本文選題：缺氧微環(huán)境 + 骨肉瘤轉(zhuǎn)移　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：研究目的：骨肉瘤是最常見的原發(fā)性骨惡性腫瘤,侵襲性高且血行轉(zhuǎn)移早。近年來隨著手術(shù)以及放化療水平的提高,骨肉瘤患者的5年生存率有所提升,但是總體治愈率仍未出現(xiàn)顯著突破。由于缺氧而引發(fā)的肺轉(zhuǎn)移是造成骨肉瘤患者死亡的一個(gè)重要原因。因此,深入研究缺氧促進(jìn)骨肉瘤轉(zhuǎn)移的分子機(jī)制,并從中發(fā)現(xiàn)可干預(yù)的靶點(diǎn),有望為臨床骨肉瘤的治療提供指導(dǎo)并推動(dòng)新型靶向抗腫瘤轉(zhuǎn)移藥物的研發(fā)。研究方法：第一部分：缺氧微環(huán)境對WSB1蛋白表達(dá)的調(diào)控作用以及WSBl與骨肉瘤惡性轉(zhuǎn)移的相關(guān)性研究本研究采用人源性骨肉瘤細(xì)胞系(KHOS/NP、U2OS以及MG63)、原代骨肉瘤細(xì)胞株(MDOS15、MDOS20)作為研究對象。(1)免疫組化分析HIF-1α與WSB1表達(dá)的相關(guān)性;(2)免疫熒光考察HIF-1α與WSB1蛋白的共定位情況;(3)Western blotting檢測WSB1蛋白水平在缺氧誘導(dǎo)以及過表達(dá)HIF-1α后的變化;(4)熒光定量PCR技術(shù)檢測WSB1 mRNA水平在缺氧誘導(dǎo)以及過表達(dá)HIF-1α后的變化;(5)雙熒光素酶報(bào)告基因檢測結(jié)合染色質(zhì)免疫共沉淀實(shí)驗(yàn)研究HIF-1α對WSB1的調(diào)控機(jī)制及兩者的結(jié)合區(qū)域;(6)Transwell實(shí)驗(yàn)檢測骨肉瘤原代細(xì)胞株在缺氧環(huán)境中遷移能力的變化；(7)采用分子克隆手段構(gòu)建野生型WSB1質(zhì)粒,制備野生型WSB1以及可敲低WSB1的慢病毒;(8)Transwell結(jié)合劃痕修復(fù)實(shí)驗(yàn)觀察骨肉瘤細(xì)胞分別在過表達(dá)野生型WSB1以及敲低WSB1后遷移能力的變化;(9)建立過表達(dá)野生型WSB1及敲低WSB1的KHOS/NP細(xì)胞的裸小鼠肺轉(zhuǎn)移模型,通過活體成像技術(shù)結(jié)合HE染色檢測骨肉瘤細(xì)胞在裸小鼠體內(nèi)的肺轉(zhuǎn)移情況。第二部分：WSB1促進(jìn)骨肉瘤轉(zhuǎn)移的分子機(jī)制研究(1)采用分子克隆手段構(gòu)建SOCS box結(jié)構(gòu)域缺失或突變的WSB1質(zhì)粒,并制備SOCS box結(jié)構(gòu)域缺失或突變的WSB1慢病毒;(2)Transwell實(shí)驗(yàn)觀察骨肉瘤細(xì)胞分別在過表達(dá)野生型WSB1、SOCS box結(jié)構(gòu)域缺失或突變情況下遷移能力的變化;(3)應(yīng)用SILAC定量蛋白質(zhì)組學(xué)分析,尋找在WSB1調(diào)控骨肉瘤轉(zhuǎn)移過程中發(fā)揮關(guān)鍵作用的蛋白;(4)免疫組化染色分析WSB1與轉(zhuǎn)移相關(guān)蛋白R(shí)hoGDI2表達(dá)的相關(guān)性;(5)免疫沉淀結(jié)合免疫熒光證明WSB1與RhoGDI2的結(jié)合;(6)免疫熒光檢測WSB1對于骨肉瘤細(xì)胞膜表面?zhèn)巫阈纬赡芰Φ挠绊?(7)Time-lapse呈像研究WSB1對于骨肉瘤細(xì)胞運(yùn)動(dòng)能力的調(diào)控作用;(8)建立過表達(dá)WSB1、同時(shí)過表達(dá)WSB1及RhoGDI2、過表達(dá)RhoGDI2的KHOS/NP細(xì)胞的裸小鼠肺轉(zhuǎn)移模型,通過熒光顯微鏡觀察結(jié)合HE染色檢測骨肉瘤細(xì)胞的體內(nèi)肺轉(zhuǎn)移情況。研究結(jié)果：第一部分：缺氧微環(huán)境對WSB1蛋白表達(dá)的調(diào)控作用以及WSB1與骨肉瘤惡性轉(zhuǎn)移的相關(guān)性研究(1) 骨肉瘤細(xì)胞中WSB1蛋白與HIF-1α蛋白表達(dá)呈高度相關(guān)采用免疫組化法檢測40例骨肉瘤患者組織切片中WSB1與HIF-1α蛋白的表達(dá)情況并作病理學(xué)評分統(tǒng)計(jì),計(jì)算得出兩者的相關(guān)系數(shù)R值為0.825,呈現(xiàn)顯著的正相關(guān)性;免疫熒光實(shí)驗(yàn)驗(yàn)證裸小鼠荷骨肉瘤移植瘤組織切片中WSB1與HIF-1α的表達(dá)呈現(xiàn)良好的共定位狀態(tài)。以上結(jié)果表明,WSB1可能是HIF-1α的靶基因。將骨肉瘤細(xì)胞系KHOS/NP、U2OS、MG63及原代骨肉瘤細(xì)胞MDOS15、MDOS20分別置于常氧及缺氧環(huán)境中培養(yǎng)24小時(shí),Western blotting檢測發(fā)現(xiàn)WSB 1蛋白表達(dá)在缺氧環(huán)境中顯著增加。在DMOG, DFX兩種缺氧造模藥物的作用下,KHOS/NP細(xì)胞中WSB1蛋白表達(dá)水平也呈現(xiàn)濃度依賴性增加。采用質(zhì)粒轉(zhuǎn)染法在KHOS/NP細(xì)胞中過表達(dá)HIF-1α蛋白,Western blotting結(jié)果顯示W(wǎng)SB1蛋白高度依賴于HIF-1α蛋白的表達(dá),而通過小分子RNA干擾技術(shù)沉默HIF-1α后,缺氧誘導(dǎo)的WSB1表達(dá)增加被顯著逆轉(zhuǎn)。以上結(jié)果表明,缺氧環(huán)境中HIF-1α蛋白介導(dǎo)了WSB1蛋白表達(dá)的增加。(2)缺氧條件下HIF-1α蛋白可轉(zhuǎn)錄激活WSB1并誘導(dǎo)其蛋白表達(dá)增加如前所述,缺氧誘導(dǎo)可上調(diào)骨肉瘤細(xì)胞中WSB1蛋白表達(dá),且WSB1蛋白與HIF-1α表達(dá)具有正相關(guān)性并呈現(xiàn)共定位狀態(tài),提示經(jīng)典的缺氧轉(zhuǎn)錄因子HIF-1α可能在這一現(xiàn)象中有重要的作用。