), and then expression of Src, p38, YAP and Cyclin D1 were detected by Western blotting. The localization of YAP was observed by immunofluorescence assay. Results UM-164 could significantly inhibited SF767 cell viability in a time-and dose-dependent manner(P<0.05), and UM-164 could inhibit colony formation of SF767 cells(P<0.01). BrdU and Ki67 incorporation assays showed the numbers of BrdU and Ki67 positive cells marked with BrdU or Ki67 were significantly reduced after the treatment with UM-164(P<0.01). FCM results indicated that UM-164 could induce SF767 cell arrest in phase G0/G1(P<0.01), but could not induce apoptosis of SF767 cells. The Western blotting showed that the expression of p-Src, p-p38, Cyclin D1 and p-YAP(S397) were significantly decreased with UM-164 treatment while the expression of Cyclin D1 was significantlyincreased(P<0.01). Immunofluorescence results showed that UM-164 induced YAP to exclude from the nucleus. Conclusion UM-164 can inhibit the proliferation of human glioma cells, and the mechanism may be related to the reduction of YAP activity by UM-164.