组蛋白甲基化酶SMYD3促进人食管鳞癌发病的表观遗传机制研究

发布时间：2018-03-02 18:28

本文选题：食管癌　切入点：表观遗传　出处：《天津医科大学》2016年博士论文　论文类型：学位论文

【摘要】：食管癌是人类常见的恶性肿瘤,位居全球六大致死性肿瘤之列。2011年国际癌症研究机构(IARC)最新统计,每年全世界有近50万食管癌新发患者,死亡人数约40万。我国是食管癌最高发的国家,发病率及死亡率均居世界首位:发病率占全球54%,以河北磁县、河南林县、山西阳城为代表的华北太行山高发区发病率可高达110/10万以上;而我国每年因食管癌死亡人数也占据世界食管癌年死亡人数52%,死亡率在我国恶性肿瘤中位居第4位。制约食管癌疗效主要有如下几方面原因:第一,食管癌的早期筛查尚无明确的方法,尚未发现明确的肿瘤标志物,由于发现不及时而错过治疗最佳时机;第二,术后的复发和转移,是食管癌患者治疗失败和死亡的主要原因,该机理尚不十分清楚,也无有效的抑制手段;第三,国内外尚未报道任何针对食管癌特异性的治疗靶点及药物。另外,我国食管癌的发病相对欧美国家有其独特性,以鳞癌为主,而非国外报道较多的腺癌,因此不能只依赖于国际报道的数据,而应针对中国食管癌发病特点来展开研究。SMYD3与许多恶性肿瘤的发生发展关系密切,能够影响肿瘤细胞的增殖、凋亡、迁移及粘附等生物学行为,如结直肠癌、肝细胞癌、胰腺癌、前列腺癌、多发性骨髓瘤、肺癌、乳腺癌、脑瘤、胃癌等。本研究前期已应用免疫组化方法在15例人食管鳞癌及癌旁组织样本检测了SMYD3蛋白表达情况,发现其表达呈浆染且在癌组织中表达水平明显增高,并发现SMYD3对抑癌基因RIZ1具有调控作用,下调SMYD3表达后RIZ1表达增高,肿瘤增殖受到抑制。本研究进一步针对143例临床大样本病例中SMYD3的表达情况进行检测,试图明确其表达水平与临床病理参数关系,如肿瘤大小、病变长度、肿瘤分化、发生部位、有无淋巴结转移、TNM分期等。另一方面,试图明确SMYD3的表达水平是否与食管鳞癌患者的生存情况相关,是否具有指示预后的作用。研究结果显示,SMYD3表达水平与中位总生存期以及中位无进展生存期均独立相关,是食管鳞癌独立的预后指标,SMYD3高表达患者较低表达患者具有较差的预后。SMYD3在癌组织高表达进一步被证实,随肿瘤TNM分期的从低到高,浆染逐渐加强,但并未发现转移淋巴结中SMYD3表达与癌组织之间的差异。另外,在对其表达水平与临床病理参数相关性分析中也未有阳性发现,差异无统计学意义。因此,SMYD3是食管鳞癌重要的预后因子,其对癌症发生和进展临床意义重大。前期研究通过shrna敲降smyd3后riz1表达水平提升,抑癌活性恢复,肿瘤增殖受到抑制。本次应用sirna及构建shrna表达载体转染细胞的方法在食管鳞癌细胞系中下调和上调smyd3表达,检测riz1表达水平,验证前期结果并评价riz1表达是否随smyd3过表达而减低?另外,通过borden小室法检测癌细胞侵袭能力变化情况;通过流式细胞仪检测细胞凋亡的改变以及细胞周期的变化。结果我们发现,当应用sirna下调smyd3表达后,riz1的mrna和蛋白表达明显提升,癌细胞的侵袭力减弱,细胞凋亡增加,细胞周期s期细胞含量增加,而g2/m期细胞含量相对减少,g0/g1期无明显变化,提示抑制smyd3后细胞增殖下降的原因可能是由于细胞阻滞于s期。相反,当过表达smyd3后riz1的表达并未发生减低,也未发现肿瘤侵袭力的增加。针对前两部分内容及前期研究结果,smyd3作为抑癌基因riz1的上游调控基因,究竟是通过哪些因素的改变而调控了riz1的表达,又是通过哪些因素引起食管鳞癌细胞生物学行为变化呢?smyd3作为组蛋白甲基化酶,可以介导h3k4甲基化修饰的改变,而组蛋白甲基化改变已被证明与多种肿瘤的发生、进展、复发、耐药等方面密切相关。因此本研究的第三部分就smyd3-riz1在食管鳞癌中具体调控机制展开研究,首先应用elisa的方法对15例小样本h3k4一甲基化、二甲基化、三甲基化进行检测,经统计发现组蛋白甲基化水平在癌组织中均高于癌旁组织。之后在食管鳞癌细胞株te13敲低和过表达smyd3,检测以上甲基化的修饰变化,结果发现当smyd3下调时h3k4二甲基化和三甲基化减低,一甲基化未发生改变,而过表达smyd3时,三种甲基化水平均无变化。由于前期已证明riz1表达减低与dna启动子高甲基化有关,因此我们对smyd3敲低后的riz1启动子甲基化再一次进行了检测,发现其水平较对照发生了部分逆转。由此揭示了食管鳞癌中smyd3调控riz1的两种表观遗传机制。第一,是通过介导h3k4二甲基化和三甲基化的增加;第二,是使riz1的dna启动子区发生高甲基化。前三部分通过实验证明了smyd3与riz1的关联和可能存在的表观调控机制。组蛋白甲基化、dna甲基化都是动态存在的,它们受到组蛋白甲基化酶与去甲基化酶、dna甲基化酶与去甲基化酶间相互作用的影响,处于某种动态平衡之中,一旦平衡被打破就可能激活相关信号通路,导致肿瘤发生。本研究的第四部分试图对此展开初步探索,首先以上述15例食管鳞癌临床标本为研究对象,针对组蛋白赖氨酸特异性去甲基化酶lsd1和jarid1的酶活性进行了检测,发现癌组织中LSD1的酶活性明显高于癌旁组织,而JARID1酶活性并未发现区别。当应用5-aza-CdR逆转DNA甲基化后,H3K4一甲基化和二甲基化水平明显减低,在1μM时最为显著,H3K4三甲基化未见明显改变;LSD1和JARID1酶活性随用药浓度变化也产生了不同的改变,LSD1活性随用药浓度增加逐渐减低,而JARID1的酶活性较对照呈现先低后高的走势。以上说明了组蛋白去甲基化酶在食管鳞癌的发生过程中也起重要作用;DNA的甲基化可以影响H3K4甲基化修饰改变,同时也影响组蛋白去甲基化酶LSD1和JARID1的活性,具体机制还有待进一步探明。
[Abstract]:Esophageal cancer is a common malignant tumor, among the world's six most deadly cancer among.2011 years of the international agency for research on cancer (IARC) to the latest statistics, the whole world has nearly 500 thousand new cases of esophageal cancer each year, about 400 thousand deaths. China is the highest incidence of esophageal cancer, incidence rate and mortality rate in the the first in the world: the incidence accounted for 54% of the world, in Hebei, Cixian, Henan, Lin County, Shanxi Yangcheng as the representative of the high incidence area of North Taihang Mountain the incidence rate can be as high as 110/10 million; and China each year due to esophageal cancer deaths also occupy the world of esophageal cancer deaths in 52%, mortality ranked fourth in malignant tumors in China control effect of esophageal cancer. There are several reasons: first, there is no clear method for early screening of esophageal cancer, has not found a clear tumor marker, because it is not timely and miss the best time for treatment; second, postoperative rehabilitation Primary and metastatic esophageal cancer patients, is the main