RanBP3L的磷酸化机制及其功能研究

发布时间：2018-04-19 19:33

  本文选题：癌症 + 细胞表皮间质转化　； 参考：《浙江大学》2017年硕士论文

【摘要】：癌症、心血管疾病、神经性疾病是威胁人类生命健康最主要的三大病症。癌症的研究是目前生物医学领域的热点。癌症是由外在因素和内在因素共同作用所引发的疾病。外在因素包括辐射、紫外线、有毒物质、抽烟、酒精等,内在因素包括DNA突变,染色体异常,抑癌基因失活,原癌基因异常活化等。在内外因素的作用下,造成细胞异常生长,在组织表面形成肿瘤。早期形成的肿瘤是良性肿瘤,发展到后期,会变成恶性肿瘤。在癌细胞转移前期,一群肿瘤表皮细胞发生表皮间质转化,转变成间充质类细胞,这群细胞逃脱胞外基质的限制,穿过毛细血管,并通过淋巴或血液系统的循环转移到其他器官,最后少数细胞发生间质表皮转化,重新变成表皮细胞,在新的位置形成新的肿瘤,即转移灶。癌细胞转移,意味着癌症已经进入中后期,病人的生存时间所剩无几。因此,表皮间质转化所引发的癌细胞转移是癌症发展过程中的重要一环。表皮间质转化(Epithelial-Mesenchymal Transition,EMT)是良性肿瘤向恶性癌症发展过程中的重要步骤。目前已知导致EMT发生的重要转录因子包括Snail、Slug、TWIST、锌指结构蛋白等。在EMT发生过程中,细胞失去表皮细胞的特性,获得间充质细胞的特性。具体表现为细胞形态变成梭形,细胞间结构变得松散,细胞失去极性。表皮细胞的标志蛋白如E-钙黏素(E-cadherin)、β-连环素(β-catenin)、闭合蛋白(Occludin)等减少,间充质细胞的标志蛋白如N-钙黏素(N-cadherin)、波形蛋白(Vimentin)、纤连蛋白(Fibronectin)等增加。实验室早期研究发现BMP(Bone morphogenetic protein)信号通路中的转运蛋白 RanBP3L(Ran-binding Protein 3 Like)能把蛋白 Smad1/5/8 从细胞核内转运到细胞质中,在此基础上,我们准备深入研究RanBP3L的其他功能。查找文献资料,我们发现 RanBP3L 的类似蛋白 RanBP3(Ran-binding Protein 3)能被 Akt/PKB 磷酸化,通过序列比对,我们发现RanBP3L同样具有Akt的识别的序列72RVRSSS77。实验确认RanBP3L的S77位点能被Akt磷酸化,并且该磷酸化修饰能被磷酸酶PPM1A去磷酸化。在乳腺上皮细胞MCF10A中稳定表达RanBP3L-WT,我们发现细胞形态变成梭形,细胞间结构变得松散,表现出间充质细胞的特性。表达RanBP3L-S77D时,间充质细胞的特性变得更加明显。表达RanBP3L-S77A时,与对照组接近,仍具有表皮细胞的特性。综上所述,我们的实验确认了 RanBP3L的S77位点能被激酶Akt磷酸化,该磷酸化修饰能被磷酸酶PPM1A去磷酸化调控。并且发现RanBP3L能促进细胞发生表皮间质转化,其作用效果与磷酸化状态有关。
[Abstract]:Cancer, cardiovascular disease and neuropathy are the three most important diseases threatening human life and health. The research of cancer is a hot spot in the field of biomedicine. Cancer is a disease caused by both external and internal factors. External factors include radiation, ultraviolet radiation, toxic substances, smoking, alcohol and so on. Intrinsic factors include DNA mutation, chromosomal abnormality, inactivation of tumor suppressor gene, abnormal activation of proto-oncogene and so on. Under the action of internal and external factors, the cells grow abnormally and form tumor on the tissue surface. Early tumors are benign tumors that develop into malignant tumors later in life. In the early stages of cancer cell metastasis, a group of tumor epidermal cells undergo epidermal interstitial transformation into mesenchymal cells, which escape the restriction of extracellular matrix and pass through the capillaries. Through the circulation of lymphatic or blood system to other organs, finally a few cells develop interstitial epidermis transformation, then become epidermal cells again, and form new tumor in the new position, that is, metastasis focus. Cancer cell metastasis means that cancer has entered the middle and late stages of the patient's survival time is running out. Therefore, the metastasis of cancer cells induced by epidermal interstitial transformation is an important link in the development of cancer. Epithelial-mesenchymal transition (EMTT) is an important step in the development of benign tumor to malignant cancer. The important transcription factors that have been known to cause the occurrence of EMT include Snail Slug TWIST, zinc finger structural protein and so on. During the development of EMT, the cells lost the characteristics of epidermal cells and obtained the characteristics of mesenchymal cells. Cell morphology becomes fusiform, intercellular structure becomes loose and cells lose polarity. The marker proteins of epidermal cells such as E-cadherin, 尾 -cateninine (Occludinin) were decreased, and those of mesenchymal cells such as N-cadherinine, vimentin and fibronectin were increased. In early laboratory studies, it was found that the transporter RanBP3L(Ran-binding Protein 3 in the BMP(Bone morphogenetic protein signaling pathway can transport Smad1/5/8 from the nucleus to the cytoplasm. On the basis of this, we are prepared to further study the other functions of RanBP3L. We found that RanBP3(Ran-binding Protein 3, a similar protein of RanBP3L, could be phosphorylated by Akt/PKB. By sequence alignment, we found that RanBP3L also had Akt recognized sequence 72 RVRSSS77. The S77 site of RanBP3L can be phosphorylated by Akt, and the phosphorylation modification can be dephosphorylated by phosphatase PPM1A. The stable expression of RanBP3L-WTin in MCF10A of breast epithelial cells was observed. We found that the morphology of the cells became fusiform and the intercellular structure became loose, showing the characteristics of mesenchymal cells. When RanBP3L-S77D was expressed, the characteristics of mesenchymal cells became more obvious. The expression of RanBP3L-S77A was similar to that of the control group and still had the characteristics of epidermal cells. In conclusion, our experiment confirmed that the S77 site of RanBP3L can be phosphorylated by kinase Akt, and the phosphorylation modification can be regulated by dephosphorylation of phosphatase PPM1A. It was also found that RanBP3L could promote epidermal mesenchymal transformation, and its effect was related to phosphorylation state.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.2
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本文编号：1774421