Wang Lijie, Han Xi, Zheng Xia, Zhou Yuanyuan, Hou Huilian, Chen Wei, Li Xu, Zhao Le
Center for Translational Medicine, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
Department of Gynecology, Lanzhou University Second Hospital, Lan Zhou 730030, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Jan 30;41(1):100-106. doi: 10.12122/j.issn.1673-4254.2021.01.14.
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.
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.
Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels ( < 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 ( < 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 ( < 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.
Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNA methylation.
探讨人参皂苷20(S)-Rg3上调卵巢癌细胞中肿瘤抑制因子冯·希佩尔-林道（VHL）基因表达的机制。
分别采用实时荧光定量PCR和蛋白质免疫印迹法检测经20(S)-Rg3处理的卵巢癌细胞系SKOV3中VHL、DNMT1、DNMT3A和DNMT3B的mRNA和蛋白质水平。使用实时荧光定量PCR检测DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理SKOV3细胞后VHL mRNA表达的变化。采用甲基化特异性PCR检测VHL基因启动子甲基化情况，并通过实时荧光定量PCR和蛋白质免疫印迹法测定未处理或经20(S)-Rg3处理的SKOV3细胞以及经20(S)-Rg3处理的过表达DNMT3A的SKOV3细胞中的VHL表达水平。通过免疫组织化学检测裸鼠皮下SKOV3细胞异种移植瘤中VHL和DNMT3A蛋白水平。
用20(S)-Rg3处理SKOV3细胞后，VHL在mRNA和蛋白质水平均显著上调，DNMT3A在mRNA和蛋白质水平均显著下调（<0.05），DNMT3B仅在mRNA水平上调，而DNMT1的mRNA和蛋白质水平均无显著变化。用2和5μmol/L的5-Aza-CdR处理细胞后，VHL mRNA表达明显增加超过3倍（<0.05）。20(S)-Rg3显著降低了VHL基因启动子区域的甲基化水平，而细胞中DNMT3A过表达可消除这种作用（<0.05）。免疫组织化学显示，在经20(S)-Rg3处理的裸鼠皮下SKOV3细胞异种移植瘤中，VHL表达显著增加，而DNMT3A表达降低。
人参皂苷20(S)-Rg3通过抑制DNMT3A介导的DNA甲基化上调卵巢癌细胞中VHL的表达。
