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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。
Methylation deactivation of tumor suppressor gene OPCML is an important mechanism of tumorigenesis. Therefore, reversing the inactivation status of tumor suppressor gene by drugs is an important strategy for tumor prevention and treatment. Studies have shown that Luteolin can inhibit DNA methyltransferase activity to some extent, but it is still not clear whether luteolin can reverse the inactivation status of OPCML. In this study, hepatoma cell line HepG2 was cultured in vitro and treated with luteolin at different concentrations. The mRNA and protein expressions of OPCML and DNMT1 were detected by real-time PCR and Western blotting respectively. Methyl-specific PCR (MSP) and DNA methyl DNA methyltransferases catalyzed the methylation and nuclei methylation in the promoter region of OPCML gene, the changes of nuclear factor Sp1 activity before and after luteolin treatment, RNA interference and lentivirus transfection To observe the role of Sp1 in methylation and CPCML expression. Finally, a nude mouse xenograft tumor model was established to observe the inhibitory effect of luteolin on xenograft tumor growth. The results showed that the expression level of OPCML in Hep G2 cells was low and the methylation level of promoter region was higher in Hep G2 cells. After treatment with 0-30 μmol / L luteolin, the expression of OPCML protein and m RNA was significantly increased, And can reduce its promoter methylation level and nuclear methylation activity. In the meantime, luteolin also inhibited Sp1 activity and DNMT1 expression in Hep G2 cells. siRNA could reverse the growth inhibitory effect of luteolin on Hep G2 cells after up-regulating the expression of Sp1, and up-regulated the expression of Sp1 in DNMT1 and decreased the expression of OPCML. In addition, luteolin also increased OPCML expression in nude xenografts and inhibited the growth of xenograft tumors. The above results indicated that luteolin may up-regulate the expression of OPCML gene by decreasing the level of intracellular methylation and eventually inhibit the growth and proliferation of Hep G2 cells.