人参皂苷20(S)-Rg3通过抑制DNMT3A介导的启动子甲基化促进卵巢癌细胞中抑癌基因VHL的表达

Ginsenoside 20(S)-Rg3 upregulates tumor suppressor VHL gene expression by suppressing DNMT3A-mediated promoter methylation in ovarian cancer cells

目的明确人参皂苷20(S)-Rg3促进卵巢癌细胞抑癌基因von Hippel-Lindau(VHL)表达的机制。方法Real-time PCR和Western blotting检测20(S)-Rg3处理前后卵巢癌细胞SKOV3中的VHL、DNA甲基转移酶DNMT1、DNMT3A和DNMT3B的mRNA和蛋白水平。Real-time PCR检测甲基转移酶抑制剂5-氮杂-2-脱氧胞苷(5-Aza-CdR)处理卵巢癌细胞SKOV3前后VHL的mRNA水平变化。甲基化特异性PCR(MSP)检测20(S)-Rg3单纯处理组和20(S)-Rg3处理且过表达DNMT3A组的VHL基因启动子区的甲基化水平,并检测VHL的mRNA和蛋白表达水平。免疫组化检测课题组前期获得的20(S)-Rg3处理组及对照组的裸鼠皮下移植瘤组织中VHL和DNMT3A的蛋白表达。结果20(S)-Rg3处理后,卵巢癌细胞SKOV3中VHL的mRNA水平升高到阴性对照细胞的2倍以上,蛋白水平亦上调(P<0.05);DNMT3A的mRNA水平和蛋白水平均下降(P<0.05),DNMT3B的mRNA水平略有升高但蛋白水平无明显变化(P>0.05),DNMT1的mRNA和蛋白水平均无变化(P>0.05)。2μmol/L和5μmol/L的5-Aza-CdR处理后,SKOV3细胞的VHL mRNA水平升高到阴性对照细胞的3倍以上(P<0.05)。20(S)-Rg3使VHL基因启动子区的甲基化水平降低(P<0.05),在20(S)-Rg3处理的同时过表达DNMT3A,则VHL基因启动子甲基化水平再次升高,同时VHLmRNA和蛋白水平均降低(P<0.05)。免疫组织显示,相对于对照组,20(S)-Rg处理组的裸鼠皮下移植瘤组织中VHL的表达上调、DNMT3A的表达下调。结论20(S)-Rg3通过抑制DNMT3A介导的启动子甲基化而促进卵巢癌细胞中VHL的表达。

Objective To explore the mechanism by which ginsenoside 20(S)-Rg3 upregulates the expression of tumor suppressor von Hippel-Lindau(VHL)gene in ovarian cancer cells.Methods Ovarian cancer cell line SKOV3 treated with 20(S)-Rg3 were examined for mRNA and protein levels of VHL,DNMT1,DNMT3A and DNMT3B by real-time PCR and Western blotting,respectively.The changes in VHL mRNA expression in SKOV3 cells in response to treatment with 5-Aza-CdR,a DNA methyltransferase inhibitor,were detected using real-time PCR.VHL gene promoter methylation was examined with methylation-specific PCR and VHL expression levels were determined with real-time PCR and Western blotting in non-treated or 20(S)-Rg3-treated SKOV3 cells and in 20(S)-Rg3-treated DNMT3A-overexpressing SKOV3 cells.VHL and DNMT3A protein levels were detected by immunohistochemistry in subcutaneous SKOV3 cell xenografts in nude mice.Results Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels(P<0.05)and upregulated DNMT3B expression only at the mRNA level,but did not cause significant changes in either the mRNA or protein level of DNMT1.Treatment of the cells with 2 and 5μmol/L 5-Aza-CdR obviously increased VHL mRNA expression by by over 3 folds(P<0.05).20(S)-Rg3 significantly decreased the methylation level in the promoter region of VHL gene,and this effect was abrogated by DNMT3A overexpression in the cells(P<0.05).Immunohistochemisty showed a significantly increased VHL expression but a lowered DNMT3A expression in subcutaneous SKOV3 cell xenografts in 20(S)-Rg3-treated nude mice.Conclusion Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNAmethylation.