WSB1蛋白在缺氧促进骨肉瘤转移进程中的作用及其机制研究

发布时间：2018-05-24 09:17

本文选题：缺氧微环境 + 骨肉瘤转移　；参考：《浙江大学》2016年博士论文

【摘要】：研究目的：骨肉瘤是最常见的原发性骨恶性肿瘤,侵袭性高且血行转移早。近年来随着手术以及放化疗水平的提高,骨肉瘤患者的5年生存率有所提升,但是总体治愈率仍未出现显著突破。由于缺氧而引发的肺转移是造成骨肉瘤患者死亡的一个重要原因。因此,深入研究缺氧促进骨肉瘤转移的分子机制,并从中发现可干预的靶点,有望为临床骨肉瘤的治疗提供指导并推动新型靶向抗肿瘤转移药物的研发。研究方法：第一部分：缺氧微环境对WSB1蛋白表达的调控作用以及WSBl与骨肉瘤恶性转移的相关性研究本研究采用人源性骨肉瘤细胞系(KHOS/NP、U2OS以及MG63)、原代骨肉瘤细胞株(MDOS15、MDOS20)作为研究对象。(1)免疫组化分析HIF-1α与WSB1表达的相关性;(2)免疫荧光考察HIF-1α与WSB1蛋白的共定位情况;(3)Western blotting检测WSB1蛋白水平在缺氧诱导以及过表达HIF-1α后的变化;(4)荧光定量PCR技术检测WSB1 mRNA水平在缺氧诱导以及过表达HIF-1α后的变化;(5)双荧光素酶报告基因检测结合染色质免疫共沉淀实验研究HIF-1α对WSB1的调控机制及两者的结合区域;(6)Transwell实验检测骨肉瘤原代细胞株在缺氧环境中迁移能力的变化；(7)采用分子克隆手段构建野生型WSB1质粒,制备野生型WSB1以及可敲低WSB1的慢病毒;(8)Transwell结合划痕修复实验观察骨肉瘤细胞分别在过表达野生型WSB1以及敲低WSB1后迁移能力的变化;(9)建立过表达野生型WSB1及敲低WSB1的KHOS/NP细胞的裸小鼠肺转移模型,通过活体成像技术结合HE染色检测骨肉瘤细胞在裸小鼠体内的肺转移情况。第二部分：WSB1促进骨肉瘤转移的分子机制研究(1)采用分子克隆手段构建SOCS box结构域缺失或突变的WSB1质粒,并制备SOCS box结构域缺失或突变的WSB1慢病毒;(2)Transwell实验观察骨肉瘤细胞分别在过表达野生型WSB1、SOCS box结构域缺失或突变情况下迁移能力的变化;(3)应用SILAC定量蛋白质组学分析,寻找在WSB1调控骨肉瘤转移过程中发挥关键作用的蛋白;(4)免疫组化染色分析WSB1与转移相关蛋白RhoGDI2表达的相关性;(5)免疫沉淀结合免疫荧光证明WSB1与RhoGDI2的结合;(6)免疫荧光检测WSB1对于骨肉瘤细胞膜表面伪足形成能力的影响;(7)Time-lapse呈像研究WSB1对于骨肉瘤细胞运动能力的调控作用;(8)建立过表达WSB1、同时过表达WSB1及RhoGDI2、过表达RhoGDI2的KHOS/NP细胞的裸小鼠肺转移模型,通过荧光显微镜观察结合HE染色检测骨肉瘤细胞的体内肺转移情况。研究结果：第一部分：缺氧微环境对WSB1蛋白表达的调控作用以及WSB1与骨肉瘤恶性转移的相关性研究(1) 骨肉瘤细胞中WSB1蛋白与HIF-1α蛋白表达呈高度相关采用免疫组化法检测40例骨肉瘤患者组织切片中WSB1与HIF-1α蛋白的表达情况并作病理学评分统计,计算得出两者的相关系数R值为0.825,呈现显著的正相关性;免疫荧光实验验证裸小鼠荷骨肉瘤移植瘤组织切片中WSB1与HIF-1α的表达呈现良好的共定位状态。以上结果表明,WSB1可能是HIF-1α的靶基因。将骨肉瘤细胞系KHOS/NP、U2OS、MG63及原代骨肉瘤细胞MDOS15、MDOS20分别置于常氧及缺氧环境中培养24小时,Western blotting检测发现WSB 1蛋白表达在缺氧环境中显著增加。在DMOG, DFX两种缺氧造模药物的作用下,KHOS/NP细胞中WSB1蛋白表达水平也呈现浓度依赖性增加。