【作者】 梁静；

【导师】 卞美璐；

【作者基本信息】 北京协和医学院 ， 妇产科学， 2011， 博士

【摘要】 E2-EPF是E2泛素结合酶家族成员之一,分子量为24KD,在多种肿瘤组织中高表达。E2泛素结合酶同E1泛素激活酶,以及E3水解酶一起,催化蛋白质泛素化,进而被蛋白酶小体降解。肿瘤抑制因子pVHL是E3泛素水解酶复合体的一部分,它可使HIF等降解。E2-EPF泛素结合酶可使pVHL发生水解,进而使得HIF-1(?)表达水平增强。HIF-1(?)作为细胞低氧应答反应中起核心作用的转录因子,能调节100多种与低氧应激下细胞适应和存活相关的靶基因。HIF-1(?)是参与肿瘤增殖、血管增生、转移等过程的重要分子。E2-EPF泛素结合酶表达水平增高,可通过pVHL-HIF-1(?)途径促进癌细胞增殖、侵袭和转移。E2-EPF在宫颈癌细胞中的作用尚未见报道,本研究旨在阐明E2-EPF在宫颈癌细胞中的表达水平和功能。主要研究内容如下：一、E2-EPF在宫颈鳞状细胞癌组织中高表达选取了宫颈鳞状细胞癌标本75例,正常宫颈组织13例为研究对象,采用实时荧光定量PCR技术检测E2-EPF mRNA表达水平,结果发现宫颈癌组织中E2-EPF mRNA水平是正常宫颈组织的4.08倍；Ⅱ期以上宫颈癌组织中E2-EPF的mRNA表达水平是Ⅰ期的2.72倍,二者均具有显著差异。由此可见,E2-EPF在宫颈癌组织中也呈高表达状态,并且与肿瘤细胞的恶性程度有一定的相关。二、宫颈癌细胞系SIHA E2-EPF低表达克隆的建立采用商品化E2-EPF shRNA质粒载体下调细胞内源性E2-EPF。通过脂质体将E2-EPFshRNA质粒载体转染入SIHA细胞,并通过嘌呤霉素筛选获得了稳定的E2-EPF低表达克隆,共筛选获得15个E2-EPF低表达克隆,mRNA水平下降可达到正常SIHA细胞的18%左右,蛋白水平降至正常水平的20%左右。三、E2-EPF下降后导致HIF-1(?)的表达下降由于HIF-1α在常氧状态下极不稳定,常规的方法不易检测到HIF-1α的存在。本实验采用荧光素酶报告系统成功的检测到微量的HIF-1α。结果显示,E2-EPF的表达与HIF-1a成正相关,低表达克隆细胞内HIF-1a水平较正常对照组明显降低。四、E2-EPF下调后对细胞周期、增殖及侵袭性生物学特性的影响采用MTT细胞增殖实验检测发现,E2-EPF低表达克隆細胞的增殖速度显著低于正常和对照细胞。软琼脂克隆形成实验发现,E2-EPF低表达克隆细胞其克隆形成能力远远低于正常和对照细胞。以流式细胞术检测细胞周期发现,E2-EPF低表达克隆细胞的GO-G1期细胞百分比显著增加,S和G2-M期(尤其是S期)细胞数减少,细胞增殖减慢。采用经典的Transwell小室研究E2-EPF对细胞侵袭能力的影响发现,E2-EPF低表达克隆细胞穿膜细胞较正常和对照细胞明显减少。本部分研究结果说明,E2-EPF下调后对SIHA细胞的细胞周期、增殖及侵袭性等生物学行为具有显著的影响。五、E2-EPF下调对放、化疗的影响细胞经0Gy,4 Gy,7 Gy,10 Gy4个不同剂量X线照射后,采用MTT法检测细胞相对数量,发现不同剂量下,E2-EPF低表达克隆细胞的存活数量和正常、对照组细胞没有显著差异。采用Annexin-V.PI联合的流式细胞术检测细胞凋亡发现,各个剂量下,细胞凋亡和死亡的数量在各组之间没有差异。说明E2-EPF下调后,对细胞的放疗耐受性没有显著影响。采用不同机理化疗药物：顺铂、紫杉醇、多烯紫杉醇、拓扑替康、依托泊苷、表阿霉素,以不同浓度分别加入各组细胞中,观察E2-EPF下调后是否对化疗药物疗效有一定影响。采用MTT法检测细胞存活的相对数量,结果发现：降调内源性E2-EPF水平,可以增加细胞对拓扑异构酶Ⅰ抑制剂——拓扑替康,拓扑异构酶Ⅱ抑制剂——依托泊苷和表阿霉素的敏感性,但是对顺铂、紫杉醇和多烯紫杉醇的作用无明显影响。以上试验说明,E2-EPF下降后增加了部分化疗药物的疗效,可以作为宫颈癌化疗联合用药的一种增效途径。六、E2-EPF下调后裸鼠成瘤能力显著下降本实验通过接种一定数量的肿瘤细胞,发现E2-EPF低表达组肿瘤生长速度减慢、瘤体大小减小。E2-EPF低表达组平均瘤体重量仅相当于对照组的1/4,这就提示E2-EPF在肿瘤的发展中具有重要的作用。体内实验进一步说明降调E2-EPF的表达可以作为晚期宫颈癌生物学治疗的重要方法。结论：E2-EPF在宫颈癌组织中成高表达状态,且其表达量的高低和临床肿瘤分级具有一定的相关性。通过RNAi下调E2-EPF的表达之后,HIF-1(?)的表达随之下调；细胞的增殖能力下降、细胞周期中Go-G1期细胞数显著增加；细胞克隆形成能力下降、侵袭力下降；裸鼠成瘤性下降；并能增强化疗药物拓扑异构酶抑制剂的疗效。E2-EPF是参与宫颈癌发生和发展的重要分子,针对E2-EPF的治疗手段可望成为宫颈癌治疗的新途径。更多还原