鑒于轉(zhuǎn)錄因子通常可在轉(zhuǎn)錄層面調(diào)控下游基因的表達(dá),我們首先采用RT-PCR法檢測缺氧環(huán)境中及過表達(dá)HIF-1α后KHOS/NP細(xì)胞內(nèi)WSB1的mRNA水平,結(jié)果顯示缺氧環(huán)境及過表達(dá)HIF-1α均可顯著上調(diào)WSB1的tnRNA水平。然后,我們通過分子克隆技術(shù)構(gòu)建包括WSB1啟動(dòng)子區(qū)域以及三個(gè)HRE結(jié)合區(qū)域(-1768位點(diǎn),-1461位點(diǎn),-339位點(diǎn))分別突變的質(zhì)粒并與HIF-1α質(zhì)粒共同轉(zhuǎn)染至細(xì)胞中,雙熒光素酶報(bào)告基因檢測結(jié)果表明HIF-1α可明顯轉(zhuǎn)錄激活WSB1基因,且HIF-1α可能是通過結(jié)合在WSB1啟動(dòng)子區(qū)域的-339位點(diǎn)實(shí)現(xiàn)對其的轉(zhuǎn)錄調(diào)控,并在染色質(zhì)免疫共沉淀實(shí)驗(yàn)中得到了進(jìn)一步確證。以上結(jié)果表明HIF-1α可轉(zhuǎn)錄激活WSB1基因并誘導(dǎo)其蛋白表達(dá)增加。(3) WSB1蛋白的表達(dá)水平與臨床骨肉瘤患者的轉(zhuǎn)移率呈正相關(guān)性通過對臨床骨肉瘤患者的癌組織及癌旁組織樣本進(jìn)行免疫組化分析發(fā)現(xiàn),WSB1在癌組織區(qū)域的陽性表達(dá)率高達(dá)88%,且多呈強(qiáng)陽性;而在癌旁組織區(qū)域中,WSB1的陽性表達(dá)率僅為32%。這一結(jié)果提示W(wǎng)SB1可作為骨肉瘤診斷的生物學(xué)標(biāo)記物,并且可能調(diào)控骨肉瘤的惡性進(jìn)展。通過回顧性調(diào)研28位骨肉瘤患者的3年臨床隨訪紀(jì)錄,發(fā)現(xiàn)WSB1的表達(dá)水平與骨肉瘤患者的轉(zhuǎn)移率呈現(xiàn)正相關(guān)性。 以上結(jié)果表明WSB1的表達(dá)水平與臨床骨肉瘤患者的轉(zhuǎn)移密切相關(guān),提示W(wǎng)SB1可能參與介導(dǎo)了缺氧促進(jìn)的骨肉瘤轉(zhuǎn)移。(4)WSB1蛋白可促進(jìn)骨肉瘤細(xì)胞的體外遷移能力制備野生型WSB1以及可敲低WSB1蛋白表達(dá)的慢病毒顆粒并感染骨肉瘤細(xì)胞株KHOS/NP以及U2OS,在Transwell以及劃痕修復(fù)實(shí)驗(yàn)中發(fā)現(xiàn)過表達(dá)WSB1蛋白能夠顯著增加兩株骨肉瘤細(xì)胞的運(yùn)動(dòng)遷移能力;而敲低WSB1則能夠逆轉(zhuǎn)缺氧促進(jìn)的骨肉瘤細(xì)胞體外遷移能力。以上結(jié)果提示W(wǎng)SB1蛋白可促進(jìn)骨肉瘤細(xì)胞的體外遷移能力。(5)WSB1蛋白可促進(jìn)骨肉瘤細(xì)胞的體內(nèi)肺轉(zhuǎn)移能力構(gòu)建過表達(dá)野生型WSB1及敲低WSB1的KHOS/NP細(xì)胞株并建立相應(yīng)的裸小鼠腫瘤肺轉(zhuǎn)移模型,micro-PET活體成像結(jié)果顯示,過表達(dá)野生型WSB1能夠顯著增加骨肉瘤細(xì)胞在裸小鼠體內(nèi)的肺部轉(zhuǎn)移能力,對肺組織切片進(jìn)行HE染色統(tǒng)計(jì)轉(zhuǎn)移灶點(diǎn)數(shù)也證實(shí)以上結(jié)果;而敲低WSB1則能夠明顯抑制骨肉瘤細(xì)胞的肺部轉(zhuǎn)移。以上結(jié)果提示W(wǎng)SB1蛋白可促進(jìn)骨肉瘤細(xì)胞的肺轉(zhuǎn)移能力。第二部分：WSB1蛋白促進(jìn)骨肉瘤轉(zhuǎn)移的分子機(jī)制研究(1)WSB1蛋白促進(jìn)腫瘤轉(zhuǎn)移的作用依賴于其自身E3酶活性WSB1蛋白主要由N端的七個(gè)WD40重復(fù)結(jié)構(gòu)域和C端的SOCS box結(jié)構(gòu)域構(gòu)成。已有研究表明,SOCS box結(jié)構(gòu)域能夠與延伸蛋白(Elongin) B和C相互結(jié)合,形成E3泛素連接酶復(fù)合物,是WSB1發(fā)揮E3酶活性的關(guān)鍵區(qū)域。我們構(gòu)建了WSBl SOCS box結(jié)構(gòu)域缺失或突變的質(zhì)粒,并與野生型WSB1進(jìn)行比較,發(fā)現(xiàn)SOCS box結(jié)構(gòu)域的缺失或突變均能夠明顯逆轉(zhuǎn)WSB1促進(jìn)骨肉瘤轉(zhuǎn)移的生物學(xué)作用。這一結(jié)果表明WSB1蛋白促進(jìn)腫瘤轉(zhuǎn)移的生物學(xué)功能依賴于其自身E3泛素連接酶活性。(2) SILAC定量蛋白質(zhì)組學(xué)分析WSB1通過其E3酶活性調(diào)控腫瘤轉(zhuǎn)移的下游底物蛋白我們對WSB1過表達(dá)及空白載體組的KHOS/NP細(xì)胞進(jìn)行SILAC定量蛋白質(zhì)組學(xué)分析,質(zhì)譜鑒定得到1078個(gè)變化三倍以上的差異蛋白。前期結(jié)果表明WSB1促進(jìn)骨肉瘤轉(zhuǎn)移的生物學(xué)效應(yīng)依賴其E3泛素連接酶活性,我們推測WSB1可能通過降解某個(gè)轉(zhuǎn)移相關(guān)蛋白而發(fā)揮這一功能�；诖�,我們對518個(gè)發(fā)生下調(diào)的蛋白進(jìn)行重點(diǎn)考察。差異分析報(bào)告顯示轉(zhuǎn)移相關(guān)蛋白R(shí)hoGDI2變化最為顯著,提示其可能作為WSB1潛在的底物蛋白介導(dǎo)了WSB1對于骨肉瘤轉(zhuǎn)移的調(diào)控過程。(3)WSB1可促進(jìn)轉(zhuǎn)移相關(guān)蛋白R(shí)hoGDI2經(jīng)由泛素蛋白酶體途徑發(fā)生降解通過免疫組化染色對骨肉瘤患者組織樣本中WSB1與RhoGDI2蛋白的表達(dá)水平進(jìn)行考察,分析發(fā)現(xiàn)兩者的相關(guān)系數(shù)R值為-0.65,表明兩者表達(dá)呈現(xiàn)負(fù)相關(guān)性。