雷公藤内酯醇对多发性骨髓瘤U266细胞组蛋白去甲基化酶的调节作用

Regulation of triptolide on histone demethylase in multiple myeloma U266 cells

目的研究雷公藤内酯醇对多发性骨髓瘤U266细胞组蛋白去甲基化酶的影响,探讨其表观遗传调控作用。方法采用MTT法检测细胞增殖活性;Annexin V-FITC/PI双标法流式细胞术检测细胞凋亡;RT-PCR法检测U266细胞组蛋白赖氨酸去甲基化酶1(LSD1)mRNA、组蛋白去甲基化酶(JMJD2B)mRNA表达的变化;激光共聚焦显微技术观察LSD1亚细胞定位情况及蛋白量的变化;Western blotting法检测在雷公藤内酯醇干预下LSD1和JMJD2B蛋白表达的变化。结果雷公藤内酯醇以剂量、时间相关方式抑制U266细胞增殖,与细胞培养24 h的IC50为153.2 nmol/L;以剂量相关方式诱导U266细胞凋亡,雷公藤内酯醇80 nmol/L作用U266细胞24 h后,细胞出现典型的凋亡形态学改变,总凋亡率达32.9%。;以剂量相关方式抑制JMJD2B蛋白的表达,同时促进LSD1蛋白表达。结论雷公藤内酯醇在抑制U266细胞增殖、诱导其凋亡的同时,明显改变LSD1、JMJD2B的表达,其诱导U266细胞凋亡和抗肿瘤效应可能与其调节LSD1、JMJD2B表达有关。

Objective To elucidate the effects of triptolide on histone demethylase in multiple myeloma U266 cell and to investigate its epigenetic regulation.Methods The inhibition of triptolide on U266 cell proliferation was studied by MTT assay.Apoptosis was evaluated by Annexin-V-FITC/PI-labeled flow cytometry.The changes of mRNA expression of histone lysine demethylase 1（LSD1） and histone demethylase JMJD2B in U266 cells were verified by RT-PCR.Furthermore,laser confocal fluorescence microscopy（LCFM） was used to observe the subcellular localization and content of protein of LSD1;Changes of protein expression in LSD1 and JMJD2B interfered by triptolide were studied by Western blotting analysis.Results Triptolide inhibited U266 cell proliferation in a dose-and time-dependent manner and its IC50of 24 h was 153.2 nmol/L.The triptolide-treated cells exhibited a significant apoptotic morphology with the total apoptosis rate of 32.9% after exposed to triptolide（80 nmol/L） for 24 h.JMJD2B was significantly depressed at protein expression,but LSD1 was upregulated at protein expression,both of them in a dose-dependent manner.Conclusion Triptolide could inhibit U266 cell proliferation,induce the apoptosis,meanwhile,significantly alter the expression of LSD1 and JMJD2B.The apoptosis induction and the antitumor effects of triptolide is probably related to the regulation of LSD1 and JMJD2B expression.