雷公藤内酯醇通过抑制SMYD3对骨髓瘤细胞凋亡及基质金属蛋白酶-9基因表达的影响

Effects of Triptolide on MMP-9 Expression and Inducing Apoptosis of Multiple Myeloma Cells by Inhibiting SMYD3

目的:探讨雷公藤内酯醇(triptolide,TPL)抑制组蛋白甲基化酶SMYD3对多发性骨髓瘤RPMI8226细胞增殖和凋亡的影响及其作用机制。方法:采用MTT法检测不同浓度(10、20、40、80、160 nmol/L)TPL作用不同时间(24、48、72 h)对RPMI8226细胞增殖的影响;应用流式细胞术检测不同浓度(40、80、160 nmol/L)TPL作用48 h后RPMI8226细胞的凋亡率;实时荧光定量PCR检测SMYD3和MMP-9基因的mRNA表达水平;Western blot法检测组蛋白H3K4me2,H3K4me3的甲基化水平。结果:TPL能够明显抑制RPMI8226细胞的增殖活性,且随作用时间延长及药物浓度增高,细胞增殖抑制率明显升高(P<0.05);不同浓度TPL作用RPMI8226细胞48 h后,随着药物浓度的增高,细胞凋亡比例逐渐增加,与空白对照组比较,差异均有统计学意义(r=0.974,P<0.05);实时荧光定量PCR结果显示,不同浓度TPL作用RPMI8226细胞48 h后,随着药物浓度的增高,SMYD3的mRNA相对表达水平呈浓度依赖性下降,与空白对照组比较,差异均有统计学意义(r=-0.882,P<0.05);siRNA-SMYD3转染RPM I8226细胞48 h后,siRNA-SM YD3组与空白对照组比较MMP-9的mRNA相对表达水平明显下降,与80nmol/L的TPL作用一致;Western blot结果显示,不同浓度的TPL作用RPM I8226细胞48 h后,随着药物浓度的增高,组蛋白H3K4me2和H3K4me3的甲基化水平呈浓度依赖性下降,分别与空白对照组比较,差异均有统计学意义(r=-0.971,r=-0.985,P<0.05);siRNA-SMYD3转染RPMI8226细胞48 h,组蛋白H3K4me2和H3K4me3的甲基化水平也明显下降,分别与siRNA阴性对照组、空白对照组比较,差异均有统计学意义(P<0.05)。结论:TPL对多发性骨髓瘤RPMI8226细胞增殖具有明显的抑制作用,并可诱导细胞凋亡,其机制与抑制SMYD3,调控组蛋白H3K4甲基化和MMP-9基因转录激活有关。

Objective： To investigate the effect of triptolide（ TPL） on proliferation and apoptosis of RPMI8226 cells and its mechanism. Methods： MTT assay was used to measure the proliferation of RPMI8226 cells after treatment with different concentration（ 10,20, 40,80 and 160 nmol/L） of TPL for different incubation time（ 24 h,48 h and 72 h）.The cell apoptosis was detected by flow cytometry,the mRNA expressions of SMYD3 and MMP-9 were measured by quantitative real-time PCR,the protein level of H3K4me2 and H3K4me3 in RPMI8226 cells was assayed by Western blot. Results： TPL inhibited RPMI8226 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose-and time-dependent manner（ P〈0. 05）,the RPMI8226 cell apoptosis was induced by treatment with 40,80 and 160 nmol/L TPL（ P〈0. 05）,the qRT-PCR showed that treatment of RPMI8226 cells with TPL downregulated the mRNA expression of SMYD3 in a dose-dependent manner（ P〈0. 05）. Compared with the blank group,the mRNA expression level of MMP-9 in RPMI8226 cells transfected by siRNA-SMYD3 was significantly depressed.Western blot showed that the protein levels of H3K4me2 and H3K4me3 were decreased in a dose-dependent manner afterTPL treatment（ P〈0. 05）. Compared with the blank group and siRNA negative group, the protein level of H3K4me2 and H3K4me3 in RPMI8226 cells transfected by siRNA-SMYD3 also were significantly depressed（ P〈0. 05）.Conclusion： TPL can significantly inhibit the proliferation of RPMI8226 cells and induce their apoptosis,which may be related to the inhibition of SMYD3 expression by TPL-down-regulating the H3K4 methylation and the activating the MMP-9 transcription.