Smad4介导的丙戊酸钠（VPA）抑制肾细胞癌上皮间质转化（EMT）的机制研究

发布时间：2018-09-06 16:07

【摘要】：[目的]肾细胞癌在全世界是非常常见的肾恶性肿瘤之一,在人肿瘤中占2-3%,透明细胞癌是最常见的类型。尽管当前医学诊疗技术的快速提高,特别是影像学诊断鉴别技术的高速发展,肾肿瘤患者的预后仍然不良。相比较非转移肾肿瘤患者的肿瘤特异性存活率(70%),转移性肾肿瘤患者5年肿瘤特异性存活率仅为27.1%,而且肾透明细胞癌倾向于转移。令人遗憾的是,肾肿瘤的转移的机制和对治疗抵抗性,我们现在仍然没有完全清楚,这里仍然需要更多研究和关注。上皮间质转化(EMT),是组织中表皮细胞向间皮细胞转换的病理生理过程,在肿瘤向邻近和远处组织器官侵袭转移过程中发挥着非常重要的作用。发生EMT过程中,表皮细胞失去它的细胞极性,以及细胞和细胞之间的紧密连接,进而获得间皮细胞的特征,使肿瘤细胞具有较强的侵袭转移能力,转移播散至远处的脏器器官。这个现象过程由肿瘤微环境中的多个复合体和生长因子所引起,TGF-β就是这样一个在肿瘤侵袭转移过程中具有促进和抑制双重作用的因子。已有研究关注TGF-β在肿瘤发生EMT的不同阶段发挥着的重要作用,但其在EMT初始反应过程的作用仍存在未明确之处。Smad4被认为是TGF-β通路和BMP信号通路的关键中间传导蛋白,研究显示Smad4蛋白的缺失使TGF-β经典的信号通路失去功能,而TGF-β从早期的肿瘤抑制作用到晚期的肿瘤促进作用。通过TGF-β信号通路的经典和非经典的途径,TGF-β因子发挥着潜在的诱导形成EMT的作用,Smad4蛋白可以作为肾肿瘤患者预后的独立因子。组蛋白乙酰化和去乙酰化是组蛋白表观学修饰一种,调控基因表达的一种机制。组蛋白乙酰化转移酶和去乙酰化酶发挥重要作用,伴随着组蛋白的乙酰化和去乙酰化,从而影响着肿瘤基因表达的沉默和激活。VPA属于Ⅰ类HDAC类抑制剂,数十年来作为经典的抗癫痫药物用于临床,研究发现VPA还具有多种的抗肿瘤潜能,具有抑制肿瘤细胞的增殖,生长和分化,以及肾癌细胞的周期,凋亡。根据目前的研究,VPA具有调控Smad4的表达和抑制肿瘤的侵袭转移的能力,鉴于Smad4在EMT过程中的重要作用,我们认为VPA可能通过调节Smmad4蛋白,进而抑制EMT过程,达到抑制肾肿瘤的侵袭转移的调控作用。[方法]:肾癌细胞系给以不同浓度、不同作用时间的VPA刺激,利用实时定量PCR技术和免疫蛋白印迹技术验证VPA作用后Smad4和EMT相关指标的改变。以过表达Smad4质粒和小干扰RNA转染肾肿瘤细胞786-0,通过免疫蛋白印记技术实验检测在上调或下调Smad4水平后,786-0细胞EMT相关蛋白指标的改变。利用免疫组化技术检测省立医院东院病理科,39例肾透明细胞癌临床标本中,Smad4,TIF1-γ和TGF-β蛋白表达量差异。[结果]:根据VPA调控EMT相关转录因子,我们的研究指出VPA抑制肾癌的EMT过程是通过改变Smad4的表达水平,Smad4经过TGF-β1的诱导,形成Smad3/4复合物从而诱导肿瘤EMT过程,从而可作为抗肿瘤的靶点抑制肾癌细胞的侵袭能力。
[Abstract]:[Objective] Renal cell carcinoma (RCC) is one of the most common renal malignancies in the world, accounting for 2-3% of all human tumors, and clear cell carcinoma is the most common type. The tumor-specific survival rate (70%) of patients with metastatic renal tumors was only 27.1% at 5 years, and clear-cell renal carcinoma tended to metastasize. Unfortunately, the mechanisms of metastasis and resistance to treatment of renal tumors are still not fully understood. More research and attention are needed here. Transforming (EMT) is a pathophysiological process in which epidermal cells transform into mesothelial cells. It plays an important role in the invasion and metastasis of tumor to adjacent and distant tissues and organs. This phenomenon is caused by multiple complexes and growth factors in the tumor microenvironment. TGF-beta is such a factor that can promote and inhibit tumor invasion and metastasis. Smad4 is considered to be a key intermediate transduction protein in the TGF-beta pathway and BMP signaling pathway. Studies have shown that the deletion of Smad4 proteins disrupts the classical signal pathway of TGF-beta and TGF-beta is suppressed by early tumors. Through the classical and non-classical pathways of the TGF-beta signaling pathway, TGF-beta plays a potential role in inducing the formation of EMT. Smad4 protein can be used as an independent prognostic factor in patients with renal tumors. Histone acetylation and deacetylation are epigenetic modifications of histones that regulate gene expression. Histone acetyltransferases and deacetylases play an important role, accompanied by histone acetylation and deacetylation, which affect the silence and activation of tumor gene expression. VPA belongs to class I HDAC inhibitors. For decades, VPA has been used as a classical antiepileptic drug in clinic. It has been found that VPA also has a variety of anti-swelling properties. According to current research, VPA has the ability to regulate the expression of Smad4 and inhibit the invasion and metastasis of tumor. Considering the important role of Smad4 in the process of EMT, we believe that VPA may inhibit the EMT process by regulating the Smmad4 protein. [Methods] Renal cancer cell lines were stimulated by VPA at different concentrations and at different time of action. The changes of Smad4 and EMT related indexes after VPA treatment were verified by real-time quantitative PCR and Western blotting. Smad4 plasmid and small interfering RNA were overexpressed and transfected into renal tumor cells 786-0. Immunohistochemistry was used to detect the expression of Smad4, TIF1-gamma and TGF-beta in 39 cases of renal clear cell carcinoma (RCC) in the Department of Pathology, East Hospital of Provincial Hospital. Our study indicates that VPA inhibits the EMT process of renal cell carcinoma by altering the expression of Smad4. Smad4 is induced by TGF-beta 1 to form Smad3/4 complex, which induces the EMT process of tumor, and can be used as an anti-tumor target to inhibit the invasion of renal cell carcinoma.
【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】：R737.11

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