本文選題：乳腺癌 + RSK4�。� 參考：《廣西醫(yī)科大學(xué)》2015年碩士論文

【摘要】：研究背景：p90核糖體S6蛋白激酶4(Ribosomal S6 protein kinase 4, RSK4)是一新發(fā)現(xiàn)的抑制乳腺癌侵襲轉(zhuǎn)移的重要因子,它能抑制乳腺癌細(xì)胞的生長(zhǎng)并誘導(dǎo)其衰老；而RSK4前體mRNA在可變剪切作用下形成可變剪接的變異體(RSK4m),并具有不同的生物學(xué)功能。迄今為止,RSK4及其變異體RSK4m在乳腺癌中作用機(jī)制所知甚少。蛋白質(zhì)是生命活動(dòng)的主要物質(zhì),蛋白質(zhì)功能的實(shí)現(xiàn),離不開與其它蛋白質(zhì)之間的相互作用�？勺兗艚邮钦{(diào)節(jié)基因表達(dá)和產(chǎn)生蛋白質(zhì)多樣性的重要機(jī)制。前期研究我們認(rèn)為RSK4及其變異體與其它蛋白通過相互作用形成不同的蛋白質(zhì)復(fù)合物后在乳腺癌發(fā)生發(fā)展中發(fā)揮重要的作用。本研究擬建立哺乳動(dòng)物體系中的串聯(lián)親和純化技術(shù),并聯(lián)合質(zhì)譜分析鑒定RSK4及其變異體相互作用蛋白質(zhì),經(jīng)生物信息學(xué)方法分析找尋RSK4及其變異體相互作用蛋白中可能存在的關(guān)鍵蛋白,初步驗(yàn)證其生物學(xué)功能。本研究通過對(duì)RSK4和其變異體RSK4m的相互作用蛋白的研究,可以為我們初步了解其獨(dú)特的蛋白功能和作用機(jī)制,從而為進(jìn)一步闡明乳腺癌發(fā)生發(fā)展機(jī)制及尋找乳腺癌治療新靶點(diǎn)開辟新思路和新途徑。研究方法：分別構(gòu)建了帶有串聯(lián)親和標(biāo)簽(兩個(gè)strep II標(biāo)簽和一個(gè)FLAG標(biāo)簽)的RSK4和RSK4m過表達(dá)載體。轉(zhuǎn)染到RSK4低表達(dá)的乳腺癌細(xì)胞株MDA-MB-231中,運(yùn)用串聯(lián)親和純化技術(shù)(tandem affinity purification, TAP),得到與RSK4或RSK4m相互作用的蛋白復(fù)合物,然后對(duì)所得的蛋白復(fù)合物進(jìn)行質(zhì)譜鑒定和分析,應(yīng)用生物信息學(xué)分析方法(GO和IPA分析),分別對(duì)RSK4和RSK4m的相互作用蛋白進(jìn)行功能注釋和繪制了蛋白-蛋白相互作用網(wǎng)絡(luò)圖。為了確認(rèn)生物信息學(xué)分析結(jié)果,我們還利用免疫共沉淀(Co-Immunoprecipitation, CO-IP)對(duì)其中PRMT5(蛋白精氨酸甲基轉(zhuǎn)移酶5)蛋白與RSK4的相互作用關(guān)系進(jìn)行驗(yàn)證。研究結(jié)果：本研究通過兩個(gè)Strep II標(biāo)簽和單個(gè)FLAG標(biāo)簽為基礎(chǔ)的串聯(lián)親和純化技術(shù)(SF-TAP)聯(lián)合運(yùn)用nano LC-MS/MS質(zhì)譜技術(shù)分別分離和鑒定出82個(gè)RSK4相關(guān)的相互作用蛋白,137個(gè)RSK4m相關(guān)的相互作用蛋白。應(yīng)用生物信息學(xué)分析方法(GO和IPA分析),分別對(duì)RSK4和RSK4m的相互作用蛋白進(jìn)行功能注釋和繪制了蛋白-蛋白相互作用網(wǎng)絡(luò)圖。GO(Gene Ontology)分析結(jié)果：RSK4 wt或RSK4m的互作蛋白參與細(xì)胞的多種生理活動(dòng),比如：tRNA代謝(tRNA metabolic process),核糖體蛋白復(fù)合物的生物合成(ribonucleoprotein complex biogenesis),氨基酸的活化(amino acid activation)和蛋白折疊(protein folding)等,這些互作蛋白絕大部分都位于細(xì)胞質(zhì)中,與RSK4本身位于細(xì)胞質(zhì),在細(xì)胞質(zhì)發(fā)揮生物功能是一致的。IPA (Ingenuity Pathway Analysis)分析結(jié)果：將鑒定到的互作蛋白經(jīng)過核心數(shù)據(jù)分析方法(Core Analyze dateset),分別得到RSK4 wt和RSK4m各自三個(gè)富集度最高的信號(hào)通路和蛋白互作網(wǎng)絡(luò)：①RSK4 wt和RSK4m主要參與了核糖體生物合成,互作蛋白組網(wǎng)絡(luò)中的PRMT5、STAT3、 HSP90AB1、ATP5B、RPL核糖體亞基系列蛋白等與細(xì)胞中核糖體生物合成密切相關(guān)；②RSK4 wt和RSK4m的互作蛋白均參與細(xì)胞前體RNA的處理,互作蛋白中的HNRNPH1、HNRNPK、HNRNPM屬于hnRNPs(heterogeneous nuclear ribonucleproteins)家族,是RNA結(jié)合蛋白,能與mRNA前體相互作用,影響mRNA前體的處理以及mRNA代謝和運(yùn)輸；③RSK4 wt和RSK4m主要參與染色體的重組與代謝,相互作用蛋白STK38、STK38L、 MOB2和DDX3X參與調(diào)節(jié)中心體的復(fù)制和有絲分裂期染色質(zhì)的重組。綜合GO分析和IPA分析的結(jié)果,表明RSK4 wt和RSK4m參與了多種細(xì)胞活動(dòng),擁有多種不同的生物學(xué)功能。RSK4m的相互作用蛋白更多,并且參與更多的生物通路。同時(shí),CO-IP證實(shí)了其中的蛋白如PRMT5與RSK4存在相互作用關(guān)系,表明PRMT5可能可以通過甲基化RSK4從而抑制RSK4對(duì)細(xì)胞生長(zhǎng)的抑制作用。研究結(jié)論：RSK4和RSK4m存在與其相互作用蛋白,相互作用蛋白復(fù)合物主要參與了核糖體生物合成、前體RNA處理和染色體的重組與代謝,并介導(dǎo)多種生物學(xué)功能調(diào)控,特別是有關(guān)細(xì)胞衰老的調(diào)控；而且RSK4和RSK4m有著不同相互作用蛋白,其調(diào)控作用及機(jī)制有可能不同,RSK4m可能比RSK4擁有更多的分子功能,但其中的具體功能尚待于進(jìn)一步研究。而其中的互作蛋白PRMT5與RSK4存在直接的相互作用關(guān)系。
[Abstract]:Background: P90 ribosome S6 protein kinase 4 (Ribosomal S6 protein kinase 4, RSK4) is an important factor in inhibiting the invasion and metastasis of breast cancer, which inhibits the growth and senescence of breast cancer cells, and RSK4 precursor mRNA forms a variant spliced variant (RSK4m) under variable shear action, and has a different birth rate. Physical function. So far, RSK4 and its variant RSK4m have little knowledge of the mechanism of action in breast cancer. Protein is the main substance of life activity. The realization of protein function can not be separated from the interaction with other proteins. Variable splicing is an important mechanism for regulating gene expression and producing egg white diversity. It is considered that RSK4 and its variants and other proteins play an important role in the development of breast cancer after interacting with other proteins by interacting with other proteins. This study aims to establish a series affinity purification technology in mammalian system, and to identify the interaction proteins of RSK4 and its variants by mass spectrometry analysis. The possible biological function of RSK4 and its variant protein was found by method analysis. The study on the interaction protein of RSK4 and its variant RSK4m could provide us a preliminary understanding