DKK2在人结直肠癌中表观遗传学调控及其抑制结直肠癌细胞生长和转移的机制研究

发布时间：2018-01-25 01:31

本文关键词： DKK2 结直肠癌 DNA甲基化 Wnt/β-catenin 抑癌基因　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：癌症是全球重大的公共卫生问题,同时也是各国最主要的致死性疾病之一。据2017年度美国国家癌症中心数据统计显示:美国所有癌症疾病中,结直肠癌(Colorectal cancer,CRC)的发病率和死亡率高居前三位[1]。而在发展中国家,癌症发病率和死亡率远高于发达国家,据2016年发表于美国CA CANCER J CLIN杂志的中国癌症数据统计显示[2]:中国癌症患者的发病和死亡人数呈不断上升的趋势,2015年大约281400名患者死于癌症,平均每天7500名患者。结直肠癌是最常见的消化道恶性肿瘤之一,随着经济发展及生活水平的改善,其发病率及死亡率有不断上升的趋势;因此,寻找新的癌症治疗方法对肿瘤病患者治疗和预防有重要意义。DKK2(Dickkopf WNT signaling pathway inhibitor 2)是WNT信号通路最常见的调控因子,其家族成员主要包含4个(DKK1,DKK2,DKK3,DKK4),均为外分泌型蛋白。其中DKK2位于染色体4q25,基因长度149 kb,编码的蛋白约28 kd。目前已有文献报道:DKK2在肾癌、卵巢癌组织中存在着不同程度的甲基化及组织表达下调,同时有抑制肿瘤细胞生长、侵袭等生物学能力[3-4];在消化道肿瘤中存在不同程度的甲基化现象[5-6],但其在结直肠癌中的功能及相关分子机制尚不清楚。因此我们推测DKK2可能作为抑癌基因(Tumor suppressor gene,TSG)参与结直肠癌的进程。本研究采用逆转录聚合酶链式反应(Reverse transcription polymerase chain reaction,RT-PCR)、甲基化特异性聚合链式反应(Methylation specific PCR,MSP)、在线数据库分析等方法分析了DKK2在结直肠癌细胞系及组织中的表达和甲基化状态;同时体外实验和体内实验检测DKK2对细胞增殖及迁移的影响,并进一步采用实时荧光定量聚合酶链式反应(Real-time fluorescence quantiattive polymerase chain reaction,qRT-PCR)、RT-PCR及蛋白印迹法(Western blot,WB)等方法检测DKK2对增殖及迁移能力影响的相关分子机制。结果发现:DKK2基因在7株(100%)结直肠癌细胞中均有不同程度的表达下调或缺失;联合去甲基化药物5-氮杂-2脱氧胞苷(5-aza-2-deoxycytidine,5-Aza-dC)及组蛋白去乙酰化酶抑制剂曲骨菌素A(Trichostatin A,TSA)处理后,DKK2信使RNA(Messenger RNA,mRNA)表达下调或沉默的细胞株(HCT116、HT-29、SW480)能恢复表达;同时MSP发现,A+T处理后细胞(HT-29),其甲基化程度有不同程度的降低,因此提示了DKK2启动子甲基化可能是导致mRNA表达水平下调的主要原因之一。体外功能实验发现:DKK2能通过Wnt/β-catenin通路抑制肿瘤的生长及迁移。综上所述,DKK2作为抑癌基因参与结直肠癌的发生发展,可能为结直肠癌早期诊断及治疗提供了相关分子依据。
[Abstract]:Cancer is a major public health problem in the world and one of the leading fatal diseases in the world. According to the National Cancer Center of the United States in 2017, all cancer diseases in the United States show that. The incidence and mortality rate of Colorectal Cancer CRC are among the top three. [1. In developing countries, cancer morbidity and mortality are much higher than in developed countries, according to Chinese cancer statistics published in 2016 in the journal CA CANCER J CLIN. [2]: the incidence and death of cancer patients in China is on the rise. In 2015, about 281400 patients died of cancer. Colorectal cancer is one of the most common malignant tumors of digestive tract. With the development of economy and the improvement of living standard, the morbidity and mortality of colorectal cancer are increasing. So... Finding new methods of cancer treatment is of great significance for the treatment and prevention of oncology. Dickkopf WNT signaling pathway inhibitor 2) is the most common regulator of WNT signaling pathway. The family members are mainly composed of four DKK1DK2DKK3DKK3DKK4, all of which are exocrine proteins, in which DKK2 is located on chromosome 4q25 and the gene length is 149kb. It has been reported that there are different degrees of methylation and down-regulation of tissue expression in renal cell carcinoma and ovarian cancer, and also inhibit the growth of tumor cells. Invasion and other biological abilities. [3-4]; there are different degrees of methylation in digestive tract tumors. [5-6, but its function and related molecular mechanism in colorectal cancer are not clear. Therefore, we speculate that DKK2 may be used as tumor suppressor gene or suppressor gene. TSGs were involved in the progression of colorectal cancer. Reverse transcriptase polymerase chain reaction (RT-PCR) was used in this study. Reverse transcription polymerase chain reaction. RT-PCR, methylation specific PCR. The expression and methylation of DKK2 in colorectal cancer cell lines and tissues were analyzed by on-line database analysis. At the same time, the effects of DKK2 on cell proliferation and migration were detected in vitro and in vivo. Furthermore, real-time fluorescence quantitative polymerase chain reaction (. Real-time fluorescence quantiattive polymerase chain reaction. QRT-PCRX RT-PCR and Western blotting were used to detect Western blot. The molecular mechanism of the effect of DKK2 on proliferation and migration was detected by WB. the results showed that the gene of 1: DKK2 was found in 7 strains. In colorectal cancer cells, the expression was down-regulated or absent to varying degrees. Combined with demethylated drug 5-aza-2-deoxycytidine 5-aza-2-deoxycytidine (5-aza-2-deoxycytidine). After treatment with 5-Aza-dC and histone deacetylase inhibitor ATrichostatin (TSA). The down-regulated or silenced DKK2 messenger RNA(Messenger mRNAs can restore the expression of HCT116 (HT-29V480). At the same time, MSP found that the methylation degree of HT-29 cells was decreased in different degree after treated with T. The results suggest that methylation of DKK2 promoter may be one of the main causes of down-regulation of mRNA expression. DKK2 can inhibit tumor growth and migration through Wnt- 尾 -catenin pathway. As a tumor suppressor gene, DKK2 is involved in the development of colorectal cancer, which may provide molecular basis for early diagnosis and treatment of colorectal cancer.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34

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