Objective: To explore the effects of hinokiol on the cell cyle and apoptosis of CNE1 nasopharyngeal carcinoma cells and the relevant molecular mechanism. Methods: The CNE1 cells were cultured in vitro and incubated with different concentrations of honokiol, and the cells were divided into blank control group, 10 μmol/L, 20 μmol/L and 40 μmol/L hinokiol treatment groups, and 10 μg/ml cisplatin group. Cell viability was determined by methylthiazolyldiphenyl- tetrazolium bromide (MTT) method, the cell cycle distribution was detected by flow cytometry, mitochondrial membrane potential was detected by mitochondrial membrane potential test kit, apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method, and the proteins expression of proliferating cell nuclear antigen (PCNA) and G1/S specific cyclin D1 (cyclin D1) were detected by immunoblotting. RNA-Seq was conducted in the hinokiol-treated cells. The mRNA expression of yes-associated protein delta (YAP) was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The proteins expression of phosphor-YAP (p-YAP) and nuclear YAP were detected by immunoblotting, the nuclear distribution of YAP protein was detected by immunofluorescence in the cells with or without treated with the mammalian STE20-like kinase 1/2 (MST1/2) inhibitor (XMU-MP-1), hinokiol, and XMU-MP-1+hinokiol. Statistical analysis of the data was conducted using GraphPad Prism 8.0 software. Resluts Compared with the control group, the cell viablity of CNE1 cells, the levels of mitochondrial membrane potential, the proteins expression of PCNA and cyclin D1 in hinokiol treatment groups were markedly decreased (all P values<0.05), while the proportion of G0/G1 phase cells and the ratio of TUNEL-positive cells were significantly increased (both P values<0.05). Transcriptome analysis showed that differential genes were mainly enriched in Wnt signaling pathway, tumor necrosis factor pathway, and Hippo signaling pathway. The mRNA level of YAP and the protein expression of YAP in the nucleus were decreased and the level of p-YAP protein was increased in cells treated with hinokiol, which were significantly different from control group (all P values<0.05). Compared with the hinokiol group, XMU-MP-1+hinokiol groups showed the decrease of p-YAP protein expression (1.157±0.076 vs 0.479±0.038, t=37.120, P<0.05), the increase of YAP protein expression in the nucleus (0.143±0.012 vs 0.425±0.031, t=29.181, P<0.05), the reduced proportion of cells in G0/G1 phase [(72.494±3.309)% vs (58.747±2.865)%, t=17.265, P<0.05], and the decrease of apoptosis ratio [(53.158±3.376)% vs (29.621±2.713)%, t=28.584, P<0.05]. Conclusion: Hinokiol can arrest the cell cycle and induce the cell apoptosis of CNE1 cells via Hippo/YAP signaling pathway.
目的： 探究和厚朴酚对CNE1鼻咽癌细胞周期和凋亡的影响及分子机制。 方法： 采用不同浓度的和厚朴酚处理CNE1细胞，噻唑蓝（methylthiazolyldiphenyl-tetrazolium bromide，MTT）法测定细胞的活性；将CNE1细胞分为空白对照组，10、20、40 μmol/L和厚朴酚处理组和10 μg/ml顺铂组。流式细胞术检测细胞周期分布，线粒体膜电位检测试剂盒检测细胞线粒体膜电位，原位末端标记（terminal deoxynucleotidyl transferase mediated dUTP nick end labeling，TUNEL）法检测细胞凋亡，免疫印迹检测细胞中增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）和细胞周期蛋白D1（G1/S specific cyclin D1，cyclin D1）蛋白表达；体外培养CNE1细胞，和厚朴酚处理后进行转录组测序，实时荧光定量反转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction，RT-qPCR）检测Yes激酶相关蛋白（yes-associated protein delta，YAP）mRNA表达，免疫印迹检测磷酸化YAP蛋白（phospho-YAP，p-YAP）以及细胞核中YAP蛋白表达水平，免疫荧光检测YAP蛋白核分布；将细胞分为对照组、哺乳动物STE20样激酶1/2（mammalian STE20-like kinase 1/2 inhibitor，MST1/2）抑制剂（XMU-MP-1）组、和厚朴酚组、XMU-MP-1+和厚朴酚组，利用免疫印迹、流式细胞术和TUNEL法观察Hippo通路在和厚朴酚影响CNE1细胞周期与凋亡中的作用。采用GraphPad Prism 8.0软件对数据进行统计学分析。 结果： 与对照组比较，和厚朴酚处理组CNE1细胞活力、线粒体膜电位、PCNA与cyclin D1蛋白表达水平显著降低（P值均<0.05），G0/G1期细胞比例和TUNEL阳性细胞率显著增加（P值均<0.05）。转录组分析结果发现，差异基因主要富集在Wnt信号通路、肿瘤坏死因子通路和Hippo信号通路等。和厚朴酚处理后，细胞中YAP mRNA表达和细胞核中YAP蛋白表达水平降低，p-YAP蛋白水平升高，较对照组差异有统计学意义（P值均<0.05）。与和厚朴酚组比较，XMU-MP-1+和厚朴酚组细胞中的p-YAP蛋白表达水平降低（1.157±0.076比0.479±0.038，t=37.120，P<0.05），细胞核中YAP的蛋白表达水平升高（0.143±0.012比0.425±0.031，t=29.181，P<0.05），G0/G1期细胞比例降低［（72.494±3.309）%比（58.747±2.865）%，t=17.265，P<0.05］、细胞凋亡水平减少［（53.158±3.376）%比（29.621±2.713）%，t=28.584，P<0.05］。 结论： 和厚朴酚能够引起CNE1鼻咽癌细胞周期阻滞，诱导细胞凋亡，这可能是通过调控Hippo/YAP信号通路来实现的。.