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

发布时间：2018-03-18 07:26

本文选题：雷公藤内醇酯　切入点：多发性骨髓瘤　出处：《中国实验血液学杂志》2017年04期 　论文类型：期刊论文

【摘要】：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(P0.05);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,P0.05);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,P0.05);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,P0.05);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(P0.05)。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。
[Abstract]:Objective: to investigate the effect of triptolide triptolide triptolide (TPL) on the proliferation and apoptosis of multiple myeloma RPMI8226 cells induced by histone methylase SMYD3. Methods: MTT assay was used to detect the effect of triptolide triptolide (TPL) on the proliferation and apoptosis of multiple myeloma RPMI8226 cells. Effects on proliferation of RPMI8226 cells; Flow cytometry was used to detect the apoptotic rate of RPMI8226 cells after 48 h treatment with different concentrations of 40,80,160 nmol/L)TPL. Real-time fluorescence quantitative PCR was used to detect the mRNA expression of SMYD3 and MMP-9 genes. Western blot assay was used to detect the methylation level of histone H3K4ME2H3K4me3.Results: TPL could significantly inhibit the proliferative activity of RPMI8226 cells and increase the drug concentration with the prolongation of time and the increase of drug concentration. The inhibition rate of cell proliferation was significantly increased (P 0.05), and the ratio of apoptosis of RPMI8226 cells increased with the increase of drug concentration after 48 h treatment with different concentrations of TPL, compared with the control group. The results of real-time fluorescence quantitative PCR showed that the relative mRNA expression of RPMI8226 cells decreased in a concentration-dependent manner with the increase of drug concentration after 48 h treatment with different concentrations of TPL, compared with the control group. The difference was statistically significant. The relative expression of MMP-9 mRNA in the siRNA-SMYD3 transfected RPM I8226 cells 48 h after transfection was significantly lower than that in the control group, which was consistent with the effect of 80 nmol / L TPL. The results showed that different concentrations of TPL treated RPM I8226 cells for 48 h. The methylation level of histone H3K4me2 and H3K4me3 decreased in a concentration-dependent manner with the increase of drug concentration, and the methylation levels of histone H3K4me2 and H3K4me3 were significantly decreased after transfection of histone H3K4me2 and H3K4me3 into RPMI8226 cells for 48 h after transfection of histone H3K4me2 and H3K4me3. Compared with siRNA negative control group and blank control group, the difference was statistically significant (P 0.05). Conclusion: TPL can significantly inhibit the proliferation of multiple myeloma RPMI8226 cells and induce apoptosis. The mechanism is related to inhibition of SMYD3, regulation of histone H3K4 methylation and transcriptional activation of MMP-9 gene.
【作者单位】：福建省人民医院福建中医药大学附属人民医院血液科;福建医科大学附属协和医院血液科福建省血液病研究所;
【基金】：福建省自然科学基金项目(2015J01327) 福建省卫计委青年科研基金(2016-1-77)
【分类号】：R733.3

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