TIF1γ介导丙戊酸钠抑制前列腺癌上皮间充质转化的机制研究

发布时间：2018-06-02 07:28

本文选题：前列腺癌 + VPA　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景前列腺癌是全球范围内男性第二大恶性肿瘤,我国前列腺癌的发病率近十年来逐年增长。在我国,超过40%的前列腺癌患者确诊时已经进入中晚期,远远高于欧美水平。雄激素剥夺治疗(Androgen deprivation therapy,ADT)是国内外晚期前列腺癌的主要治疗方式,但其有效期一般仅能维持18-24个月。在ADT治疗失效之后,疾病将发展为去势抵抗性前列腺癌(Castrateresistant prostate cancer,CRPC),部分患者的肿瘤甚至进一步转移至前列腺以外的其它器官。针对转移性去势抵抗性前列腺癌虽然可以选择新型雄激素生物合成酶抑制剂治疗,但作用有限。因此,寻求更多更有效的治疗方法是目前研究的方向。上皮间充质转化(Epithelial-mesenchymal transition,EMT)在肿瘤的发生发展过程中起到重要的调控作用,在肿瘤细胞的EMT过程中,上皮细胞逐渐失去了本身的特性,侵入上皮基底膜并通过进入血液循环系统获得了浸润周围组织或远处转移的能力。除了失去胞间链接外,发生EMT的细胞逐渐失去了其细胞极性,上皮标记物诸如E钙黏蛋白减少,而N钙黏蛋白等间充质细胞标记物逐渐增多,因此,具有此种特性的细胞更易向邻近组织浸润性生长或发生远处转移。肿瘤相关的EMT并不是单一的过程,肿瘤细胞除获得了迁移和侵袭能力外,还具备了复杂且全面地参与表观遗传重组、新陈代谢和分化等能力。在前列腺癌病人肿瘤进展过程中,EMT在前列腺癌由原位细胞向转移性细胞的转化发展过程中起到了非常重要的作用,此类前列腺癌细胞失去了本身的上皮特性并获得了间充质细胞的特性。EMT同样也促进了去势抵抗性前列腺癌的发展过程。EMT过程受体内多个信号通路的调节,其中转化生长因子β(Transforming growth factor β,TGF-β)信号通路起主要作用,SMAD4 蛋白作为TGF-β信号通路信号传递过程中重要的载体蛋白,被认为是前列腺癌的促癌因子。E3 泛素连接酶-转录中介因子 1γ(Transcriptional intermediary factor1 γ,TIF1 γ)被认为通过单泛素化修饰SMAD4蛋白,促使其与磷酸化的SMAD3蛋白解离,参与了 TGF-β/SMAD蛋白信号系统的调节过程。本课题组的前期相关研究显示,丙戊酸钠(Valproic acid,VPA)通过抑制SMAD4蛋白的表达逆转了前列腺癌EMT过程。在本课题中,我们收集了山东省立医院泌尿微创中心2012年以来收治的20例接受腹腔镜前列腺癌根治术患者的前列腺癌组织及癌旁组织,应用免疫组化技术分析SMAD4及TIF1 γ蛋白在前列腺癌患者与正常人之间的不同表达水平以及各蛋白与临床病理变量的关系;体外实验部分,我们运用了免疫组化、免疫共沉淀、免疫印迹等技术,证实了 TIF1γ对SMAD4蛋白的单泛素化修饰作用是介导VPA抑制前列腺癌EMT过程的具体分子机制。研究目的1.应用生物信息学分析前列腺癌相关蛋白在前列腺癌患者与正常人之间的不同表达水平以及各蛋白与临床病理变量的关系。2.制作20例前列腺癌组织及其癌旁组织标本的石蜡切片,应用免疫组化技术检测SMAD4及TIF1 γ蛋白的表达情况。3.体内、外实验部分,分别培养PC3细胞及LNCaP细胞,构建荷瘤小鼠模型,并用VPA处理,检测TIF1 γ、SMAD4及EMT过程中相关标记蛋白在不同处理组的表达量,探讨VPA抑制前列腺癌EMT过程的具体机制。研究方法1.应用石蜡包埋切片技术处理临床采集的20例前列腺癌患者的前列腺癌组织及癌旁组织标本,应用免疫组化技术检测TIF1γ、SMAD4蛋白在不同组织标本中表达量的差异,结合患者随访资料,应用统计学方法分析目的蛋白在前列腺癌患者与正常人之间不同表达水平以及各蛋白与临床病理变量的关系,探讨目的蛋白的表达与患者预后的关系,以期寻找新的前列腺癌预后指标和潜在的治疗靶点。2.采用免疫印迹、免疫共沉淀及免疫组化等分子生物学技术检测前列腺癌细胞及荷瘤小鼠瘤体在VPA处理组与对照组中相关蛋白的表达情况,探讨VPA调控TIF1 γ抑制前列腺癌细胞EMT过程及其与单泛素化SMAD4蛋白之间的关系。研究结果1.VPA升高了野生型PC3/LNCaP细胞内单泛素化SMAD4蛋白水平,但无法影响SMAD4点突变的PC3/LNCaP细胞内单泛素化SMAD4蛋白的水平。2.VPA通过调控TIF1 γ蛋白抑制前列腺癌细胞EMT过程。3.VPA抑制了荷瘤小鼠瘤体的生长。4.前列腺癌组织内TIF11γ与SMAD4蛋白含量呈负相关,免疫组化结果显示前列腺癌组织内TIF1 γ含量明显低于癌旁组织,SMAD4含量与上述结果相反。5.体内实验结果显示,相较于对照组,VPA处理组中TIF1γ表达量升高,而SMAD4蛋白表达量下降。研究结论VPA通过加速SMAD3/SMAD4复合物的核内解离过程,降低核内SMAD3/SMAD4复合物含量,从而抑制前列腺癌细胞EMT过程,TIF1γ对SMAD4的单泛素化修饰作用是VPA扣动这一过程的"扳机"。研究意义本研究证实了 TIF1 γ介导VPA抑制前列腺癌EMT过程的具体分子机制,旨在证明VPA治疗进展期前列腺癌的可能性,丰富了进展期前列腺癌的治疗策略,并为临床用药提供了较为详实的统计学依据。
[Abstract]:Background prostate cancer is the second major male malignant tumor around the world. The incidence of prostate cancer in China has increased year by year in the past ten years. In China, more than 40% of the prostate cancer patients have entered the middle and late stages, far higher than the European and American levels. Androgen deprivation therapy (Androgen deprivation therapy, ADT) is advanced at home and abroad. The main treatment of adenocarcinoma, but the duration of its validity is generally only 18-24 months. After the failure of ADT treatment, the disease will develop to Castrateresistant prostate cancer (CRPC), some of the patients may even be further transferred to other organs other than the prostate. Although adenocarcinoma can choose the new androgen biosynthesis inhibitor therapy, the effect is limited. Therefore, it is the current research direction to seek more and more effective treatment