本文選題：前列腺癌 + EZH2　； 參考：《東南大學(xué)》2016年博士論文

【摘要】：前列腺癌是男性泌尿生殖系統(tǒng)常見的惡性腫瘤。其發(fā)病率有著明顯的地理和種族差異。在美國(guó),前列腺癌是男性發(fā)病率最高的惡性腫瘤,據(jù)"Cancer Statistics,2016"最新研究報(bào)道,2016年全美將有180,890例前列腺癌新發(fā)病例,將有26,120名患者死于前列腺癌。在歐洲,預(yù)計(jì)2016年因前列腺癌致死病例達(dá)75,800,死亡人數(shù)位居男性惡性腫瘤致死人數(shù)第三位。在我國(guó),隨著人口老齡化形勢(shì)的加劇,前列腺癌的發(fā)病率逐年上升。根據(jù)國(guó)家癌癥中心數(shù)據(jù),2009年我國(guó)前列腺癌發(fā)病率為9.92/10萬；。'Cancer Statistics in China,2015"最新數(shù)據(jù)顯示,2015年我國(guó)前列腺癌發(fā)病率激增至60.3/10萬,死亡率達(dá)26.6/10萬。前列腺癌已成為威脅中國(guó)男性健康的主要疾病之一。目前,根治性前列腺切除術(shù)和外放射治療是治愈局限性前列腺癌的主要方法。但是,對(duì)于已有遠(yuǎn)處轉(zhuǎn)移、不適宜手術(shù)或治愈性治療后復(fù)發(fā)(或轉(zhuǎn)移)等情況,通常選擇內(nèi)分泌治療。研究表明,80%以上的患者可通過雄激素剝奪治療獲得疾病的緩解,但經(jīng)過14-30個(gè)月的中位時(shí)間后,幾乎所有患者的病變都將逐漸發(fā)展為去勢(shì)抵抗性前列腺癌(Castration-Resistant Prostate Cancer, CRPC)�；颊咭坏┌l(fā)生去勢(shì)抵抗和遠(yuǎn)處轉(zhuǎn)移,預(yù)后較差。雖然多種藥物包括多西紫杉醇、阿比特龍、恩雜魯胺等可用于CRPC的治療,但其療效仍然有限。CRPC已成為晚期前列腺癌患者主要的死亡原因。因此,探討前列腺癌的進(jìn)展機(jī)制,尋找新的分子靶點(diǎn),對(duì)延緩疾病進(jìn)程和降低晚期腫瘤的死亡率具有重大作用。過去的十年里,人們發(fā)現(xiàn)在前列腺癌的惡性進(jìn)程中有大量的表觀遺傳分子的異常改變。Polycomb repressive complex2 (PRC2復(fù)合體)是由多個(gè)蛋白組合而成的表觀調(diào)控復(fù)合體。EZH2是PRC2復(fù)合體的重要催化亞基。它含有一個(gè)活性的SET催化結(jié)構(gòu)域,該結(jié)構(gòu)域內(nèi)的甲基轉(zhuǎn)移酶可以被外源甲基基團(tuán)識(shí)別并通過共價(jià)反應(yīng)的形式結(jié)合,催化組蛋白H3的第9和27位賴氨酸發(fā)生三甲基化(H3K9me3和H3K27me3)。研究表明,EZH2需要和EED和SUZ12兩個(gè)PRC2家族成員協(xié)同完成以上酶促反應(yīng)。此外,PRC2的另外兩個(gè)核心成員RBBP4和RBBP7可以直接與組蛋白結(jié)合,通過催化甲基化抑制基因的轉(zhuǎn)錄活性。近年來,研究發(fā)現(xiàn)EZH2在乳腺癌、膀胱癌、胃癌、肺癌、肝癌等腫瘤組織中異常高表達(dá)且預(yù)示著較差的預(yù)后。在前列腺癌相關(guān)研究中,人們發(fā)現(xiàn)EZH2在轉(zhuǎn)移的前列腺癌組織中較局限性前列腺癌異常高表達(dá)。進(jìn)一步研究發(fā)現(xiàn),EZH2通過催化H3K27me3,沉默ADRB2和CDH1基因的表達(dá),從而促進(jìn)腫瘤細(xì)胞的侵襲能力。然而,有關(guān)EZH2在前列腺癌進(jìn)展過程中不同階段和不同病理級(jí)別的表達(dá)情況和潛在的生物學(xué)功能,以及EZH2是否參與調(diào)控與前列腺癌進(jìn)展相關(guān)的非編碼RNA信號(hào)通路仍需進(jìn)一步深入探索。本項(xiàng)研究綜合運(yùn)用臨床大樣本分析、生物信息學(xué)、基因芯片、細(xì)胞功能實(shí)驗(yàn)、分子生物學(xué)實(shí)驗(yàn)及動(dòng)物在體實(shí)驗(yàn)等手段深入研究EZH2在前列腺癌進(jìn)展中的生物學(xué)行為及其潛在的分子機(jī)制。在本課題第一部分中,我們首先借助GEO數(shù)據(jù)庫(kù)和課題組前期收集的前列腺正常和腫瘤組織樣本,從RNA表達(dá)和蛋白表達(dá)兩個(gè)層面分析EZH2在正常前列腺組織和前列腺癌組織中的表達(dá)情況；并進(jìn)一步細(xì)化EZH2在前列腺癌不同病理級(jí)別和不同進(jìn)展階段的表達(dá)情況,同時(shí)利用隨訪數(shù)據(jù)分析EZH2與前列腺癌患者預(yù)后之間的關(guān)系。隨后,運(yùn)用RNA干擾手段下調(diào)前列腺癌細(xì)胞中的EZH2,通過流式細(xì)胞儀、酶標(biāo)儀、克隆形成實(shí)驗(yàn)、XF糖酵解壓力測(cè)試盒及裸鼠在體成瘤等實(shí)驗(yàn)技術(shù)檢測(cè)EZH2對(duì)前列腺癌細(xì)胞周期、增殖、糖代謝、成瘤等生物學(xué)行為的影響。結(jié)果顯示：1)與前列腺正常組織和良性增生組織相比,EZH2在前列腺癌組織中的表達(dá)顯著上升：且EZH2在去勢(shì)抵抗性前列腺癌組織中的表達(dá)顯著高于激素依賴性前列腺癌；2)EZH2在Gleason7分的前列腺癌組織中的表達(dá)顯著高于Gleason≤7的前列腺癌組織。3)體外細(xì)胞功能實(shí)驗(yàn)表明,利用EZH2-shRNA敲低EZH2的表達(dá)后,前列腺癌細(xì)胞的增殖能力和克隆形成能力受到明顯抑制,細(xì)胞周期阻滯于G1期,細(xì)胞的有氧糖酵解水平明顯下降。4)裸鼠皮下成瘤實(shí)驗(yàn)表明,EZH2敲低組的腫瘤生長(zhǎng)速率較對(duì)照組明顯減慢；至觀察終點(diǎn),EZH2敲低組的腫瘤體積顯著小于對(duì)照組。微小RNA(microRNAs, miRNA)近10年來始終是腫瘤研究領(lǐng)域的熱點(diǎn)。miRNA的信號(hào)傳遞體系包括多種形式,其本身接受上游轉(zhuǎn)錄調(diào)控分子的信號(hào),向下游則可通過識(shí)別特定的目標(biāo)mRNA的3’端非編碼區(qū)域(3'untranslated region,3’UTR),在轉(zhuǎn)錄后水平通過促進(jìn)靶mRNA的降解和(或)抑制翻譯過程而發(fā)揮調(diào)控基因表達(dá)的作用。