通過Westren blotting檢測發(fā)現(xiàn),在WSB1過表達(dá)的情況下RhoGDI2蛋白水平顯著下調(diào)。但是,當(dāng)WSB1的SOCS box結(jié)構(gòu)域缺失后,其下調(diào)RhoGDI2的作用即被逆轉(zhuǎn),提示W(wǎng)SB1可通過其E3泛素連接酶活性負(fù)性調(diào)控RhoGDI2o在蛋白合成抑制劑CHX的作用下,WSB1能夠加速RhoGDI2的降解,表明WSB1下調(diào)RhoGDI2可能是通過促進(jìn)其降解而實(shí)現(xiàn)。而在蛋白酶體抑制劑MG132的作用下,WSB1下調(diào)RhoGDI2的現(xiàn)象被逆轉(zhuǎn),提示W(wǎng)SB1降解RhoGDI2的過程可能經(jīng)由泛素—蛋白酶體通路。免疫熒光實(shí)驗(yàn)表明WSB1蛋白與RhoGDI2蛋白呈現(xiàn)較明顯的共定位現(xiàn)象,免疫沉淀結(jié)果顯示W(wǎng)SB1與RhoGDI2蛋白能夠直接發(fā)生結(jié)合,并且WSB1能夠促進(jìn)RhoGDI2的多聚泛素化修飾。以上結(jié)果均表明WSB1可促進(jìn)轉(zhuǎn)移相關(guān)蛋白R(shí)hoGDI2經(jīng)由泛素—蛋白酶體途徑發(fā)生降解。(4)WSB1可激活RhoGDI2下游信號(hào)通路我們接著考察了WSB1對RhoGDI2調(diào)控的Rho信號(hào)通路的影響。通過pull-down實(shí)驗(yàn)檢測發(fā)現(xiàn),過表達(dá)WSB1能夠激活RhoGTPases家族蛋白成員Rac1。 Rac1蛋白的主要生物學(xué)功能為調(diào)控細(xì)胞骨架重構(gòu)從而促進(jìn)細(xì)胞運(yùn)動(dòng)。因此,我們通過免疫熒光實(shí)驗(yàn)考察KHOS/NP細(xì)胞骨架的變化。結(jié)果顯示,過表達(dá)WSB1能夠促進(jìn)肌動(dòng)蛋白Actin的多聚化并促進(jìn)細(xì)胞膜表面?zhèn)巫愕男纬?從而增強(qiáng)細(xì)胞的運(yùn)動(dòng)遷移能力。以上結(jié)果表明,WSB1可通過激活RhoGDI2下游Rho信號(hào)通路增加腫瘤細(xì)胞的運(yùn)動(dòng)潛能。(5)過表達(dá)RhoGDI2能逆轉(zhuǎn)WSB1的轉(zhuǎn)移促進(jìn)功能我們構(gòu)建了過表達(dá)WSB1、過表達(dá)RhoGDI2及同時(shí)過表達(dá)WSB1、與RhoGDI2的KHOS/NP細(xì)胞。通過體外Transwell實(shí)驗(yàn)檢測發(fā)現(xiàn),過表達(dá)RhoGDI2能夠逆轉(zhuǎn)WSB1促進(jìn)骨肉瘤細(xì)胞遷移的現(xiàn)象。進(jìn)一步,我們構(gòu)建上述KHOS/NP細(xì)胞的裸小鼠肺轉(zhuǎn)移模型,體式熒光顯微鏡觀察肺部轉(zhuǎn)移灶點(diǎn)結(jié)合組織切片的HE染色的結(jié)果表明,過表達(dá)RhoGDI2能夠顯著逆轉(zhuǎn)WSB1促進(jìn)骨肉瘤細(xì)胞體內(nèi)肺轉(zhuǎn)移的現(xiàn)象。研究結(jié)論：缺氧條件下,HIF-1α蛋白可轉(zhuǎn)錄激活E3泛素連接酶家族蛋白WSB1并誘導(dǎo)其蛋白表達(dá)增加,而缺氧激活的WSB1則通過其E3連接酶活性促進(jìn)底物蛋白R(shí)hoGDI2經(jīng)由泛素蛋白酶體途徑發(fā)生降解,進(jìn)而激活RhoGDI2下游信號(hào)通路,促進(jìn)肌動(dòng)蛋白Actin多聚化程度的增加和細(xì)胞偽足的形成,從而增強(qiáng)腫瘤細(xì)胞的運(yùn)動(dòng)潛能,最終介導(dǎo)了缺氧促進(jìn)的骨肉瘤轉(zhuǎn)移過程。我們的研究發(fā)現(xiàn)了缺氧促腫瘤轉(zhuǎn)移的全新信號(hào)通路HIF-1α-WSB1-RhoGDI2,可能成為繼HIF-1α-VEGF以外的另一重要缺氧調(diào)控腫瘤惡性演進(jìn)的信號(hào)通路,豐富了缺氧微環(huán)境促進(jìn)腫瘤轉(zhuǎn)移的理論體系。探索基于缺氧調(diào)控WSB1等E3連接酶活性以干預(yù)腫瘤轉(zhuǎn)移的新領(lǐng)域,并為治療骨肉瘤缺氧轉(zhuǎn)移提供了新靶點(diǎn)和新思路。
[Abstract]:Objective: osteosarcoma is the most common primary bone malignant tumor with high invasive and early metastasis. In recent years, with the improvement of operation and radiotherapy and chemotherapy, the 5 year survival rate of osteosarcoma patients has improved, but the overall cure rate has not been significantly broken. The lung metastasis caused by hypoxia is the death of the osteosarcoma patients. This is an important cause of death. Therefore, the molecular mechanism of anoxia to promote osteosarcoma metastasis is studied, and the targeted targets are found, which can provide guidance for the treatment of clinical osteosarcoma and promote the development of new target antitumor drugs. The first part: the regulation effect of hypoxia microenvironment on the expression of WSB1 