methods. Epithelial-mesenchymal transition (EMT) plays an important role in the development of tumor, and the EMT over the tumor cells is over. During the process, the epithelial cells gradually lose their own characteristics, invade the epithelial basement membrane and get the ability to infiltrate the surrounding tissues or distant metastases through the blood circulation system. In addition to the loss of intercellular links, EMT cells gradually lose their cell polarity, epithelial markers such as E calcium mucin and N calcium mucin. As a result, the cells with this characteristic are more susceptible to infiltrating or distant metastasis to adjacent tissues. The tumor related EMT is not a single process. In addition to the ability to migrate and invasiveness, the tumor cells have a complex and comprehensive participation in epigenetic reorganization, metabolism and differentiation. In the process of cancer progression in patients with prostate cancer, EMT plays a very important role in the transformation and development of prostate cancer from in situ cells to metastatic cells. This kind of prostate cancer cells lose their own epithelial properties and obtain the characteristics of mesenchymal cells.EMT, which also promote the development of castration resistance to prostate cancer. There are several signaling pathways within the.EMT process receptor, in which TGF beta (Transforming growth factor beta, TGF- beta) signaling pathway plays a major role, and SMAD4 protein is an important carrier protein in the signaling process of TGF- beta signaling. It is considered to be a cancer promoting factor.E3 ubiquitin ligase transcription mediator for Prost adenocarcinoma. Factor 1 gamma (Transcriptional intermediary factor1 gamma, TIF1 gamma) is considered to modify the SMAD4 protein by single ubiquitination to dissociate the phosphorylated SMAD3 protein and participate in the regulation process of the TGF- beta /SMAD protein signal system. Previous studies in this group showed that the sodium valproate (Valproic acid, VPA) could inhibit the SMAD4 protein. The expression reverses the EMT process of prostate cancer. In this subject, we collected 20 cases of prostate cancer tissue and para cancer tissue in patients with laparoscopic radical prostatectomy since 2012 in the urinary minimally invasive center of Shangdong Province-owned Hospital. The immunohistochemical technique was used to analyze the SMAD4 and TIF1 gamma protein in patients with prostate cancer and normal people. The relationship between different levels of expression and the relationship between the protein and the clinicopathological variables; in vitro experiment part, we used immunohistochemistry, immunoblotting and immunoblotting techniques to confirm that the single ubiquitination modification of TIF1 gamma to SMAD4 protein is a specific molecular mechanism to mediate the EMT process of prostate cancer by VPA. Objective 1. application of Biology Informatics analysis of the different expression levels of prostate cancer related proteins in the prostate cancer patients and normal people and the relationship between the protein and the clinicopathological variables.2. made paraffin sections of 20 cases of prostate cancer tissue and its