of its unique protein function and mechanism, so as to further elucidate the development of breast cancer. Mechanism and new approaches for finding new targets for breast cancer treatment. Research methods: RSK4 and RSK4m overexpression vectors with tandem affinity labels (two strep II tags and one FLAG tag) were constructed respectively. The transfected to RSK4 low expression breast cancer cell line MDA-MB-231, using serial affinity purification technology (tandem affinity PU). Rification, TAP), the protein complexes interacting with RSK4 or RSK4m were obtained, and then the protein complexes were identified and analyzed by mass spectrometry. The interaction proteins of RSK4 and RSK4m were explained by bioinformatics analysis method (GO and IPA analysis), and the protein protein interaction network diagram was plotted and plotted for the interaction proteins of RSK4 and RSK4m. As a result of bioinformatics analysis, we also used Co-Immunoprecipitation (CO-IP) to verify the interaction between PRMT5 (protein arginine methyltransferase 5) protein and RSK4. The results of this study were based on the tandem affinity purification technology (SF-TAP) based on two Strep II tags and single FLAG tags. 82 RSK4 related interacting proteins and 137 RSK4m related interacting proteins were isolated and identified by nano LC-MS/MS mass spectrometry. The interaction proteins of RSK4 and RSK4m were annotated and the protein protein interaction network diagram.GO (Gene) was plotted by bioinformatics analysis method (GO and IPA analysis). Ontology) analysis results: the interaction proteins of RSK4 WT or RSK4m participate in various physiological activities of the cells, such as the tRNA metabolism (tRNA metabolic process), the biosynthesis of the ribosomal protein complex (ribonucleoprotein complex biogenesis), the activation of the amino acids (amino) and protein folding, and so on. Most of the proteins are located in the cytoplasm, and RSK4 itself is located in the cytoplasm, and the.IPA (Ingenuity Pathway Analysis) analysis is consistent with the biological function of the cytoplasm. The identified mutual protein, through the core data analysis method (Core Analyze dateset), obtains the three highest concentration of RSK4 WT and RSK4m respectively. Signal pathway and protein interaction network: (1) RSK4 WT and RSK4m are mainly involved in ribosome biosynthesis, PRMT5, STAT3, HSP90AB1, ATP5B, RPL ribosome series proteins are closely related to the biosynthesis of ribosome biosynthesis in the cell network, and both RSK4 WT and RSK4m interaction proteins are involved in the treatment of cell precursor RNA. HNRNPH1, HNRNPK, and HNRNPM belong to the hnRNPs (heterogeneous nuclear ribonucleproteins) family, which are RNA binding proteins that interact with the mRNA precursors, affect the processing of mRNA precursors and mRNA metabolism and transport. To regulate the replication of the centrosome and the recombination of chromatin during mitosis. Combined with the results of GO analysis and IPA analysis, it shows that RSK4 WT and RSK4m are involved in a variety of cell activities, with a variety of different biological functions,.RSK4m interaction proteins, and more biological pathways. Meanwhile, CO-IP has confirmed the proteins such as PRMT5. The interaction with RSK4 indicates that PRMT5 may be able to inhibit the inhibitory effect of RSK4 on cell growth by methylation of RSK4. Conclusions: RSK4 and RSK4m exist with their interacting proteins. The interaction protein complex is mainly involved in ribosome biosynthesis, precursor RNA treatment and chromosome recombination and metabolism, and mediates A variety of biological functions and regulation, especially the regulation of cell senescence, and RSK4 and RSK4m have different interacting proteins, and their regulatory roles and mechanisms may be different. RSK4m may have more molecular functions than RSK4, but the specific functions of which are yet to be further studied. The mutual protein PRMT5 and RSK4 exist directly. The interaction between them.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 李田田;劉明;魏鑒騰;吳寧;王翠翠;林秀坤;;RSK4基因表達(dá)及其對(duì)U87細(xì)胞增殖的影響[J];中國(guó)神經(jīng)腫瘤雜志;2011年02期