隨著系統(tǒng)生物學(xué)研究的開展,miRNA的研究也從單一功能走向整體綜合的水平。大量報(bào)道證實(shí)miRNA表達(dá)譜特征與腫瘤的診斷、分期、分級(jí)、進(jìn)展、預(yù)后及治療存在相關(guān)性,是腫瘤基因調(diào)控網(wǎng)絡(luò)中的重要樞紐分子。作為轉(zhuǎn)錄抑制因子,EZH2可沉默下游諸多靶基因,而miRNA也是一大類可被EZH2調(diào)控的靶向分子。在本課題第二部分中,我們首先利用基因表達(dá)譜芯片數(shù)據(jù)預(yù)測(cè)可能參與EZH2調(diào)控細(xì)胞周期和代謝的相關(guān)基因,結(jié)果顯示,E2F3和HK2兩個(gè)基因與EZH2的表達(dá)顯著正相關(guān)；隨后,借助miRNA表達(dá)譜芯片技術(shù)篩選EZH2相關(guān)miRNA差異譜。結(jié)果顯示,EZH2敲低后miR-200和miR-181兩個(gè)家族中的部分成員表達(dá)顯著上升。利用蛋白免疫印跡和熒光定量PCR技術(shù)檢測(cè)發(fā)現(xiàn),EZH2敲低后H3K27Me3的表達(dá)水平顯著降低,miR-200c和miR-181b的表達(dá)明顯上調(diào)；熒光素酶報(bào)告基因?qū)嶒?yàn)證實(shí)EZH2敲低后miR-200c和miR-181b基因啟動(dòng)子區(qū)域的轉(zhuǎn)錄活性增強(qiáng)。進(jìn)一步通過生物信息學(xué)預(yù)測(cè)、蛋白免疫印跡和熒光素酶報(bào)告基因?qū)嶒?yàn)發(fā)現(xiàn),E2F3和HK2分別是miR-200c和miR-181b的直接靶基因,提示miR-200c和miR-181b可以介導(dǎo)EZH2對(duì)E2F3和HK2的正向調(diào)控作用。為了進(jìn)一步驗(yàn)證EZH2對(duì)前列腺癌細(xì)胞周期和糖代謝的影響是通過miR-200c和miR-181b介導(dǎo)所實(shí)現(xiàn)的,我們?cè)O(shè)計(jì)了相應(yīng)的回復(fù)實(shí)驗(yàn)。在對(duì)照組和EZH2敲低組的基礎(chǔ)上增加sh-EZH2+As-miR-200c組和sh-EZH2+As-miR-181b組。結(jié)果顯示sh-EZH2+As-miR-200c組較sh-EZH2單獨(dú)轉(zhuǎn)染組的細(xì)胞周期進(jìn)程出現(xiàn)回復(fù)效應(yīng),且sh-EZH2引起的E2F3表達(dá)下降可以被As-miR-200c部分逆轉(zhuǎn)；另一方面,sh-EZH2+As-miR-181b組較sh-EZH2單獨(dú)轉(zhuǎn)染組的細(xì)胞糖酵解能力也顯示回復(fù)效應(yīng),且sh-EZH2引起的HK2表達(dá)下降可以被As-miR-181b部分逆轉(zhuǎn)；再次證明miR-200c和miR-181b是EZH2對(duì)細(xì)胞周期和糖代謝調(diào)控作用的中介分子。綜上所述,本課題綜合運(yùn)用臨床大樣本、生物信息學(xué)分析、細(xì)胞功能檢測(cè)、高通量篩選、miRNA/mRNA和蛋白/DNA互作檢測(cè)(熒光素酶報(bào)告基因)、熒光定量PCR等方法,對(duì)前列腺癌細(xì)胞中EZH2的功能及機(jī)制進(jìn)行了系列探索。研究發(fā)現(xiàn)EZH2能夠調(diào)節(jié)前列腺癌細(xì)胞增殖、周期、糖代謝等生物學(xué)行為,并且完成了EZH2/miRNAs信號(hào)軸及其下游信號(hào)通路的實(shí)驗(yàn)構(gòu)建。這些結(jié)果為后續(xù)深入研究miRNAs介導(dǎo)下的EZH2調(diào)控分子網(wǎng)絡(luò)奠定了基礎(chǔ),也為尋找前列腺癌分子診斷和預(yù)后評(píng)估的生物靶點(diǎn)和探索前列腺癌發(fā)生及演進(jìn)機(jī)制并優(yōu)化治療策略提供了理論依據(jù)。
[Abstract]:Prostate cancer is a common malignant tumor in the male genitourinary system. Its incidence has obvious geographical and racial differences. In the United States, prostate cancer is the highest incidence of malignant tumors in men. According to the latest research of "Cancer Statistics, 2016", there will be 180890 new cases of prostate cancer in the United States in 2016, and 26120 patients will die. Prostate cancer. In Europe, the death toll of prostate cancer in 2016 is expected to reach 75800, and the death toll is third in the number of male malignant tumors. In China, the incidence of prostate cancer is rising year by year with the aging of the population. According to the National Cancer Center, the incidence of prostate cancer in China is 9.92/10 million in 2009;.'C Ancer Statistics in China, 2015 "the latest data show that the incidence of prostate cancer in China surged to 60.3/10 million in 2015, with a mortality rate of 26.6/10 million. Prostate cancer has become one of the major diseases that threaten Chinese male health. Currently, radical prostatectomy and external radiation therapy are the main methods to