本文選題：SPSB3 + SNAIL　； 參考：《北京協(xié)和醫(yī)學院》2016年博士論文

【摘要】：上皮間質轉換(Epithelial-Mesenchymal Transition,EMT)在腫瘤的發(fā)生與轉移過程中發(fā)揮著重要的作用,它賦予腫瘤上皮細胞間質樣的特性,使得腫瘤細胞具有更強的運動能力和侵襲能力。SNAIL是EMT轉變過程中最重要的調控因子,在腫瘤的發(fā)生發(fā)展中發(fā)揮了關鍵的調節(jié)作用,因此它的表達受到轉錄水平、翻譯水平和翻譯后水平等多方面的精密調控。在翻譯后水平,SNAIL主要受到泛素化的修飾。多聚泛素化調節(jié)了SNAIL通過泛素-蛋白酶體途徑的降解。而具有底物特異性的E3泛素連接酶是調節(jié)SNAIL多聚泛素化與蛋白穩(wěn)定性的關鍵因子。真核細胞擁有超過五百種E3連接酶負責蛋白質的多聚泛素化修飾,每個E3連接酶可調節(jié)多個蛋白質底物的泛素化,每個蛋白質底物也通常受到多個E3連接酶的調控。研究表明轉錄因子SNAIL在細胞中極不穩(wěn)定,半衰期只有15分鐘,因此泛素化調節(jié)是影響SNAIL在細胞內表達水平的一個重要因素。到目前為止,若干E3泛素連接酶已經被證實能夠參與調節(jié)SNAIL的泛素化修飾和蛋白酶體途徑降解,鑒于細胞內蛋白質泛素化修飾的多樣性與復雜性,我們利用基于熒光素酶報告基因的方法篩選了E3連接酶siRNA文庫,以期發(fā)現(xiàn)影響SNAIL泛素化修飾和蛋白酶體途徑降解的新的E3連接酶。通過篩選文庫,我們發(fā)現(xiàn)SOCS box家族E3泛素連接酶SPSB3調節(jié)了SNAIL的多聚泛素化和蛋白酶體降解。首先我們通過免疫共沉淀的實驗方法證實了SPSB3和SNAIL之間存在著很強的相互作用,接著利用放線菌酮追蹤實驗以及泛素化實驗證明了SPSB3能夠特異性的識別并結合SNAIL,從而介導SNAIL的多聚泛素化和蛋白酶體途徑降解。我們特別關注了SPSB3對于細胞功能的影響。由于SNAIL在腫瘤細胞中主要通過促進EMT的轉變來促進腫瘤細胞的遷移和侵襲,我們選擇了遷移能力比較強的人食管癌細胞系KYSE30,發(fā)現(xiàn)過表達SPSB3大大減弱了人食管癌細胞系KYSE30的侵襲和轉移能力。在4T1細胞原位肺轉移動物模型中,我們發(fā)現(xiàn)SPSB3的過表達能夠逆轉SNAIL導致的4T1細胞的原位肺轉移。其次,考慮到SNAIL能夠通過調節(jié)細胞干性影響腫瘤發(fā)生,我們在T24 H-Ras轉化的永生化人乳腺上皮細胞MCFCA1a中過表達SPSB3來研究SPSB3介導的SNAIL泛素化降解對腫瘤發(fā)生的影響,發(fā)現(xiàn)SPSB3的表達確實降低了MCFCA1a細胞的克隆形成能力。為更加全面地探討SPSB3對于體內功能的影響,我們將MCFCA1a以及過表達SPSB3的MCFCA1a細胞原位注射于裸鼠皮下,發(fā)現(xiàn)SPSB3的過表達減弱了腫瘤細胞的成瘤能力。總之,我們通過體內和體外實驗證明了SPSB3能夠通過降解SNAIL的方式降低腫瘤的發(fā)生和侵襲轉移能力。隨后,我們研究了SPSB3泛素化降解SNAIL的具體分子機制。我們將一系列的SNAIL的缺失突變體與SPSB3做免疫共沉淀實驗,發(fā)現(xiàn)SNAIL的絲氨酸富集結構域SRD對于兩者的相互作用至關重要。在SNAIL的SRD結構域含有6個絲氨酸位點。GSK3β通過調節(jié)SNAIL SRD結構域內6個絲氨酸位點的磷酸化調節(jié)了SPSB3對SNAIL的泛素化修飾。因此,SPSB3介導的SNAIL泛素化依賴于GSK3β調節(jié)的SNAIL磷酸化。此外我們發(fā)現(xiàn)TNFα作為影響SNAIL蛋白穩(wěn)定性的上游信號參與調節(jié)了SPSB3的表達,因此TNFα至少部分通過降低SPSB3的表達來提高SNAIL的穩(wěn)定性。最后,我們在組織標本中研究了SPSB3的表達情況,發(fā)現(xiàn)兩者在組織標本中的表達成負相關。一方面,在食管癌的配對標本中,我們發(fā)現(xiàn)SPSB3在腫瘤組織中的表達明顯低于對應的正常組織,而且SPSB3的表達與腫瘤的分化程度密切相關。另一方面,在卵巢癌組織芯片中,SPSB3和SNAIL的表達也呈現(xiàn)負相關的關系。食管癌以及卵巢癌組織標本中的結果,一定程度上反應了SPSB3與SNAIL之間的調節(jié)關系以及二者在腫瘤發(fā)生發(fā)展中的作用�？傊�,我們的研究從尋找新的調節(jié)SNAIL泛素化降解的E3連接酶出發(fā),發(fā)現(xiàn)E3連接酶SPSB3依賴于GSK3β對SNAIL的磷酸化介導SNAIL的泛素化修飾和蛋白酶體途徑降解。SPSB3通過調節(jié)SNAIL的泛素化降解抑制了腫瘤的發(fā)生發(fā)展和侵襲轉移。我們的工作一定程度上揭示了腫瘤發(fā)生發(fā)展和侵襲轉移的具體機制,為腫瘤的臨床治療提供了理論依據(jù)。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R730.2

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