组蛋白甲基化酶抑制剂对人淋巴瘤Raji细胞生存、凋亡及细胞周期的影响

肖丽容; 侯宸; 郑仕洁; 郭波; 陈大年; 闫乃红

组蛋白甲基化酶抑制剂对人淋巴瘤Raji细胞生存、凋亡及细胞周期的影响

Effects of Histone Methyltransferase Inhibitors on the Survival, Apoptosis and Cell Cycle of Raji Cells

摘要

摘要:
目的研究组蛋白甲基化酶Zeste基因增强子同源物2（enhancer of Zeste homolog 2，EZH2）的3种抑制剂（UNC1999、DZNep及GSK343）对人B细胞非霍奇金淋巴瘤Raji细胞的影响。
方法 PCR扩增EZH2基因16（Y641）和18（A677）外显子，Sanger测序检测其突变情况；蛋白质免疫印迹法（Western blot）检测正常成年人淋巴细胞及Raji细胞EZH2的表达；使用不同浓度的UNC1999、DZNep及GSK343处理Raji细胞，CCK-8技术检测Raji细胞的生存情况；流式细胞术检测Raji细胞的凋亡情况和细胞周期变化；Western blot检测药物处理后EZH2及H3K27 me3的表达。
结果 Sanger测序显示Raji细胞不携带EZH2基因Y641和A677突变位点。Western blot显示，相比正常成年人淋巴细胞，Raji细胞EZH2蛋白高表达。CCK-8法结果显示UNC1999、DZNep及GSK343对Raji细胞均有抑制生存的作用，DZNep抑制能力最弱。流式细胞术凋亡检测显示UNC1999、DZNep及GSK343促进Raji细胞的凋亡，UNC1999作用强于GSK343与DZNep；3种抑制剂均阻滞Raji细胞于G₁/G₀期，UNC1999组细胞周期阻滞最显著。Western blot显示UNC1999和GSK343抑制EZH2组蛋白甲基化酶的活性，降低H3K27 me3的表达。
结论 EZH2抑制剂可通过降低H3K27 me3的表达抑制Raji细胞生存，促进细胞凋亡，改变细胞周期。UNC1999作用效果优于GSK343及DZNep，且EZH2抑制剂对Raji细胞的作用不依赖于EZH2的突变。

Abstract:
Objective To determine the effects of three histone methylase inhibitors UNC1999, DZNep and GSK343 on the survival, apoptosis and cell cycle of non-hodgkin's lymphoma Raji cells.
Methods PCR amplified 16 and 18 exons of enhancer of zeste homolog 2 (EZH2) gene were detected. The expression of EZH2 in normal adult lymphocytes and Raji cells was detected by Western blot. The Raji cells were treated by UNC1999, DZNep and GSK343, followed by CCK-8 assays analyzing cell survival, flow cytometry detecting cell apoptosis and cell cycle, and Western blot detecting the expressions of EZH2 and H3K27 me3.
Results The Sanger sequencing results showed that the Raji cells did not carry Y641 and A677 mutation sites of EZH2. The Western blot results showed high expressions of EZH2 in the Raji cells. The results of CCK-8 showed that UNC1999, DZNep and GSK343 inhibited cell survival, and the weakest effect was from DZNep. The flow cytometric assay showed that UNC1999, DZNep and GSK343 promoted apoptosis of the Raji cells, and the effect of UNC1999 was stronger than that of GSK343 and DZNep. The cell cycle was arrested at phase G₁/G₀ after treatment of the Raji cells with the three inhibitors, with UNC1999 triggering the most significant changes. The Western blot showed that UNC1999 and GSK343 inhibited the histone methylase activity of EZH2 and significantly reduced the expression of H3K27 me3.
Conclusion EZH2 inhibitors can inhibit cell survival, promote cell apoptosis and arrest cell cycle at phase G₁/G₀ of Raji cells through reducing the expression of H3K27me3. UNC1999 has a stronger effect than GSK343 and DZNep.

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