SNAIL泛素化调节在肿瘤发生发展中的作用研究

发布时间：2018-05-24 19:39

本文选题：SPSB3 + SNAIL　；参考：《北京协和医学院》2016年博士论文

【摘要】：上皮间质转换(Epithelial-Mesenchymal Transition,EMT)在肿瘤的发生与转移过程中发挥着重要的作用,它赋予肿瘤上皮细胞间质样的特性,使得肿瘤细胞具有更强的运动能力和侵袭能力。SNAIL是EMT转变过程中最重要的调控因子,在肿瘤的发生发展中发挥了关键的调节作用,因此它的表达受到转录水平、翻译水平和翻译后水平等多方面的精密调控。在翻译后水平,SNAIL主要受到泛素化的修饰。多聚泛素化调节了SNAIL通过泛素-蛋白酶体途径的降解。而具有底物特异性的E3泛素连接酶是调节SNAIL多聚泛素化与蛋白稳定性的关键因子。真核细胞拥有超过五百种E3连接酶负责蛋白质的多聚泛素化修饰,每个E3连接酶可调节多个蛋白质底物的泛素化,每个蛋白质底物也通常受到多个E3连接酶的调控。研究表明转录因子SNAIL在细胞中极不稳定,半衰期只有15分钟,因此泛素化调节是影响SNAIL在细胞内表达水平的一个重要因素。到目前为止,若干E3泛素连接酶已经被证实能够参与调节SNAIL的泛素化修饰和蛋白酶体途径降解,鉴于细胞内蛋白质泛素化修饰的多样性与复杂性,我们利用基于荧光素酶报告基因的方法筛选了E3连接酶siRNA文库,以期发现影响SNAIL泛素化修饰和蛋白酶体途径降解的新的E3连接酶。通过筛选文库,我们发现SOCS box家族E3泛素连接酶SPSB3调节了SNAIL的多聚泛素化和蛋白酶体降解。首先我们通过免疫共沉淀的实验方法证实了SPSB3和SNAIL之间存在着很强的相互作用,接着利用放线菌酮追踪实验以及泛素化实验证明了SPSB3能够特异性的识别并结合SNAIL,从而介导SNAIL的多聚泛素化和蛋白酶体途径降解。我们特别关注了SPSB3对于细胞功能的影响。由于SNAIL在肿瘤细胞中主要通过促进EMT的转变来促进肿瘤细胞的迁移和侵袭,我们选择了迁移能力比较强的人食管癌细胞系KYSE30,发现过表达SPSB3大大减弱了人食管癌细胞系KYSE30的侵袭和转移能力。在4T1细胞原位肺转移动物模型中,我们发现SPSB3的过表达能够逆转SNAIL导致的4T1细胞的原位肺转移。其次,考虑到SNAIL能够通过调节细胞干性影响肿瘤发生,我们在T24 H-Ras转化的永生化人乳腺上皮细胞MCFCA1a中过表达SPSB3来研究SPSB3介导的SNAIL泛素化降解对肿瘤发生的影响,发现SPSB3的表达确实降低了MCFCA1a细胞的克隆形成能力。为更加全面地探讨SPSB3对于体内功能的影响,我们将MCFCA1a以及过表达SPSB3的MCFCA1a细胞原位注射于裸鼠皮下,发现SPSB3的过表达减弱了肿瘤细胞的成瘤能力。总之,我们通过体内和体外实验证明了SPSB3能够通过降解SNAIL的方式降低肿瘤的发生和侵袭转移能力。随后,我们研究了SPSB3泛素化降解SNAIL的具体分子机制。我们将一系列的SNAIL的缺失突变体与SPSB3做免疫共沉淀实验,发现SNAIL的丝氨酸富集结构域SRD对于两者的相互作用至关重要。在SNAIL的SRD结构域含有6个丝氨酸位点。GSK3β通过调节SNAIL SRD结构域内6个丝氨酸位点的磷酸化调节了SPSB3对SNAIL的泛素化修饰。因此,SPSB3介导的SNAIL泛素化依赖于GSK3β调节的SNAIL磷酸化。此外我们发现TNFα作为影响SNAIL蛋白稳定性的上游信号参与调节了SPSB3的表达,因此TNFα至少部分通过降低SPSB3的表达来提高SNAIL的稳定性。最后,我们在组织标本中研究了SPSB3的表达情况,发现两者在组织标本中的表达成负相关。一方面,在食管癌的配对标本中,我们发现SPSB3在肿瘤组织中的表达明显低于对应的正常组织,而且SPSB3的表达与肿瘤的分化程度密切相关。另一方面,在卵巢癌组织芯片中,SPSB3和SNAIL的表达也呈现负相关的关系。食管癌以及卵巢癌组织标本中的结果,一定程度上反应了SPSB3与SNAIL之间的调节关系以及二者在肿瘤发生发展中的作用。总之,我们的研究从寻找新的调节SNAIL泛素化降解的E3连接酶出发,发现E3连接酶SPSB3依赖于GSK3β对SNAIL的磷酸化介导SNAIL的泛素化修饰和蛋白酶体途径降解。SPSB3通过调节SNAIL的泛素化降解抑制了肿瘤的发生发展和侵袭转移。我们的工作一定程度上揭示了肿瘤发生发展和侵袭转移的具体机制,为肿瘤的临床治疗提供了理论依据。
[Abstract]:Epithelial-Mesenchymal Transition (EMT) plays an important role in the development and metastasis of tumor. It endows the tumor epithelial cells with the characteristics of interstitial like, so that the tumor cells have stronger movement ability and invasion ability.SNAIL is the most important regulatory factor in the EMT transformation process, and the occurrence and development of the tumor. It plays a key regulatory role, so its expression is regulated by many aspects such as transcriptional level, translation level and post translation level. At the post translation level, SNAIL is mainly modified by ubiquitination. Polyubiquitination regulates the degradation of SNAIL through the ubiquitin proteasome pathway. The E3 ubiquitin connection with substrate specificity Enzyme is a key factor in regulating SNAIL polyubiquitination and protein stability. Eukaryotic cells have more than five hundred E3 ligase responsible for polyubiquitination of proteins. Each E3 ligase regulates the ubiquitination of multiple protein substrates, and each protein substrate is usually regulated by multiple E3 ligase. Research indicates the transcription factor SN AIL is extremely unstable in cells with a half-life of only 15 minutes, so ubiquitination is an important factor affecting the expression level of SNAIL in cells. Up to now, a number of E3 ubiquitin ligases have been proved to be able to participate in the regulation of ubiquitination and proteasome degradation of SNAIL, in view of the ubiquitination of intracellular proteins Diversity and complexity, we screened the E3 ligase siRNA Library Based on the luciferase reporter gene method to discover new E3 ligases that affect the degradation of SNAIL ubiquitination and proteasome pathway. By screening the library, we found that the SOCS box family E3 ubiquitin linked enzyme SPSB3 regulates SNAIL polyubiquitination and protein. Enzyme degradation. First of all, we confirmed