木犀草素通过抑制肝癌细胞Sp1活性上调抑癌基因OPCML表达

Luteolin Reactivates the Expression of Tumor Suppressor OPCML by Inhibiting the Activity of Sp1 in Hepatocarcinoma Cells

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。

Inactivation of the tumor suppressor OPCML by methylation is an important mechanism for the occurrence of tumor, thus the use of drugs to reverse the inactivation of tumor suppressor gene is an important strategy for cancer prevention and treatment. Studies have shown that Luteolin could inhibit the activity of DNA methyltransferase, but whether Luteolin could reverse the inactived status of OPCML is still unknown. In present study, the liver cancer cell line HepG2 was cultured in vitro, and then treated with different concentrations of Luteolin. The expressions ofmRNA and protein of OPCML and DNMT1 were respectively measured by Real-time quantitative PCR and Western blotting; Methyl specificity PCR （MSP） and DNA methyltransferases catalytic experiments were used to analyze the methylation of OPCML in gene promoter domain and the methylated activityin cell nucleus. We used ELISA to analyze activity changes of nuclear transcription factor Spl before and after treatment with Luteolin; RNA interference and Lentiviral transfection were used to observe the role of Spl in the mediation methylation and the expression of CPCML. Finally, the nude mice xenograft tumor animal model was established to observe the inhibitory effect of Luteolin on heterograft tumor growth. Our results suggested that the expression of OPCML was very low in HepG2 cells, and the methylation level was high in its promotor domain. The treatment of 0-30 ixmol/L Luteolin could significantly enhance the expressions of OPCML protein and mRNA, and reduce methylation level in promotor domain and methylated actvity in the nucleus. Meanwhile, the activity of Spl in HepG2 cells and the expression of DNMT1 could be inhibited by Luteolin. After siRNA was interfered with OPCML, the growth inhibition effect of HepG2 cells created by Luteolin could be reversed, and after up-regulating the Sp1 expression in cell, the DNMT1 expression increased while the OPCML expression decreased. Furthermore, Luteolin could up-regulate OPCML expression in nude mice xenograft tumor and inhibit the growth of xenograft tumor. The results suggested that Luteolin could increase the expression of OPCML by down-regulating methylated status in cells, and ultimately inhibit the growth and proliferation of HepG2 cells.