protein. The study of the correlation between WSBl and malignant metastasis of osteosarcoma, we used human osteosarcoma cell lines (KHOS/NP, U2OS and MG63), primary osteosarcoma cell line (MDOS15, MDOS20) as the research object. (1) immunohistochemical analysis of the correlation between HIF-1 A and WSB1 expression; (2) the co localization of HIF-1 alpha and WSB1 protein by immunofluorescence; (3) Weste The changes of WSB1 protein level after hypoxia induction and overexpression of HIF-1 alpha were detected by RN blotting; (4) fluorescence quantitative PCR technique was used to detect the changes of WSB1 mRNA level after hypoxia induction and over expression of HIF-1 a; (5) double luciferase reporter gene detection combined with chromatin immuno sedimentation experiment to study the regulation mechanism and both of HIF-1 alpha to WSB1. Combined area; (6) Transwell test was used to detect the changes in the migration ability of osteosarcoma primary cells in anoxic environment; (7) the wild type WSB1 plasmids were constructed by molecular cloning, and wild type WSB1 and lentivirus knockdown low WSB1 were prepared. (8) Transwell combined with scratch repair experiments observed that the osteosarcoma cells were overexpressed in the wild type WSB1, respectively. And the change of migration ability after knocking down WSB1; (9) a nude mouse lung metastasis model expressing wild type WSB1 and KHOS/NP cells knocking low WSB1 was established. The lung metastasis of osteosarcoma cells in nude mice was detected by living imaging technique combined with HE staining. The second part: molecular mechanism of promoting osteosarcoma metastasis (1) using molecule Cloning of WSB1 plasmid with deletion or mutation of SOCS box domain and preparation of WSB1 lentivirus with deletion or mutation in SOCS box domain; (2) Transwell experiments were conducted to observe the changes in the migratory energy of osteosarcoma cells in the expression of wild type WSB1, SOCS box domain deletion or mutation, and (3) SILAC quantitative proteomics analysis, To find the key proteins that play a key role in the process of WSB1 regulation of osteosarcoma; (4) immunohistochemical staining analysis of the correlation between WSB1 and the expression of metastasis associated protein RhoGDI2; (5) immunofluorescence combined with immunofluorescence to prove the combination of WSB1 and RhoGDI2; (6) the effect of WSB1 on the formation ability of the membrane surface of osteosarcoma cell surface; (7) T Ime-lapse presented as a study of the regulation of WSB1 on osteosarcoma cell motility; (8) the lung metastases in nude mice that overexpressed WSB1, WSB1 and RhoGDI2, and RhoGDI2 expressed KHOS/NP cells were detected by fluorescence microscopy and HE staining to detect the