采用质粒转染法在KHOS/NP细胞中过表达HIF-1α蛋白,Western blotting结果显示WSB1蛋白高度依赖于HIF-1α蛋白的表达,而通过小分子RNA干扰技术沉默HIF-1α后,缺氧诱导的WSB1表达增加被显著逆转。以上结果表明,缺氧环境中HIF-1α蛋白介导了WSB1蛋白表达的增加。(2)缺氧条件下HIF-1α蛋白可转录激活WSB1并诱导其蛋白表达增加如前所述,缺氧诱导可上调骨肉瘤细胞中WSB1蛋白表达,且WSB1蛋白与HIF-1α表达具有正相关性并呈现共定位状态,提示经典的缺氧转录因子HIF-1α可能在这一现象中有重要的作用。鉴于转录因子通常可在转录层面调控下游基因的表达,我们首先采用RT-PCR法检测缺氧环境中及过表达HIF-1α后KHOS/NP细胞内WSB1的mRNA水平,结果显示缺氧环境及过表达HIF-1α均可显著上调WSB1的tnRNA水平。然后,我们通过分子克隆技术构建包括WSB1启动子区域以及三个HRE结合区域(-1768位点,-1461位点,-339位点)分别突变的质粒并与HIF-1α质粒共同转染至细胞中,双荧光素酶报告基因检测结果表明HIF-1α可明显转录激活WSB1基因,且HIF-1α可能是通过结合在WSB1启动子区域的-339位点实现对其的转录调控,并在染色质免疫共沉淀实验中得到了进一步确证。以上结果表明HIF-1α可转录激活WSB1基因并诱导其蛋白表达增加。(3) WSB1蛋白的表达水平与临床骨肉瘤患者的转移率呈正相关性通过对临床骨肉瘤患者的癌组织及癌旁组织样本进行免疫组化分析发现,WSB1在癌组织区域的阳性表达率高达88%,且多呈强阳性;而在癌旁组织区域中,WSB1的阳性表达率仅为32%。这一结果提示WSB1可作为骨肉瘤诊断的生物学标记物,并且可能调控骨肉瘤的恶性进展。通过回顾性调研28位骨肉瘤患者的3年临床随访纪录,发现WSB1的表达水平与骨肉瘤患者的转移率呈现正相关性。以上结果表明WSB1的表达水平与临床骨肉瘤患者的转移密切相关,提示WSB1可能参与介导了缺氧促进的骨肉瘤转移。(4)WSB1蛋白可促进骨肉瘤细胞的体外迁移能力制备野生型WSB1以及可敲低WSB1蛋白表达的慢病毒颗粒并感染骨肉瘤细胞株KHOS/NP以及U2OS,在Transwell以及划痕修复实验中发现过表达WSB1蛋白能够显著增加两株骨肉瘤细胞的运动迁移能力;而敲低WSB1则能够逆转缺氧促进的骨肉瘤细胞体外迁移能力。以上结果提示WSB1蛋白可促进骨肉瘤细胞的体外迁移能力。(5)WSB1蛋白可促进骨肉瘤细胞的体内肺转移能力构建过表达野生型WSB1及敲低WSB1的KHOS/NP细胞株并建立相应的裸小鼠肿瘤肺转移模型,micro-PET活体成像结果显示,过表达野生型WSB1能够显著增加骨肉瘤细胞在裸小鼠体内的肺部转移能力,对肺组织切片进行HE染色统计转移灶点数也证实以上结果;而敲低WSB1则能够明显抑制骨肉瘤细胞的肺部转移。以上结果提示WSB1蛋白可促进骨肉瘤细胞的肺转移能力。第二部分：WSB1蛋白促进骨肉瘤转移的分子机制研究(1)WSB1蛋白促进肿瘤转移的作用依赖于其自身E3酶活性WSB1蛋白主要由N端的七个WD40重复结构域和C端的SOCS box结构域构成。已有研究表明,SOCS box结构域能够与延伸蛋白(Elongin) B和C相互结合,形成E3泛素连接酶复合物,是WSB1发挥E3酶活性的关键区域。我们构建了WSBl SOCS box结构域缺失或突变的质粒,并与野生型WSB1进行比较,发现SOCS box结构域的缺失或突变均能够明显逆转WSB1促进骨肉瘤转移的生物学作用。这一结果表明WSB1蛋白促进肿瘤转移的生物学功能依赖于其自身E3泛素连接酶活性。(2) SILAC定量蛋白质组学分析WSB1通过其E3酶活性调控肿瘤转移的下游底物蛋白我们对WSB1过表达及空白载体组的KHOS/NP细胞进行SILAC定量蛋白质组学分析,质谱鉴定得到1078个变化三倍以上的差异蛋白。