| Prostate cancer is a common malignant tumor in male genito-urinary system. Its incidence has obvious geographical and ethnic differences.In the United States, prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer mortalities in men. As reported by"cancer statistics,2016", in the United States, approximately 180,890 men will be diagnosed with prostate cancer in the year of 2016. Moreover, about 26,120 patients will died from prostate cancer in this year. In the European, about 75,800 patients will died from prostate cancer in 2016. Its mortality will rank the third in all male malignant tumor.Due to populations age, the morbidity of prostate cancer increases in our country year by year. According to the data from national cancer center, the incidence of prostate cancer was 9.92/100000 in the year of 2009. As reported by"Cancer Statistics in China,2015",its incidence and mortality rate in China increased to 60.3/100000 and 26.6/100000,respectively.It has become a major health concern in the older male population in our country. At present, radical prostatectomy and external radiation therapy are main methods of cure for localized prostate cancer. Additionally, hormonal therapy is also an important treatment method for some complicated cases such as patients with distant metastasis or recurrence after treatment.More than 80% of the patients can alleviate disease through androgen deprivation therapy. But after 14-30 months, almost all patients with lesions will be gradually transform to castration resistant prostate cancer(CRPC), which leads to very poor prognosis. Although a variety of drugs are use for CRPC patients, including docetaxel, abiraterone, and enzalutamide etc, their curative effects are still limited. CRPC has become the main cause of death in patients with advanced prostate cancer. It is necessary to further study tumor progression mechanism and novel molecular targets.Over the past decade it has been discovered that a wide spectrum of genetic as well as epigenetic aberrations are associated with prostate cancer development, many of which are connected to EZH2 dysregulation.EZH2 is the catalytic member of the PRC2 complex.It comprises a SET domain which is recognized as the signature of methyltransferases as it provides the active site for the covalent methylation reaction, resulting in trimethylation (me3) of histone 3 (H3) at lysine 27 (K27), as well as at lysine 9 (K9) albeit to a much lesser extent.EZH2 alone exhibits no intrinsic enzymatic function, in order to be catalytically active it must interact with at least two proteins, embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12).These three proteins, with the addition of the histone binding proteins retinoblastoma binding protein 4 (RBBP4) and RBBP7, form the core components of PRC2.In recent years, studies find that EZH2 is dysregulated in breast cancer, bladder cancer, gastric cancer, lung cancer, liver cancer and so on, which indicate poor prognosis. People also find there is higher expression in metastatic prostate cancer tissues than in localized ones. The study finds that EZH2 promote the invasive ability of tumor cells through catalyzing H3K27me3 and then inhibiting the expression of ADRB2 and CDH1. However, it is not clear the expression of EZH2 in prostate cancer of different stages and different pathological levels. In addition, we do not know the potential biological function of EZH2 and its relationship with non-coding RNA signaling pathways. In this study, we further explore EZH2’s biological behavior in prostate cancer progression and its potential molecular mechanisms by a series of methods including clinical sample analysis, bioinformatics, microarray, cell function experiments, molecular biology experiments and animal experiments in vivo.In the first part of this project, we employed the database of GEO and clinical samples from our hospital to analyze the expression of EZH2 in normal prostate tissues and prostate cancer tissues in different stages and different pathological levels, and then focused on the relationship between EZH2 and patient’s outcome.In addition, we downregulated EZH2 of prostate cancer cells respectively, and estimated the degree to which EZH2 exerts influence on cell cycle, proliferation, glucose metabolism capacity, and colony formation etc. The result indicated that 1) EZH2 expression was elevated in prostate cancer tissues compared to normal prostate tissues. Further, EZH2 expression significantly increased in CRPC than androgen dependent prostate cancer (ADPC).2) EZH2 expression was significantly elevated inGleason>7 prostate cancer tissues compared to Gleason≤7 ones.3) EZH2 depletion inhibited proliferation, colony formation, cell cycle process, aerobic glycolysis level in prostate cells.4) EZH2 depletion significantly slowed tumor growth rate than the control group in nude mouse tumor xenograft experiment.MicroRNAs (miRNAs) have been attracting increasing attention in the field of tumor research for the past decade. It receives transcription regulation of molecular upstream, and passes the signals through targeting specific mRNA 3’-untranslated region(3’UTR), which is regarded as post-transcriptional regulation of gene expression. With the development of systematic biology, the research on miRNA evolves as well, upgrades from single functional to a holistic level. A large number of reports demonstrate that unique miRNA expression profile is relevant to the diagnosis, staging, grading, progression, and prognosis of tumor, functioning as a significant hub molecular in the network of tumor regulation. As a transcription repressors, EZH2 is able to silence numerous genes, to which miRNA belongs.In the second part of this project, we first used gene expression profile microarray to analyze the metabolism and cell cycle related genes which were correlated with EZH2.The results showed that the expression of E2F3 and HK2 was positively correlated with EZH2 expression. Then, EZH2 related miRNAs were screened by miRNA expression profile microarray.The results indicated that EZH2 depletion increased the expression of some miR-181 and miR-200 family members. Through western blot and quantitative PCR technology, we demonstrated that reduction of EZH2 inhibited H3K27Me3 expression, followed by upregulation of miR-181b and miR-200c. Luciferase reporter assays showed that EZH2 inversely modulated miR-181b and miR-200c at transcriptional level. Further study indicated that E2F3 and HK2 were direct targets of miR-200c andmiR-181b respectively. Moreover, decreased miR-200c expression largely abrogated the effect of sh-EZH2 on E2F3 expression and E2F3-induced cell cycle progression. Similarly, the effect of sh-EZH2 on glucose metabolism was abrogated when miR-181b expression was decreased. Moreover, Western blot assay showed that the cells co-transfected with miR-181b ASO and sh-EZH2 significantly increased HK2 expression in comparison with the single sh-EZH2 group. These results indicate that miRNAs serve as important mediators between EZH2 and downstream pathways.In overview, this project applied clinical sample analysis, high throughput screening (miRNA microarray)、bioinformatics, miRNA/mRNA and protein/DNA interaction test (luciferase reporter gene test)、real-time PCR etc to explore the function and mechanism of EZH2 in prostate cancer systematically. We discover that EZH2 is able to regulate cell cycle and glucose metabolism in prostate cancer, acquire the EZH2 related miRNA profile and construct EZH2/miRNAs axis and its downstream pathway in prostate cancer. All these accomplishments establish a foundation for the further research on miRNA mediated EZH2-related molecule network. We look forward to revealing the network model, providing novel targets and contributing tothe optimizing treatment strategies for molecular targeted therapy in prostate cancer. |
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