pulmonary metastases in osteosarcoma cells. The first part was the result of the study. The effect of hypoxia microenvironment on the expression of WSB1 protein and the correlation between WSB1 and malignant metastasis of osteosarcoma (1) the expression of WSB1 protein in osteosarcoma cells was highly correlated with the expression of HIF-1 alpha protein. The expression of WSB1 and HIF-1 a protein in the tissue sections of 40 osteosarcoma patients was detected by immunohistochemistry and the pathological score was used as a pathological scoring system. It was calculated that the correlation coefficient R was 0.825, showing a significant positive correlation, and the immunofluorescence test showed that the expression of WSB1 and HIF-1 alpha in the transplanted tumor tissue section of the nude mice showed a good co localization state. The results showed that WSB1 might be the target gene of HIF-1 alpha. The osteosarcoma cell line was KHOS/NP, U2OS, MG63 and the original. The cells of osteosarcoma MDOS15, MDOS20 were cultured for 24 hours in the environment of normoxic and anoxic, and Western blotting detection found that the expression of WSB 1 protein increased significantly in the anoxic environment. The expression of WSB1 protein in KHOS/NP cells also increased in a concentration dependent manner under the action of DMOG, DFX and two kinds of hypoxia model drugs. Plasmid transfection method was used. HIF-1 alpha protein was overexpressed in KHOS/NP cells, and Western blotting results showed that WSB1 protein was highly dependent on the expression of HIF-1 alpha protein, and the increase of WSB1 expression induced by hypoxia was significantly reversed by small molecule RNA interference technique. The above results showed that HIF-1 alpha protein mediated the increase of WSB1 protein expression in anoxic environment (2 Under the condition of hypoxia, HIF-1 alpha protein can transcriptional activation of WSB1 and induce the increase of its protein expression, as mentioned earlier. Hypoxia induction can up regulate the expression of WSB1 protein in osteosarcoma cells, and the WSB1 protein has a positive correlation with HIF-1 alpha expression and presents a co localization state, suggesting that the classical hypoxia transcription factor HIF-1 alpha may be important in this phenomenon. In view of the fact that transcription factors normally regulate the expression of downstream genes at the transcriptional level, we first detect the mRNA level of WSB1 in KHOS/NP cells in anoxic environment and over expression of HIF-1 a by RT-PCR method. The results show that the hypoxia environment and overexpression of HIF-1 alpha can significantly increase the tnRNA level of the WSB1. Then, we construct the molecular cloning technique. The