cure localized prostate cancer. Endocrine therapy is usually selected for cases of distant metastasis, unsuitable surgery or cure after treatment. Studies have shown that more than 80% of the patients can get the remission of the disease through androgen deprivation treatment, but after 14-30 months of median time, several of the patients' lesions will gradually develop to castrated resistance. Castration-Resistant Prostate Cancer (CRPC). Patients have a poor prognosis in the event of castration and distant metastasis. Although a variety of drugs, including docetaxel, amieton, and eexuramine, can be used for the treatment of CRPC, the curative effect still limited to.CRPC has become the main cause of death in advanced prostate cancer patients. To explore the progress mechanism of prostate cancer and find new molecular targets, it is important to delay the disease process and reduce the mortality of advanced cancer. In the past ten years, people found that there are a large number of abnormal changes in epigenetic molecular.Polycomb repressive complex2 (PRC2 complex) in the malignant process of prostate cancer. The apparent regulatory complex.EZH2 formed by a protein combination is an important catalytic subunit of the PRC2 complex. It contains a active SET catalytic domain. The methyltransferase within the domain can be identified by the foreign methyl group and combined in the form of covalent reaction, which catalyzes the formation of ninth and twenty-seventh - bit lysine (H3K9m) of the protein H3 (H3K9m E3 and H3K27me3). Studies have shown that EZH2 needs to cooperate with two PRC2 family members of EED and SUZ12 to complete the above enzyme reaction. In addition, the other two core members of PRC2, RBBP4 and RBBP7, can directly bind to histone by catalyzing methylation to inhibit the transcriptional activity of the gene. In recent years, EZH2 has been found to be in breast, bladder, gastric, and lung cancer. Abnormal high expression in tumor tissues such as liver cancer indicates poor prognosis. In the study of prostate cancer, it is found that EZH2 is highly expressed in the metastatic prostate cancer tissues. Further studies have found that EZH2 can promote the invasion of tumor cells by catalyzing H3K27me3 to silence the expression of ADRB2 and CDH1 genes. However, the expression and potential biological functions of EZH2 during the progression of prostate cancer, as well as the potential biological functions of different stages, and whether EZH2 is involved in the regulation of the progression of prostate cancer, still need to be further explored. Study on the biological behavior and potential molecular mechanisms of EZH2 in the progress of prostate cancer. In the first part of this topic, we first used the GEO database and the prostatic normal and tumor tissue samples collected by the project group, The expression of EZH2 in normal prostate tissues and prostate cancer tissues was analyzed from