paracancerous tissue specimens. The expression of SMAD4 and TIF1 gamma protein was detected by immunohistochemical technique in.3.. The PC3 cells and LNCaP cells were cultured, and the tumor bearing mice model was constructed, and the expression of related marker proteins in the process of TIF1 gamma, SMAD4 and EMT in different treatment groups was detected by VPA treatment. The specific mechanism of VPA inhibition of EMT process in prostate cancer was investigated. Method 1. the study method of paraffin embedded section was used to deal with the 20 cases of clinical collection. The differences in the expression of TIF1 gamma and SMAD4 protein in different tissue specimens were detected by immunohistochemical technique in the prostate cancer tissues and para cancer tissues of the patients with adenocarcinoma. The different expression levels of the target protein in the patients with prostate cancer and the normal people were analyzed by the statistical method, and the protein and Clinicopathology were analyzed by the statistical method. The relationship between the expression of the variable and the relationship between the expression of the target protein and the prognosis of the patient in order to find a new prognostic indicator and a potential therapeutic target for prostate cancer,.2. using immunoblotting, immunoblotting and immunohistochemistry to detect the related proteins in the VPA treatment group and the control group in the tumor bearing mice and the tumor bearing mice. The relationship between VPA regulation of TIF1 gamma inhibition of EMT process and the relationship with mono ubiquitin SMAD4 protein in prostate cancer cells was investigated. Results 1.VPA increased the level of ubiquitin SMAD4 protein in wild PC3/LNCaP cells, but could not affect the level of mono ubiquitin SMAD4 protein in PC3/LNCaP cells of SMAD4 point mutation. The inhibitory effect of TIF1 gamma protein on the EMT process of prostate cancer cell.3.VPA inhibits the growth of the tumor of tumor bearing mice and the TIF11 gamma content in the.4. prostate cancer tissue is negatively correlated. The immunohistochemical results show that the content of TIF1 gamma in the prostate cancer tissue is significantly lower than that in the para cancerous tissue, and the SMAD4 content is contrary to the results in the experimental results of the above results. Compared to the control group, the expression of TIF1 gamma in the VPA treatment group increased and the expression of SMAD4 protein decreased. Conclusion VPA can inhibit the EMT process in the prostate cancer cells by accelerating the intra nuclear dissociation process of SMAD3/SMAD4 complex and thus inhibiting the EMT process in the prostate cancer cells. The single ubiquitination modification of TIF1 gamma is the process of VPA deduction. The study confirms the specific molecular mechanism of TIF1 gamma mediated VPA inhibition of prostate cancer EMT process, which aims to prove the possibility of the treatment of advanced prostate cancer by VPA, enriching the treatment strategy of advanced prostate cancer and providing a more detailed statistical basis for clinical use of drugs.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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