表观遗传沉默NKD2表达激活Wnt信号通路促进乳腺癌增殖

发布时间：2018-02-16 19:25

本文关键词： NKD2 DNA甲基化 Wnt信号通路表观遗传学乳腺癌　出处：《中国人民解放军医学院》2015年博士论文　论文类型：学位论文

【摘要】：背景：乳腺癌是全世界女性中最常见的恶性肿瘤,并且死亡率位居第二位。在中国,乳腺癌的发病率较西方国家低。这种差异可能与生活习惯及环境因素有关,表观遗传改变常常受到环境因素的影响。异常的基因甲基化在乳腺癌中频繁发生,与乳腺癌的发生发展相关。Naked cuticle homolog 1 (NKD1) 和 2 (NKD2)是果蝇naked cuticle基因在哺乳动物的同源基因。NKD1位于染色体16q12.1, NKD2位于染色体5p15.3。已发现许多肿瘤包括乳腺癌中,这些区域频繁发生杂合性缺失(Loss of heterozygosity, LOH)。NKD1和NKD2已被报道通过与Dishevelled相互作用负调控经典Wnt信号通路。在人和鼠的骨肉瘤细胞中发现NKD2可以抑制肿瘤的增殖和迁移,且在恶性胶质瘤细胞中NKD2发生启动子区的甲基化改变。目的：研究NKD2在乳腺癌中的表观遗传改变及其在乳腺癌中的作用和作用机制。方法：本研究在6株乳腺癌细胞系和68例乳腺癌组织中,采用甲基化特异PCR,半定量RT-PCR,免疫组织化学分析NKD2在细胞系和组织中的甲基化状态及表达水平的改变。MTT,克隆形成实验和流式细胞术的方法分析NKD2对乳腺癌细胞增殖、侵袭迁移能力的影响。双荧光素酶实验，western blot和siRNA的方法,并结合裸鼠成瘤模型探讨NKD2对乳腺癌的作用及其在Wnt信号通路中的作用机制。结果：NKD2启动子区在MDA-MB-231和MDA-MB-468细胞中呈甲基化状态,且其表达缺失,经5-AZA处理后表达恢复。在MCF7, ZR75-1, BT474和T47D细胞中NKD2呈低表达,启动子区呈半甲基化状态,经5-AZA处理后表达增加。在原发性乳腺癌组织中,NKD2的甲基化率为51.4%(35/68),其甲基化与NKD2的表达沉默或降低有关(p0.05)。体内、外实验发现NKD2可以抑制乳腺癌细胞的增殖。NKD2诱导G0/1期阻滞并且抑制Wnt信号通路的活性。结语,NKD2在乳腺癌中频繁发生甲基化,NKD2的表达受启动子区甲基化调控。NKD2通过抑制Wnt信号通路而抑制乳腺癌细胞的生长。
[Abstract]:Background: breast cancer is the most common malignant tumor among women in the world, with the second highest mortality rate. In China, the incidence of breast cancer is lower than that in western countries. This difference may be related to lifestyle and environmental factors. Epigenetic changes are often affected by environmental factors. Abnormal gene methylation occurs frequently in breast cancer. Naked cuticle homolog 1 and 2 NKD2) are the homologous genes of naked cuticle gene in Drosophila melanogaster. NKD1 is located on chromosome 16q12.1 and NKD2 is located at chromosome 5p15.3.Many tumors including breast cancer have been found. Loss of heterozygosity occurs frequently in these regions. LOH).NKD1 and NKD2 have been reported to negatively regulate classical Wnt signaling pathways through interaction with Dishevelled. NKD2 has been found to inhibit tumor proliferation and migration in human and mouse osteosarcoma cells. And methylation of NKD2 promoter in malignant glioma cells. Objective: to study the epigenetic change of NKD2 in breast cancer and its role and mechanism in breast cancer. Methods: this study was carried out in 6 breast cancer strains. Cell lines and 68 breast cancer tissues, Methylation specific PCR, semi-quantitative RT-PCR, immunohistochemical analysis of methylation status and expression level of NKD2 in cell lines and tissues, clone formation assay and flow cytometry were used to analyze the proliferation of NKD2 in breast cancer cells. Methods of Western blot and siRNA for double luciferase assay, The effect of NKD2 on breast cancer and its mechanism in Wnt signaling pathway were studied in nude mice. Results: the promoter region of NKD2 D 2 was methylated in MDA-MB-231 and MDA-MB-468 cells, and its expression was absent. In MCF7, ZR75-1, BT474 and T47D cells, the expression of NKD2 was low and the promoter was semi-methylated. In primary breast cancer, the methylation rate of NKD2 was 51.4% 35 / 68, and its methylation was related to the silencing or lowering of NKD2 expression. It was found that NKD2 could inhibit the proliferation of breast cancer cells. NKD2 induced G0 / 1 arrest and inhibited the activity of Wnt signaling pathway. The expression of NKD2 was regulated by promoter methylation in breast cancer. The growth of breast cancer cells was inhibited by inhibiting Wnt signaling pathway.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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