本文選題：食管癌　切入點：表觀遺傳　出處：《天津醫(yī)科大學》2016年博士論文　論文類型：學位論文

【摘要】：食管癌是人類常見的惡性腫瘤,位居全球六大致死性腫瘤之列。2011年國際癌癥研究機構(IARC)最新統(tǒng)計,每年全世界有近50萬食管癌新發(fā)患者,死亡人數(shù)約40萬。我國是食管癌最高發(fā)的國家,發(fā)病率及死亡率均居世界首位:發(fā)病率占全球54%,以河北磁縣、河南林縣、山西陽城為代表的華北太行山高發(fā)區(qū)發(fā)病率可高達110/10萬以上;而我國每年因食管癌死亡人數(shù)也占據世界食管癌年死亡人數(shù)52%,死亡率在我國惡性腫瘤中位居第4位。制約食管癌療效主要有如下幾方面原因:第一,食管癌的早期篩查尚無明確的方法,尚未發(fā)現(xiàn)明確的腫瘤標志物,由于發(fā)現(xiàn)不及時而錯過治療最佳時機;第二,術后的復發(fā)和轉移,是食管癌患者治療失敗和死亡的主要原因,該機理尚不十分清楚,也無有效的抑制手段;第三,國內外尚未報道任何針對食管癌特異性的治療靶點及藥物。另外,我國食管癌的發(fā)病相對歐美國家有其獨特性,以鱗癌為主,而非國外報道較多的腺癌,因此不能只依賴于國際報道的數(shù)據,而應針對中國食管癌發(fā)病特點來展開研究。SMYD3與許多惡性腫瘤的發(fā)生發(fā)展關系密切,能夠影響腫瘤細胞的增殖、凋亡、遷移及粘附等生物學行為,如結直腸癌、肝細胞癌、胰腺癌、前列腺癌、多發(fā)性骨髓瘤、肺癌、乳腺癌、腦瘤、胃癌等。本研究前期已應用免疫組化方法在15例人食管鱗癌及癌旁組織樣本檢測了SMYD3蛋白表達情況,發(fā)現(xiàn)其表達呈漿染且在癌組織中表達水平明顯增高,并發(fā)現(xiàn)SMYD3對抑癌基因RIZ1具有調控作用,下調SMYD3表達后RIZ1表達增高,腫瘤增殖受到抑制。本研究進一步針對143例臨床大樣本病例中SMYD3的表達情況進行檢測,試圖明確其表達水平與臨床病理參數(shù)關系,如腫瘤大小、病變長度、腫瘤分化、發(fā)生部位、有無淋巴結轉移、TNM分期等。另一方面,試圖明確SMYD3的表達水平是否與食管鱗癌患者的生存情況相關,是否具有指示預后的作用。研究結果顯示,SMYD3表達水平與中位總生存期以及中位無進展生存期均獨立相關,是食管鱗癌獨立的預后指標,SMYD3高表達患者較低表達患者具有較差的預后。SMYD3在癌組織高表達進一步被證實,隨腫瘤TNM分期的從低到高,漿染逐漸加強,但并未發(fā)現(xiàn)轉移淋巴結中SMYD3表達與癌組織之間的差異。另外,在對其表達水平與臨床病理參數(shù)相關性分析中也未有陽性發(fā)現(xiàn),差異無統(tǒng)計學意義。因此,SMYD3是食管鱗癌重要的預后因子,其對癌癥發(fā)生和進展臨床意義重大。前期研究通過shrna敲降smyd3后riz1表達水平提升,抑癌活性恢復,腫瘤增殖受到抑制。本次應用sirna及構建shrna表達載體轉染細胞的方法在食管鱗癌細胞系中下調和上調smyd3表達,檢測riz1表達水平,驗證前期結果并評價riz1表達是否隨smyd3過表達而減低?另外,通過borden小室法檢測癌細胞侵襲能力變化情況;通過流式細胞儀檢測細胞凋亡的改變以及細胞周期的變化。結果我們發(fā)現(xiàn),當應用sirna下調smyd3表達后,riz1的mrna和蛋白表達明顯提升,癌細胞的侵襲力減弱,細胞凋亡增加,細胞周期s期細胞含量增加,而g2/m期細胞含量相對減少,g0/g1期無明顯變化,提示抑制smyd3后細胞增殖下降的原因可能是由于細胞阻滯于s期。相反,當過表達smyd3后riz1的表達并未發(fā)生減低,也未發(fā)現(xiàn)腫瘤侵襲力的增加。針對前兩部分內容及前期研究結果,smyd3作為抑癌基因riz1的上游調控基因,究竟是通過哪些因素的改變而調控了riz1的表達,又是通過哪些因素引起食管鱗癌細胞生物學行為變化呢?smyd3作為組蛋白甲基化酶,可以介導h3k4甲基化修飾的改變,而組蛋白甲基化改變已被證明與多種腫瘤的發(fā)生、進展、復發(fā)、耐藥等方面密切相關。因此本研究的第三部分就smyd3-riz1在食管鱗癌中具體調控機制展開研究,首先應用elisa的方法對15例小樣本h3k4一甲基化、二甲基化、三甲基化進行檢測,經統(tǒng)計發(fā)現(xiàn)組蛋白甲基化水平在癌組織中均高于癌旁組織。之后在食管鱗癌細胞株te13敲低和過表達smyd3,檢測以上甲基化的修飾變化,結果發(fā)現(xiàn)當smyd3下調時h3k4二甲基化和三甲基化減低,一甲基化未發(fā)生改變,而過表達smyd3時,三種甲基化水平均無變化。由于前期已證明riz1表達減低與dna啟動子高甲基化有關,因此我們對smyd3敲低后的riz1啟動子甲基化再一次進行了檢測,發(fā)現(xiàn)其水平較對照發(fā)生了部分逆轉。由此揭示了食管鱗癌中smyd3調控riz1的兩種表觀遺傳機制。第一,是通過介導h3k4二甲基化和三甲基化的增加;第二,是使riz1的dna啟動子區(qū)發(fā)生高甲基化。前三部分通過實驗證明了smyd3與riz1的關聯(lián)和可能存在的表觀調控機制。組蛋白甲基化、dna甲基化都是動態(tài)存在的,它們受到組蛋白甲基化酶與去甲基化酶、dna甲基化酶與去甲基化酶間相互作用的影響,處于某種動態(tài)平衡之中,一旦平衡被打破就可能激活相關信號通路,導致腫瘤發(fā)生。本研究的第四部分試圖對此展開初步探索,首先以上述15例食管鱗癌臨床標本為研究對象,針對組蛋白賴氨酸特異性去甲基化酶lsd1和jarid1的酶活性進行了檢測,發(fā)現(xiàn)癌組織中LSD1的酶活性明顯高于癌旁組織,而JARID1酶活性并未發(fā)現(xiàn)區(qū)別。當應用5-aza-CdR逆轉DNA甲基化后,H3K4一甲基化和二甲基化水平明顯減低,在1μM時最為顯著,H3K4三甲基化未見明顯改變;LSD1和JARID1酶活性隨用藥濃度變化也產生了不同的改變,LSD1活性隨用藥濃度增加逐漸減低,而JARID1的酶活性較對照呈現(xiàn)先低后高的走勢。以上說明了組蛋白去甲基化酶在食管鱗癌的發(fā)生過程中也起重要作用;DNA的甲基化可以影響H3K4甲基化修飾改變,同時也影響組蛋白去甲基化酶LSD1和JARID1的活性,具體機制還有待進一步探明。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.1

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