cause of treatment failure and death, the mechanism is not very clear, no inhibition of effective means; third, have not been reported for any esophageal cancer specific therapeutic targets and drugs at home and abroad. In addition, the incidence of esophageal cancer in China which is unique to Europe and the United States the country, mainly squamous cell carcinoma, but not reported more adenocarcinoma, it is not only dependent on the reported data, and should be closely related to the development of Chinese for incidence of esophageal cancer of.SMYD3 and many malignant tumors, can affect tumor cell proliferation, apoptosis, migration and adhesion of biological behavior. Such as colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, multiple myeloma, lung cancer, breast cancer, brain cancer, gastric cancer. This study previously by immunohistochemical method in 15 cases of esophageal squamous cell carcinoma and paracancerous tissue samples The expression of SMYD3 protein, we found that the expression of a pulp and the expression level increased significantly, and found that SMYD3 has a role in the regulation of tumor suppressor gene RIZ1 and down-regulation of SMYD3 expression after the increased expression of RIZ1 in tumor proliferation. This study further for the expression of SMYD3 in 143 cases was detected in large sample cases, trying to clear the relationship between its expression and clinical pathological parameters, such as tumor size, tumor length, tumor differentiation, location, lymph node metastasis and TNM stage. On the other hand, trying to clear the SMYD3 expression level is associated with the survival of patients with esophageal squamous cell carcinoma, whether can indicate the prognosis. Results showed that the expression level of SMYD3 and the median overall survival and median progression free survival were independent prognostic indicators of esophageal squamous cell carcinoma, is independent of the high expression of SMYD3 in patients with low expression in patients with Have a poor prognosis of.SMYD3 in cancer tissues of high expression was further confirmed, with the tumor TNM stage from low to high, the pulp gradually strengthened, but did not find a difference between the expression of SMYD3 in lymph node metastasis and cancer. In addition, the expression level and clinicopathological correlation analysis also found no positive parameters on it the difference was not statistically significant. Therefore, SMYD3 is an important prognostic factor in esophageal squamous cell carcinoma, the cancer occurrence and development is of great clinical significance. Previous research by shRNA on the expression level of RIZ1 stable after SMYD3 tumor suppressor activity recovery, tumor proliferation was inhibited. The application of siRNA and the way to construct shRNA expression vector transfected cells in esophageal squamous cell carcinoma cell lines downregulated and upregulated expression of SMYD3 to detect the expression level of RIZ1, to validate the result and evaluate whether the expression of RIZ1 with overexpression of SMYD3 decreased? In addition, detected by Borden assay. Cancer cell invasion ability changes; changes by flow cytometry to detect apoptosis and the change of cell cycle. Results we found that when siRNA was used to downregulate SMYD3 expression, protein expression of mRNA and RIZ1 significantly, decreased cancer cell invasion, cell apoptosis, increase of cells in the S phase of the cell cycle, and the cells in g2/m phase decreased, g0/g1 phase did not change significantly, suggesting that inhibition of SMYD3 cell proliferation after