木犀草素通过抑制肝癌细胞Sp1活性上调抑癌基因OPCML表达

发布时间：2018-02-03 14:37

本文关键词： 木犀草素阿片类结合蛋白/细胞黏附样分子肝癌　出处：《基因组学与应用生物学》2017年07期 　论文类型：期刊论文

【摘要】：抑癌基因OPCML的甲基化失活是肿瘤发生的重要机制,因此采用药物逆转抑癌基因的失活状态是肿瘤防治的重要策略。研究表明,Luteolin可在一定程度上抑制DNA的甲基化转移酶(methyltransferase)的活性,但木犀草素是否能逆转OPCML的失活状态目前尚不明确。本研究通过体外培养肝癌细胞系HepG2,用不同浓度木犀草素处理后,采用实时定量PCR和Western blotting分别检测OPCML、DNMT1的m RNA和蛋白表达;甲基特异性PCR(MSP)和DNA甲基转移酶(DNA methyltransferases)催化实验分析OPCML基因启动子区域甲基化及细胞核中甲基化活性;ELISA分析木犀草素处理前后对核转录因子Sp1活性的变化;RNA干扰及慢病毒转染等方法观察Sp1在介导甲基化及CPCML表达中的作用。最后建立裸鼠异种移植瘤动物模型,观察木犀草素对异种移植瘤生长的抑制作用。结果显示,Hep G2细胞基础状态下OPCML表达水平较低,其启动子区域甲基化水平较高,而经0~30μmol/L木犀草素处理后,能显著增强OPCML蛋白和m RNA的表达,并能降低其启动子甲基化水平以及细胞核中甲基化活性。同时,木犀草素也能抑制Hep G2细胞中Sp1的活性以及DNMT1的表达。si RNA干扰OPCML后,可逆转木犀草素对Hep G2细胞的生长抑制效应,上调细胞内Sp1表达后,同时伴有DNMT1表达增加及OPCML表达降低。此外,木犀草素也能上调裸鼠异种移植瘤中OPCML表达并抑制移植瘤生长的生长。以上结果表明木犀草素可能通过降低细胞内甲基化水平而上调OPCML基因表达,最终抑制Hep G2细胞生长增殖。
[Abstract]:The methylation inactivation of tumor suppressor gene OPCML is an important mechanism of tumorigenesis. Therefore, reversing the inactivation of tumor suppressor gene by drug is an important strategy for the prevention and treatment of tumor. Luteolin can inhibit the activity of DNA methyltransferase to some extent. However, it is not clear whether luteolin can reverse the inactivation of OPCML. In this study, hepatoma cell line HepG2 was cultured in vitro and treated with different concentrations of luteolin. The m RNA and protein expression of OPC MLN DNMT1 were detected by real-time quantitative PCR and Western blotting, respectively. Methyl-specific DNA methyltransferases and DNA methyltransferases. The promoter region methylation and nuclear methylation activity of OPCML gene were analyzed by catalytic experiments. The changes of Sp1 activity of nuclear transcription factor before and after luteolin treatment were analyzed by ELISA. RNA interference and lentivirus transfection were used to observe the role of Sp1 in mediating methylation and CPCML expression. The inhibitory effect of luteolin on the growth of xenografts was observed. The results showed that the expression of OPCML was low and the methylation level of the promoter region was higher in Hep G2 cells. After treated with 0 ~ 30 渭 mol/L luteolin, the expression of OPCML protein and m RNA were significantly increased, and the methylation level of promoter and the methylation activity in the nucleus were decreased. Luteolin also inhibited the activity of Sp1 in Hep G2 cells and the expression of DNMT1. Si RNA interfered with OPCML. Luteolin can reverse the inhibitory effect of luteolin on the growth of Hep G2 cells, and increase the expression of DNMT1 and decrease the expression of OPCML after up-regulating the expression of Sp1. Luteolin also up-regulated the expression of OPCML and inhibited the growth of xenografts in nude mice. These results suggest that luteolin may up-regulate the OPCML gene table by reducing intracellular methylation level. Da. Finally, the growth and proliferation of Hep G2 cells were inhibited.
【作者单位】：南华大学附属第二医院;
【基金】：国家自然科学基金(81071965)资助
【分类号】：R735.7
【正文快照】： *通讯作者,qinyingnan@163.comLuteolin Reactivates the Expression of Tumor Suppressor OPCML byInhibiting the Activity of Sp1 in Hepatocarcinoma CellsLi Xia Feng Yingjuan Wang Zhenggen Zhu Lihui Jiang Maibo Li Guoqing Qin Yingnan*The Second Affiliated Hospi

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