[关键词]
[摘要]
目的 探讨吴茱萸碱对人肝癌细胞株BEL-7402凋亡的影响并阐明其发挥作用的可能机制。方法 CCK-8法检测吴茱萸碱对BEL-7402细胞增殖的影响;Hoechst 33258染色观察吴茱萸碱对BEL-7402细胞凋亡现象的影响;流式细胞术观察吴茱萸碱对BEL-7402细胞凋亡和周期的影响;实时荧光定量PCR(qRT-PCR)和Western blotting实验检测Hippo-YAP通路中哺乳动物STE20样蛋白激酶1(MST1)、MST2、大肿瘤抑制因子1(LATS1)和Yes相关蛋白(YAP)在正常人肝细胞株HL-7702与BEL-7402细胞中的表达差异及吴茱萸碱对BEL-7402细胞这些关键基因表达水平的影响;免疫荧光实验观察吴茱萸碱对BEL-7402细胞中YAP表达水平的影响。结果 吴茱萸碱对BEL-7402细胞增殖活力具有明显的抑制作用(P<0.05),呈浓度和时间依赖性;Hoechst 33258染色显示吴茱萸碱能够引起BEL-7402细胞出现核固缩、核边集等凋亡的典型形态学改变。流式细胞术结果提示吴茱萸碱能够诱导BEL-7402细胞出现凋亡数目增多和G2/M期阻滞(P<0.01);qRT-PCR和Western blotting检测结果提示,与HL-7702细胞相比,MST2和LATS1在BEL-7402细胞中的表达明显下调(P<0.05),YAP则明显升高(P<0.05);吴茱萸碱能够激活Hippo信号通路,分别上调MST2、LATS1和抑制YAP在BEL-7402细胞中的表达水平(P<0.05)。免疫荧光结果显示吴茱萸碱能够显著抑制BEL-7402细胞中过表达的YAP荧光强度(P<0.01)。结论 吴茱萸碱能诱导BEL-7402细胞的凋亡,其机制可能是通过激活Hippo信号通路,进而抑制下游关键基因YAP的表达。
[Key word]
[Abstract]
Objective To investigate the effects and the underlying mechanisms of evodiamine (EVO) on apoptosis of hepatocellular carcinoma (HCC) BEL-7402 cells. Methods Cell counting kit-8 (CCK-8) assay was used to detect the effect of EVO on proliferation activity of BEL-7402 cells. Hoechst 33258 staining was used for observing morphological changes of apoptosis. The cell apoptosis and cycle distribution were analyzed by flow cytometry. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay were used to detect the expression levels of key genes from Hippo-YAP pathway in HL-7702 and BEL-7402 cells, including mammalian STE20-like protein kinase 1/2 (MST1/2), large tumor suppressor 1 (LATS1), and Yes-associated protein (YAP), then to examine the effect of EVO on the expression levels of these genes in BEL-7402 cells. The effect of EVO on the expression of YAP in hepatocellular carcinoma BEL-7402 cells was observed by immunofluorescence assay. Results The proliferation of BEL-7402 cells were significantly inhibited by EVO in a dose- and time-dependent manners. Hoechst 33258 staining showed that EVO induced BEL-7402 cell typical apoptotic morphology, such as nuclear chromatin concentration and edge accumulation. Besides, flow cytometry tests showed that BEL-7402 cell apotosis were increased and cell cycle arrested in G2/M phase after being treated with EVO (P < 0.01). qRT-PCR and Western blotting showed that the expression level of MST2 and LATS1 were lower in BEL-7402 cell (P < 0.05), while the transcription and protein levels of YAP were significantly higher (P < 0.05). EVO could activate Hippo signal pathway, upregulate the expression of MST2 and LATS1 and then inhibit the expression of YAP in BEL-7402 cell (P < 0.05). Immunofluorescence assay also validated that EVO would significantly inhibit the overexpression of YAP in BEL-7402 cell (P < 0.01). Conclusion EVO can induce the apoptosis of BEL-7402 cells, which may be through activating Hippo signal pathway and then down-regulate the expression of YAP.
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[基金项目]
国家自然科学基金面上项目(31271368);重庆市教委重点项目(KJZD-K201802701)