前期结果表明WSB1促进骨肉瘤转移的生物学效应依赖其E3泛素连接酶活性,我们推测WSB1可能通过降解某个转移相关蛋白而发挥这一功能。基于此,我们对518个发生下调的蛋白进行重点考察。差异分析报告显示转移相关蛋白RhoGDI2变化最为显著,提示其可能作为WSB1潜在的底物蛋白介导了WSB1对于骨肉瘤转移的调控过程。(3)WSB1可促进转移相关蛋白RhoGDI2经由泛素蛋白酶体途径发生降解通过免疫组化染色对骨肉瘤患者组织样本中WSB1与RhoGDI2蛋白的表达水平进行考察,分析发现两者的相关系数R值为-0.65,表明两者表达呈现负相关性。通过Westren blotting检测发现,在WSB1过表达的情况下RhoGDI2蛋白水平显著下调。但是,当WSB1的SOCS box结构域缺失后,其下调RhoGDI2的作用即被逆转,提示WSB1可通过其E3泛素连接酶活性负性调控RhoGDI2o在蛋白合成抑制剂CHX的作用下,WSB1能够加速RhoGDI2的降解,表明WSB1下调RhoGDI2可能是通过促进其降解而实现。而在蛋白酶体抑制剂MG132的作用下,WSB1下调RhoGDI2的现象被逆转,提示WSB1降解RhoGDI2的过程可能经由泛素—蛋白酶体通路。免疫荧光实验表明WSB1蛋白与RhoGDI2蛋白呈现较明显的共定位现象,免疫沉淀结果显示WSB1与RhoGDI2蛋白能够直接发生结合,并且WSB1能够促进RhoGDI2的多聚泛素化修饰。以上结果均表明WSB1可促进转移相关蛋白RhoGDI2经由泛素—蛋白酶体途径发生降解。(4)WSB1可激活RhoGDI2下游信号通路我们接着考察了WSB1对RhoGDI2调控的Rho信号通路的影响。通过pull-down实验检测发现,过表达WSB1能够激活RhoGTPases家族蛋白成员Rac1。 Rac1蛋白的主要生物学功能为调控细胞骨架重构从而促进细胞运动。因此,我们通过免疫荧光实验考察KHOS/NP细胞骨架的变化。结果显示,过表达WSB1能够促进肌动蛋白Actin的多聚化并促进细胞膜表面伪足的形成,从而增强细胞的运动迁移能力。以上结果表明,WSB1可通过激活RhoGDI2下游Rho信号通路增加肿瘤细胞的运动潜能。(5)过表达RhoGDI2能逆转WSB1的转移促进功能我们构建了过表达WSB1、过表达RhoGDI2及同时过表达WSB1、与RhoGDI2的KHOS/NP细胞。通过体外Transwell实验检测发现,过表达RhoGDI2能够逆转WSB1促进骨肉瘤细胞迁移的现象。进一步,我们构建上述KHOS/NP细胞的裸小鼠肺转移模型,体式荧光显微镜观察肺部转移灶点结合组织切片的HE染色的结果表明,过表达RhoGDI2能够显著逆转WSB1促进骨肉瘤细胞体内肺转移的现象。研究结论：缺氧条件下,HIF-1α蛋白可转录激活E3泛素连接酶家族蛋白WSB1并诱导其蛋白表达增加,而缺氧激活的WSB1则通过其E3连接酶活性促进底物蛋白RhoGDI2经由泛素蛋白酶体途径发生降解,进而激活RhoGDI2下游信号通路,促进肌动蛋白Actin多聚化程度的增加和细胞伪足的形成,从而增强肿瘤细胞的运动潜能,最终介导了缺氧促进的骨肉瘤转移过程。我们的研究发现了缺氧促肿瘤转移的全新信号通路HIF-1α-WSB1-RhoGDI2,可能成为继HIF-1α-VEGF以外的另一重要缺氧调控肿瘤恶性演进的信号通路,丰富了缺氧微环境促进肿瘤转移的理论体系。探索基于缺氧调控WSB1等E3连接酶活性以干预肿瘤转移的新领域,并为治疗骨肉瘤缺氧转移提供了新靶点和新思路。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738.1

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