木犀草素对乳腺癌BT474细胞增殖、侵袭的影响及机制研究

发布时间：2018-05-10 08:57

本文选题：木犀草素 + 乳腺癌　；参考：《南方医科大学》2017年博士论文

【摘要】：[背景与目的]乳腺癌发病率逐年增高,机制仍欠清晰。木犀草素可抑制多种肿瘤的发生、进展,乳腺癌中报道少。抑癌基因失活是肿瘤发生重要机制,采用药物逆转是肿瘤防治重要策略。OPCML抑癌基因甲基化失活可致肿瘤发生、进展。本研究旨在探讨木犀草素对乳腺癌发生、发展的影响,对其中OPCML抑癌基因表达的影响,并探讨其在细胞增殖、侵袭中的作用机制。[方法]1.体外培养乳腺癌细胞系BT474,用不同浓度木犀草素孵育后,采用CCK-8、划痕及Transwell小室实验观察木犀草素对BT474细胞的增殖、迁移、侵袭能力的影响;建立裸鼠异种移植瘤模型,观察不同浓度木犀草素对移植瘤生长的影响。2.木犀草素处理BT474细胞或移植瘤裸鼠,实时定量PCR和Western blot分别检测不同浓度木犀草素对BT474细胞及裸鼠异种移植瘤中OPCML mRNA和蛋白表达影响;采用siRNAOPCML转染BT474细胞,观察siRNA干扰前后能否影响木犀草素对BT474细胞的生长抑制作用;构建OPCML表达载体并转染BT474细胞,观察其能否影响细胞的生长增殖。3采用甲基化特异性PCR法检测木犀草素处理前后OPCML启动子区域甲基化水平,同时测定木犀草素处理前后细胞内甲基化及转录因子Sp1活性;qPCR、Western blot检测木犀草素对DNMT1 mRNA及蛋白表达的影响;转染使BT474细胞过表达Sp1,观察其对木犀草素处理后OPCML表达、启动子甲基化以及DNMT1 mRNA表达的影响;通过流式细胞术检测BT474细胞的凋亡情况以及细胞周期的变化,凋亡相关分子r-H2AX磷酸化情况,Caspase-8的活化以及cPARP的水平。[结果](1)采用不同浓度木犀草素处理BT474细胞后,可显著抑制乳腺癌细胞的增殖、侵袭与迁移,呈一定的浓度与时间依赖性;同时,木犀草素也可抑制裸鼠皮下肿瘤的形成和生长。(2)木犀草素可上调乳腺癌细胞中、乳腺癌异种移植瘤中OPCML mRNA及蛋白的表达;siRNA沉默OPCML后可减弱木犀草素对BT474细胞增殖、侵袭与迁移的抑制作用;此外,细胞内OPCML过表达可抑制细胞增殖、侵袭、迁移;(3)木犀草素可抑制OPCML启动子区域DNA甲基化;并能下调细胞内以及移植瘤中甲基化活性;木犀草素能下调细胞内以及移植瘤中DNMT1 mRNA及蛋白的表达,木犀草素能下调细胞内SP1的活性;过表达Sp1,木犀草素对DNMT1的抑制作用、对DNA甲基化的抑制作用、对OPCML表达的诱导作用都减弱;木犀草素可诱导BT474细胞凋亡;诱导BT474细胞r-H2A磷酸化,促caspase-8活化,上调细胞内cPARP水平。[结论]:木犀草素可通过抑制OPCML基因的启动子区域的甲基化并上调其表达水平,从而诱导乳腺癌细胞周期阻滞和凋亡,抑制乳腺癌细胞增殖、迁移、侵袭。
[Abstract]:Background and objective: the incidence of breast cancer is increasing year by year and the mechanism is still unclear. Luteolin inhibits the occurrence and progression of multiple tumors, and is rarely reported in breast cancer. Inactivation of tumor suppressor gene is an important mechanism of tumorigenesis. Drug reversal is an important strategy for prevention and treatment of cancer. The methylation inactivation of OPCML suppressor gene can lead to tumorigenesis and progress. The purpose of this study was to investigate the effect of luteolin on the occurrence and development of breast cancer and the expression of OPCML tumor suppressor gene, and to explore the mechanism of luteolin in cell proliferation and invasion. [methods] 1. Breast cancer cell line BT474 was cultured in vitro. After incubation with luteolin of different concentrations, the effects of luteolin on the proliferation, migration and invasion of BT474 cells were observed by CCK-8, scratch and Transwell chamber experiments. To observe the effect of luteolin at different concentrations on the growth of transplanted tumor. The effects of luteolin on the expression of OPCML mRNA and protein in BT474 cells and xenografts of nude mice were detected by real-time quantitative PCR and Western blot. SiRNAOPCML was used to transfect BT474 cells. The effects of luteolin on the growth inhibition of BT474 cells were observed before and after siRNA interference, and the expression vector of OPCML was constructed and transfected into BT474 cells. The effect of luteolin on cell growth and proliferation was observed. Methylation specific PCR assay was used to detect the methylation level of OPCML promoter region before and after luteolin treatment. At the same time, the intracellular methylation and Sp1 activity of transcription factor Sp1 were measured before and after luteolin treatment. The effect of luteolin on the expression of DNMT1 mRNA and protein was detected by Western blot, and the expression of OPCML was observed after BT474 cells were overexpressed by transfection. The effects of promoter methylation and DNMT1 mRNA expression, apoptosis and cell cycle changes of BT474 cells, the activation of Caspase-8 and the level of cPARP were detected by flow cytometry. [results] after treated with luteolin at different concentrations, BT474 cells could significantly inhibit the proliferation, invasion and migration of breast cancer cells in a dose-dependent and time-dependent manner. Luteolin can also inhibit the formation and growth of subcutaneous tumor in nude mice. Luteolin can up-regulate the expression of OPCML mRNA and protein in breast cancer xenografts. After silencing OPCML, luteolin can attenuate the proliferation of BT474 cells by luteolin. In addition, overexpression of OPCML in cells inhibited cell proliferation, invasion and migration (3) luteolin inhibited DNA methylation in OPCML promoter region, and down-regulated methylation activity in cells and xenografts. Luteolin can down-regulate the expression of DNMT1 mRNA and protein in cells and transplanted tumors, luteolin can down-regulate the activity of SP1 in cells, over-express Sp1, inhibit DNMT1 and DNA methylation. Luteolin could induce apoptosis of BT474 cells, phosphorylation of r-H2A, activation of caspase-8 and up-regulation of cPARP in BT474 cells. [conclusion] luteolin can induce cell cycle arrest and apoptosis by inhibiting methylation and up-regulation of promoter region of OPCML gene, thus inhibiting proliferation, migration and invasion of breast cancer cells.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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