plasmids, including the WSB1 promoter region and the three HRE binding regions (-1768, -1461, -339), were separately transfected into the cells with the HIF-1 alpha plasmid. The results of the double luciferase reporter gene detection showed that HIF-1 a could obviously transcribe the activation of the WSB1 gene, and HIF-1 alpha might be the -33 by binding to the WSB1 promoter region. The transcriptional regulation of the 9 loci was further confirmed in the chromatin immunoprecipitation experiment. The results showed that HIF-1 alpha could activate the WSB1 gene transcriptional and induce the increase of its protein expression. (3) the expression level of WSB1 protein is positively correlated with the metastasis rate of clinical osteosarcoma patients through the cancer group of patients with clinical osteosarcoma. The immunohistochemical analysis of the tissue and para cancerous tissue samples showed that the positive expression rate of WSB1 in the region of cancer tissue was as high as 88%, and the positive expression of WSB1 was only 32%.. The result suggested that WSB1 could be used as a biological marker for the diagnosis of osteosarcoma and may regulate the malignant progression of osteosarcoma. The 3 year clinical follow-up records of 28 patients with osteosarcoma showed a positive correlation between the expression level of WSB1 and the metastasis rate of osteosarcoma patients. The above results showed that the expression level of WSB1 was closely related to the metastasis of the patients with osteosarcoma, suggesting that WSB1 may be involved in the metastasis of osteosarcoma mediated by hypoxia. (4) WSB1 protein. In vitro migration ability of osteosarcoma cells can be promoted to prepare wild type WSB1, lentivirus particles expressed by WSB1 protein and KHOS/NP and U2OS. In Transwell and scratch repair experiments, the expression of WSB1 protein can significantly increase the movement and migration ability of two osteosarcoma cells, while knocking low WSB1 The above results suggest that WSB1 protein can promote the ability of osteosarcoma cells to migrate in vitro. (5) WSB1 protein can promote the capacity of osteosarcoma cells in vivo to express wild type WSB1 and knock low WSB1 KHOS/NP cells and establish corresponding lung metastasis of nude mice. The results of micro-PET in vivo imaging showed that overexpression of wild type WSB1 could significantly increase the lung metastasis ability of osteosarcoma cells in nude mice. The number of HE staining points for lung tissue sections also confirmed the above results, while low WSB1 could significantly inhibit the pulmonary metastasis of osteosarcoma cells. The above results suggest that WSB1 Protein can promote the lung metastasis ability of osteosarcoma cells. Second part: WSB1 protein promotes the molecular mechanism of osteosarcoma metastasis (1) the role of WSB1 protein to promote tumor metastasis depends on its own E3 enzyme activity WSB1 protein mainly composed of seven WD40 duplication domains in N terminal and SOCS box domain of SOCS at the C end. The present study shows that SOCS box The domain can be combined with extended protein (Elongin) B and C to form E3 ubiquitin ligase complex, which is the key area for WSB1 to play E3 enzyme activity. We constructed the WSBl SOCS box domain deletion or mutation plasmid, and compared with the wild type WSB1, found that the deletion or mutation of SOCS box structure can obviously reverse the promotion of promotion. The biological function of osteosarcoma metastasis. This result shows that the biological function of WSB1 protein to promote tumor metastasis depends on its own E3 ubiquitin ligase activity. (2) SILAC quantitative proteomic analysis of WSB1 through its E3 enzyme activity regulating the downstream substrate protein of tumor metastasis, we have the WSB1 overexpression and KHOS/NP cells in the blank carrier group. SILAC quantitative proteomic analysis showed that 1078 different proteins were identified by mass spectrometry. The previous results showed that the biological effects of WSB1 on the metastasis of osteosarcoma depended on their E3 ubiquitin ligase activity. We speculate that WSB1 may play this function by degrading a transfer related protein. Based on this, we have 518 hair. The difference analysis showed that the change of RhoGDI2 was most significant, suggesting that it may act as a potential substrate protein of WSB1 to mediate the regulation of WSB1 on osteosarcoma metastasis. (3) WSB1 can promote the degradation of transfer related protein RhoGDI2 via the ubiquitin proteasome pathway through the immune group The expression of WSB1 and RhoGDI2 protein in the tissue samples of osteosarcoma patients was examined by chemical staining. The correlation coefficient R value of the two was -0.65, indicating that the expression of the two was negative correlation. The level of RhoGDI2 protein was significantly down regulated by Westren blotting detection, but the SOCS box structure of WSB1 was found. After domain deletion, the role of its down-regulation of RhoGDI2 is reversed, suggesting that WSB1 can modulate RhoGDI2o through its E3 ubiquitin ligase activity negatively to regulate the degradation of RhoGDI2 by WSB1 under the action of protein synthesis inhibitor CHX, indicating that WSB1 down regulation RhoGDI2 may be achieved by promoting its degradation, and WS under the action of proteasome inhibitor MG132. The phenomenon of B1 down regulation of RhoGDI2 was reversed, suggesting that the process of WSB1 degradation of RhoGDI2 