the two levels of RNA expression and protein expression, and the expression of EZH2 in different pathological and progressive stages of prostate cancer was further refined, and the relationship between EZH2 and the prognosis of prostate cancer patients was analyzed by follow up data. Then, RN was used. A interfering method downregulated the EZH2 in prostate cancer cells. The effects of EZH2 on the biological behavior of prostate cancer cell cycle, proliferation, glucose metabolism, tumor formation were detected by flow cytometer, enzyme labeling apparatus, clone formation experiment, XF glycolysis pressure test box and nude mice in vivo tumorigenesis. The results showed that: 1) and normal prostate tissue and benign prostatic cancer. The expression of EZH2 in the prostate cancer tissue was significantly higher than that of the hyperplastic tissue, and the expression of EZH2 in the castrated resistant prostate cancer tissues was significantly higher than that of the hormone dependent prostate cancer; 2) the expression of EZH2 in the prostate cancer tissues of the Gleason7 score was significantly higher than that of the.3 in the prostate cancer tissue with Gleason < < 7. The proliferation and clonogenic ability of prostate cancer cells were significantly inhibited by EZH2-shRNA knockout EZH2, the cell cycle was blocked at G1, and the level of aerobic glycolysis decreased significantly by.4) in nude mice, the tumor growth rate in the EZH2 knockout group was significantly slower than that in the control group; to the observation point, EZH The volume of tumor in the 2 knockout group was significantly smaller than that of the control group. RNA (microRNAs, miRNA) has been a hot spot in the field of cancer research for the last 10 years. The signal transmission system of.MiRNA includes a variety of forms, which itself receives the signal of the upstream transcriptional regulators, and the downstream is the 3 'end non coding region of the specific target mRNA (3'untransla). Ted region, 3 'UTR) play the role of regulating gene expression by promoting the degradation of target mRNA and (or) inhibiting the translation process at post transcriptional level. With the development of systematic biology, the study of miRNA has also moved from single function to overall level. A large number of the characteristics of the miRNA expression profiles and the diagnosis, staging, classification of the tumor are reported. Progress, prognosis and treatment are the key elements in the tumor gene regulation network. As a transcriptional inhibitor, EZH2 can silence a number of target genes downstream, and miRNA is a large category of target molecules controlled by EZH2. In the second part of this project, we first use gene expression chip data to predict the possibility of participation in EZH 2 regulating the related genes of cell cycle and metabolism, the results showed that the two genes of E2F3 and HK2 were positively