南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (1): 100-106.doi: 10.12122/j.issn.1673-4254.2021.01.14

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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

王莉洁，韩 曦，郑 霞，周园园，侯惠莲，陈 葳，李 旭，赵 乐   

  • 出版日期:2021-01-26 发布日期:2021-01-26

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

  • Online:2021-01-26 Published:2021-01-26

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摘要：

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）。2 μmol/L和5 μmol/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VHL mRNA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。

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Abstract:

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 5 μmol/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 DNA methylation.

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