the strong interaction between SPSB3 and SNAIL by the method of immunoprecipitation, and then using Actinomycate tracing experiments and ubiquitination experiments proved that SPSB3 can identify specifically and combine SNAIL, which mediates the polyubiquitination of SNAIL and proteasome pathway degradation. Special attention is paid to the effect of SPSB3 on cell function. Since SNAIL promotes the migration and invasion of tumor cells by promoting the transformation of EMT in tumor cells, we choose the human esophageal cancer cell line, KYSE30, which has a relatively strong migration ability. It is found that the expression of SPSB3 greatly reduces the invasion and metastasis of the human esophageal cancer cell line KYSE30. In the animal model of 4T1 cell in situ lung metastasis, we found that the overexpression of SPSB3 can reverse the in situ lung metastasis of 4T1 cells induced by SNAIL. Secondly, we consider that SNAIL can affect the occurrence of tumor by regulating cell stem nature, and we overexpress SPSB3 in the immortalized human mammary gland cell MCFCA1a of the T24 H-Ras transformation to study SPSB3 The effect of SNAIL ubiquitination on the carcinogenesis of MCFCA1a cells was found to reduce the clone formation ability of MCFCA1a cells. In order to explore the effect of SPSB3 on the function of SPSB3 in a more comprehensive way, we injected the MCFCA1a and the SPSB3 MCFCA1a cells in the nude mice subcutaneously, and found that the overexpression of SPSB3 weakened the tumor finer. In conclusion, we have demonstrated that SPSB3 can reduce the occurrence and invasion and metastasis of the tumor by degradation of SNAIL in vivo and in vitro. Then, we studied the specific molecular mechanism of SPSB3 ubiquitination to degrade SNAIL. We have a series of SNAIL deficiency mutants and SPSB3 immunoprecipitation experiments. The SNAIL serine enriched domain SRD is very important for the interaction of the two. The SRD domain of SNAIL contains 6 serine loci.GSK3 beta that regulates the ubiquitination of SNAIL by the phosphorylation of 6 serine loci within the SNAIL SRD domain. Therefore, SNAIL ubiquitination mediated by SPSB3 depends on GSK3 beta regulation AIL phosphorylation. In addition, we found that TNF alpha as the upstream signal affecting the stability of SNAIL protein participates in the regulation of the expression of SPSB3. Therefore, TNF alpha at least partly improves the stability of SNAIL by reducing the expression of SPSB3. Finally, we study the expression of SPSB3 in tissue specimens, and find that the expression of the two in the tissue specimen is negative. On the one hand, in the paired specimens of esophageal cancer, we found that the expression of SPSB3 in the tumor tissue is significantly lower than that of the corresponding normal tissue, and the expression of SPSB3 is closely related to the degree of differentiation of the tumor. On the other hand, the expression of SPSB3 and SNAIL is also negatively correlated in the ovarian cancer tissue microarray. The results in the specimen, to some extent, reflect the regulatory relationship between SPSB3 and SNAIL and the role of the two in the development of the tumor. In conclusion, our study, starting from the search for a new E3 ligase that regulates the ubiquitination of SNAIL, found that the E3 ligase SPSB3 is dependent on the ubiquitination of SNAIL by the phosphorylation of SNAIL by GSK3 beta. Proteasome pathway degradation of.SPSB3 inhibits the occurrence, development and invasion and metastasis of tumor by regulating the ubiquitination degradation of SNAIL. Our work reveals the specific mechanism of tumor development and invasion and metastasis to some extent, and provides a theoretical basis for the clinical treatment of tumor.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2

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