2 韋薇;韓璐璐;楊華偉;劉劍侖;;慢病毒介導(dǎo)的RSK4對(duì)乳腺癌MDA-MB-231細(xì)胞增殖的影響[J];廣西醫(yī)科大學(xué)學(xué)報(bào);2013年03期

3 付國(guó)慶;范林妮;朱瑾;王璐;呂楊;劉一雄;何芙蓉;喬少誼;張磊;邵晨;王哲;;Rsk-4蛋白在胰腺腫瘤中的表達(dá)及其與臨床病理的關(guān)系[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2010年06期

4 朱沙;張春輝;李鮮菊;;從基因組學(xué)到功能蛋白質(zhì)組學(xué)的研究[J];生物學(xué)雜志;2007年01期

5 朱佳;劉劍侖;韋薇;韓璐璐;;RNA干擾RSK4基因表達(dá)對(duì)裸鼠移植瘤生長(zhǎng)與轉(zhuǎn)移影響觀察[J];中華腫瘤防治雜志;2013年24期

6 孫玉朝;王忠臣;;腎細(xì)胞癌患者臨床病理特征與核糖體S6激酶4表達(dá)的相關(guān)性[J];中國(guó)老年學(xué)雜志;2015年01期

7 魏衍全;孫世琪;孫德惠;郭慧琛;;串聯(lián)親和純化中應(yīng)用不同標(biāo)簽組合的研究進(jìn)展[J];中國(guó)獸醫(yī)科學(xué);2012年09期

相關(guān)博士學(xué)位論文 前1條

1 趙健;AGR2基因促肝癌轉(zhuǎn)移的研究和HDGF相互作用蛋白的鑒定[D];復(fù)旦大學(xué);2011年

，

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