correlated with the expression of EZH2. Subsequently, the EZH2 related miRNA difference spectrum was screened by miRNA expression chip technology. The results showed that the expression of partial members in the two families of miR-200 and miR-181 after EZH2 knockout was significantly increased. And the fluorescence quantitative PCR assay showed that the expression level of H3K27Me3 decreased significantly after the EZH2 knockout, and the expression of miR-200c and miR-181b was obviously up. The luciferase reporter gene experiment confirmed that the transcriptional activity of the miR-200c and miR-181b gene promoter region was enhanced after the EZH2 knockout. Further, the bioinformatics prediction, the protein immunoblotting and the protein immunoblotting were further predicted. The luciferase reporter gene experiment found that E2F3 and HK2 are the direct target genes of miR-200c and miR-181b, suggesting that miR-200c and miR-181b can mediate the positive regulation of EZH2 on E2F3 and HK2. In order to further verify the effect of EZH2 on the cell cycle of prostate cancer and glucose metabolism, it is realized through miR-200c and miR-181b mediation. In the control group and the EZH2 knockout group, the sh-EZH2+As-miR-200c group and the sh-EZH2+As-miR-181b group were added to the control group. The results showed that the cell cycle process of the sh-EZH2+As-miR-200c group had a response to the cell cycle process in the sh-EZH2 alone transfection group, and the sh-EZH2 induced decline of the E2F3 expression could be reversed by the As-miR-200c part; In the sh-EZH2+As-miR-181b group, the glycolytic ability of the cells in the sh-EZH2 alone transfected group also showed the response effect, and the decrease of the HK2 expression caused by sh-EZH2 could be reversed by the As-miR-181b part. Again, miR-200c and miR-181b were intermediaries of the regulation of EZH2 to cell cycle and glucose metabolism. Large samples, bioinformatics analysis, cell function detection, high throughput screening, miRNA/mRNA and protein /DNA interaction detection (luciferase reporter gene), fluorescence quantitative PCR and other methods have been used to explore the function and mechanism of EZH2 in prostate cancer cells. It is found that EZH2 can regulate the proliferation, cycle, and glucose metabolism of prostate cancer cells. Biological behavior, and completed the experimental construction of the EZH2/miRNAs signal axis and its downstream signal pathway. These results provide a basis for further study of the EZH2 regulatory molecular network mediated by miRNAs, the biological targets for the diagnosis and prognosis evaluation of prostate cancer, and the exploration and optimization of the mechanism of the occurrence and evolution of prostate cancer. The treatment strategy provides a theoretical basis.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.25

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本文編號(hào)：1839247