EZH2通过靶向抑制miR-200c和miR-181b促进前列腺癌细胞增殖和糖代谢

发布时间：2018-05-03 16:33

本文选题：前列腺癌 + EZH2　；参考：《东南大学》2016年博士论文

【摘要】：前列腺癌是男性泌尿生殖系统常见的恶性肿瘤。其发病率有着明显的地理和种族差异。在美国,前列腺癌是男性发病率最高的恶性肿瘤,据"Cancer Statistics,2016"最新研究报道,2016年全美将有180,890例前列腺癌新发病例,将有26,120名患者死于前列腺癌。在欧洲,预计2016年因前列腺癌致死病例达75,800,死亡人数位居男性恶性肿瘤致死人数第三位。在我国,随着人口老龄化形势的加剧,前列腺癌的发病率逐年上升。根据国家癌症中心数据,2009年我国前列腺癌发病率为9.92/10万；。'Cancer Statistics in China,2015"最新数据显示,2015年我国前列腺癌发病率激增至60.3/10万,死亡率达26.6/10万。前列腺癌已成为威胁中国男性健康的主要疾病之一。目前,根治性前列腺切除术和外放射治疗是治愈局限性前列腺癌的主要方法。但是,对于已有远处转移、不适宜手术或治愈性治疗后复发(或转移)等情况,通常选择内分泌治疗。研究表明,80%以上的患者可通过雄激素剥夺治疗获得疾病的缓解,但经过14-30个月的中位时间后,几乎所有患者的病变都将逐渐发展为去势抵抗性前列腺癌(Castration-Resistant Prostate Cancer, CRPC)。患者一旦发生去势抵抗和远处转移,预后较差。虽然多种药物包括多西紫杉醇、阿比特龙、恩杂鲁胺等可用于CRPC的治疗,但其疗效仍然有限。CRPC已成为晚期前列腺癌患者主要的死亡原因。因此,探讨前列腺癌的进展机制,寻找新的分子靶点,对延缓疾病进程和降低晚期肿瘤的死亡率具有重大作用。过去的十年里,人们发现在前列腺癌的恶性进程中有大量的表观遗传分子的异常改变。Polycomb repressive complex2 (PRC2复合体)是由多个蛋白组合而成的表观调控复合体。EZH2是PRC2复合体的重要催化亚基。它含有一个活性的SET催化结构域,该结构域内的甲基转移酶可以被外源甲基基团识别并通过共价反应的形式结合,催化组蛋白H3的第9和27位赖氨酸发生三甲基化(H3K9me3和H3K27me3)。研究表明,EZH2需要和EED和SUZ12两个PRC2家族成员协同完成以上酶促反应。此外,PRC2的另外两个核心成员RBBP4和RBBP7可以直接与组蛋白结合,通过催化甲基化抑制基因的转录活性。近年来,研究发现EZH2在乳腺癌、膀胱癌、胃癌、肺癌、肝癌等肿瘤组织中异常高表达且预示着较差的预后。在前列腺癌相关研究中,人们发现EZH2在转移的前列腺癌组织中较局限性前列腺癌异常高表达。进一步研究发现,EZH2通过催化H3K27me3,沉默ADRB2和CDH1基因的表达,从而促进肿瘤细胞的侵袭能力。然而,有关EZH2在前列腺癌进展过程中不同阶段和不同病理级别的表达情况和潜在的生物学功能,以及EZH2是否参与调控与前列腺癌进展相关的非编码RNA信号通路仍需进一步深入探索。本项研究综合运用临床大样本分析、生物信息学、基因芯片、细胞功能实验、分子生物学实验及动物在体实验等手段深入研究EZH2在前列腺癌进展中的生物学行为及其潜在的分子机制。在本课题第一部分中,我们首先借助GEO数据库和课题组前期收集的前列腺正常和肿瘤组织样本,从RNA表达和蛋白表达两个层面分析EZH2在正常前列腺组织和前列腺癌组织中的表达情况；并进一步细化EZH2在前列腺癌不同病理级别和不同进展阶段的表达情况,同时利用随访数据分析EZH2与前列腺癌患者预后之间的关系。随后,运用RNA干扰手段下调前列腺癌细胞中的EZH2,通过流式细胞仪、酶标仪、克隆形成实验、XF糖酵解压力测试盒及裸鼠在体成瘤等实验技术检测EZH2对前列腺癌细胞周期、增殖、糖代谢、成瘤等生物学行为的影响。