the cause of the decline may be due to cell cycle arrest in S phase. On the contrary, when after overexpression of SMYD3 RIZ1 expression was not impaired, also found no increased invasiveness of tumors. In the first two parts and the previous research results. SMYD3 as the upstream regulatory genes of tumor suppressor gene RIZ1, whether through which factors change and regulation of the expression of RIZ1, and by what factors caused by esophageal squamous cell carcinoma cell behavior changes SMYD3? As a histone methyltransferase, mediated by H3K4 methylation modification, and group change of histone methylation has been shown with a variety of tumor occurrence, progression, recurrence, closely related to resistance and so on. So the third part of the study of smyd3-riz1 in esophageal squamous cell carcinoma specific regulatory mechanism research methods the first application of ELISA in 15 cases of small sample H3K4 two methylation, methylation, methylation was detected by statistics found that histone methylation levels were higher than the adjacent tissues in cancer tissues. After knockdown and overexpression of SMYD3 in esophageal squamous cell carcinoma cell line te13, detection of the methylation modification. The results showed that when the SMYD3 cut H3K4 two methylation and methylation reduced methylation is not changed, and the expression of SMYD3, no change in three methylation level. Because of the early RIZ1 has been shown to reduce expression of DNA promoter Hypermethylation, so we SMYD3 knockdown of RIZ1 promoter methylation were detected again, found the levels than the control has been partially reversed. This reveals two epigenetic mechanisms of SMYD3 in esophageal squamous cell carcinoma and the regulation of RIZ1. First, the two is to increase the methylation and trimethyl mediated by H3K4; second, is to make RIZ1 DNA hypermethylation of the promoter region. The three part is proved through experiments on regulation mechanism of SMYD3 is associated with RIZ1 and possible. Histone methylation and DNA methylation are dynamic, they are histone methyltransferase and demethylase, DNA methylase and to influence the interaction between methylation enzyme, in a dynamic balance. Once the balance is broken can activate signaling pathways leading to tumorigenesis. The fourth part of this study attempts to launch the preliminary exploration, First of all the 15 cases of esophageal squamous cell carcinoma clinical samples as the research object, the enzyme activity of histone lysine specific demethylase LSD1 and jarid1 were detected, found that the enzyme activity of LSD1 in cancer tissues was significantly higher than that of adjacent tissues, and the enzyme activity of JARID1 did not find a difference. When using 5-aza-CdR reverse DNA methylation after a two H3K4 methylation and methylation level was significantly decreased in 1 M was the most significant, H3K4 trimethylation had no obvious change; LSD1 and JARID1 activity changes with the drug concentration had different changes, the activity of LSD1 increased with the drug concentration gradually decreased, while the enzyme activity of JARID1 compared with the control show the trend of low to high. The above described histone demethylase also plays an important role in carcinogenesis of esophageal squamous cell carcinoma; DNA methylation can affect H3K4 methylation changes also affect histone methylation enzyme The activity of LSD1 and JARID1 and the specific mechanism still need to be further explored.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1

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