may be via the ubiquitin proteasome pathway. Immunofluorescence experiments show that the WSB1 protein and RhoGDI2 protein present a more obvious co localization phenomenon. The results of immunoprecipitation show that WSB1 and RhoGDI2 protein can be directly associated with the protein, and WSB1 can promote the polymerization of RhoGDI2. Ubiquitination. The above results all indicate that WSB1 promotes the degradation of RhoGDI2 via ubiquitin proteasome pathway. (4) WSB1 activates the downstream signal pathway of RhoGDI2. We then examine the effect of WSB1 on RhoGDI2 regulated Rho signaling pathways. Through pull-down assay detection, overexpressed WSB1 can activate RhoGTPase. The main biological function of the s family protein member Rac1. Rac1 protein is to regulate cytoskeleton remodeling and promote cell movement. Therefore, we investigate the changes in the cytoskeleton of KHOS/NP by immunofluorescence experiments. The results show that overexpression of WSB1 can promote the aggregation of actin Actin and promote the formation of the cell membrane surface pseudo foot, thus increasing the formation of the cell membrane surface pseudo foot. The above results suggest that WSB1 can increase the motility potential of tumor cells by activating the downstream Rho signaling pathway of RhoGDI2. (5) overexpression of RhoGDI2 can reverse the metastasis promoting function of WSB1, we construct WSB1, express RhoGDI2 and simultaneously overexpress WSB1, and RhoGDI2 KHOS/NP cells. Through in vitro Transwel L test showed that overexpression of RhoGDI2 could reverse the phenomenon that WSB1 promoted osteosarcoma cell migration. Further, we constructed the lung metastasis model of the nude mice of the above KHOS/NP cells. The results of HE staining of the lung metastasis foci combined with the tissue section showed that the overexpression of RhoGDI2 could significantly reverse the WSB1 promotion of bone meat. Conclusion: under hypoxia, HIF-1 alpha protein can transcriptional activation of E3 ubiquitin ligase family protein WSB1.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R738.1

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6 汪偉;陳文直;周潔敏;白玲;劉文英;葉慧義;;高強(qiáng)度聚焦超聲治療骨肉瘤的臨床應(yīng)用研究[A];中華醫(yī)學(xué)會(huì)超聲醫(yī)學(xué)新進(jìn)展學(xué)術(shù)會(huì)議論文匯編[C];2004年

7 曹慶選;李文華;曹來賓;夏寶樞;;中藥治愈骨肉瘤的中醫(yī)研究與影像學(xué)對照[A];第十次全國中西醫(yī)結(jié)合影像學(xué)術(shù)研討會(huì)暨全國中西醫(yī)結(jié)合影像學(xué)研究與診斷學(xué)習(xí)班資料匯編[C];2009年

8 王英;肖艦;陳曉華;;骨肉瘤病人癌性疼痛的護(hù)理體會(huì)[A];全國腫瘤護(hù)理學(xué)術(shù)交流暨專題講座會(huì)議論文匯編[C];2003年

9 陳秋;王達(dá)輝;閔若良;馬巍;;大段異體骨治療兒童骨肉瘤的初步應(yīng)用[A];2005'中國修復(fù)重建外科論壇論文匯編[C];2005年

10 劉巧慧;韓莎莎;;骨肉瘤保肢患者的心理護(hù)理[A];全國外科、神經(jīng)內(nèi)外科護(hù)理學(xué)術(shù)交流暨專題講座會(huì)議論文匯編[C];2006年

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