结果显示：1)与前列腺正常组织和良性增生组织相比,EZH2在前列腺癌组织中的表达显著上升：且EZH2在去势抵抗性前列腺癌组织中的表达显著高于激素依赖性前列腺癌；2)EZH2在Gleason7分的前列腺癌组织中的表达显著高于Gleason≤7的前列腺癌组织。3)体外细胞功能实验表明,利用EZH2-shRNA敲低EZH2的表达后,前列腺癌细胞的增殖能力和克隆形成能力受到明显抑制,细胞周期阻滞于G1期,细胞的有氧糖酵解水平明显下降。4)裸鼠皮下成瘤实验表明,EZH2敲低组的肿瘤生长速率较对照组明显减慢；至观察终点,EZH2敲低组的肿瘤体积显著小于对照组。微小RNA(microRNAs, miRNA)近10年来始终是肿瘤研究领域的热点。miRNA的信号传递体系包括多种形式,其本身接受上游转录调控分子的信号,向下游则可通过识别特定的目标mRNA的3’端非编码区域(3'untranslated region,3’UTR),在转录后水平通过促进靶mRNA的降解和(或)抑制翻译过程而发挥调控基因表达的作用。随着系统生物学研究的开展,miRNA的研究也从单一功能走向整体综合的水平。大量报道证实miRNA表达谱特征与肿瘤的诊断、分期、分级、进展、预后及治疗存在相关性,是肿瘤基因调控网络中的重要枢纽分子。作为转录抑制因子,EZH2可沉默下游诸多靶基因,而miRNA也是一大类可被EZH2调控的靶向分子。在本课题第二部分中,我们首先利用基因表达谱芯片数据预测可能参与EZH2调控细胞周期和代谢的相关基因,结果显示,E2F3和HK2两个基因与EZH2的表达显著正相关；随后,借助miRNA表达谱芯片技术筛选EZH2相关miRNA差异谱。结果显示,EZH2敲低后miR-200和miR-181两个家族中的部分成员表达显著上升。利用蛋白免疫印迹和荧光定量PCR技术检测发现,EZH2敲低后H3K27Me3的表达水平显著降低,miR-200c和miR-181b的表达明显上调；荧光素酶报告基因实验证实EZH2敲低后miR-200c和miR-181b基因启动子区域的转录活性增强。进一步通过生物信息学预测、蛋白免疫印迹和荧光素酶报告基因实验发现,E2F3和HK2分别是miR-200c和miR-181b的直接靶基因,提示miR-200c和miR-181b可以介导EZH2对E2F3和HK2的正向调控作用。为了进一步验证EZH2对前列腺癌细胞周期和糖代谢的影响是通过miR-200c和miR-181b介导所实现的,我们设计了相应的回复实验。在对照组和EZH2敲低组的基础上增加sh-EZH2+As-miR-200c组和sh-EZH2+As-miR-181b组。结果显示sh-EZH2+As-miR-200c组较sh-EZH2单独转染组的细胞周期进程出现回复效应,且sh-EZH2引起的E2F3表达下降可以被As-miR-200c部分逆转；另一方面,sh-EZH2+As-miR-181b组较sh-EZH2单独转染组的细胞糖酵解能力也显示回复效应,且sh-EZH2引起的HK2表达下降可以被As-miR-181b部分逆转；再次证明miR-200c和miR-181b是EZH2对细胞周期和糖代谢调控作用的中介分子。综上所述,本课题综合运用临床大样本、生物信息学分析、细胞功能检测、高通量筛选、miRNA/mRNA和蛋白/DNA互作检测(荧光素酶报告基因)、荧光定量PCR等方法,对前列腺癌细胞中EZH2的功能及机制进行了系列探索。研究发现EZH2能够调节前列腺癌细胞增殖、周期、糖代谢等生物学行为,并且完成了EZH2/miRNAs信号轴及其下游信号通路的实验构建。这些结果为后续深入研究miRNAs介导下的EZH2调控分子网络奠定了基础,也为寻找前列腺癌分子诊断和预后评估的生物靶点和探索前列腺癌发生及演进机制并优化治疗策略提供了理论依据。
[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.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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