【Abstract】 E2-EPF ubiquitin carrier protein is a 24-kDa protein that is a member of the E2 family of ubiquitin-conjugating enzymes, which has been showed to be highly expressed in many tumor tissues. E2-EPF, E1 ubiquitin-activating enzyme and an E3 ubiquitin ligase, catalyze the addition of ubiquitin to substrate proteins. Polyubiquitinylation of the substrate protein can target that protein for proteasome dependent destruction. Von Hippel-Lindau (VHL) tumor suppressor gene product is a part of an active E3 ubiquitin ligase complex. In the presence of oxygen, hypoxiainducible factor(HIF) is targeted for destruction by the E3 ubiquitin ligase complex containing the VHL tumor suppressor protein (pVHL).E2-EPF target pVHL for proteosomal degradation under normoxic conditions resulting in the stabilization of hypoxia inducible factor 1α(HIF-1α). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that controls the expression of a battery of more than 100 target genes whose protein are expressed in responses to cellular and systemic hypoxia. HIF-1αhas been showed to be associated with increased tumor growth, angiogenesis and tumor metastasis. Thereafter, increased expression of E2-EPF in multiple cancers was reported to promote tumor growth, invasion and metastasis by the pVHL-HIF-1αpathway.Role of E2-EPF in cervical tumors has not been reported until now. In this study, our objectives are to explore the expression level of E2-EPF in cervical tumor and its function. The followings are studied:E2-EPF is overexpressed in cervical tumor specimensTotally,13 normal cervical tissues, and 75 cervical carcinoma tissues were analysed. Relative expression levels of were determined by a Taqman real-time PCR system. E2-EPF was significantly overexpressed in tumors relative to normal tissues. The average relative expression level in tumors is 4.08 folds of the normal tissue and expression level of E2-EPF in tumor tissues of clinical stage ofⅡ-Ⅳis 2.72 folds of tumor tissues in stage I. Both are statistically significant. Therefore E2-EPF is also high expressed in cervical cancer, and its expression level is associated with the clinical stage of tumor.Establishment of E2-EPF low expression cloneCommercial available E2-EPF shRNA vector was used to down-regulate the expression of E2-EPF. E2-EPF shRNA plasmid vector was transfected into the SIHA cell line with lipofectamine reagent and the low expression clone was screened by puromycin selection.15 low expression clones were obtained. The lowest expression level of E2-EPF of the obtained clones was 18% of the normal in mRNA and 20% of the normal in protein respectively.Decreased expression of E2-EPF resulting in lower expression of HIF-1αBecause HIF-1αis extremely unstable in normoxia, it is hard to detect its protein level by routine method. In this section, we use the HIF1 Luciferase Reporter Vector to detect the presence of HIF1. Results showed that the expression level of E2-EPF was positively correlated with the HIF-1αexpression level. In the E2-EPF low expression clone, HIF-1αexpression was significantly lower than the normal and control cells.Effects of E2-EPF knock down on cell cycle, cell growth and invasionThe cell growth of E2-EPF low expression clone was significantly lower than the normal and control cells detected by MTT cell proliferation kit. In the soft agarose clone formation assay, the clones formed by the E2-EPF low expression clone cell were much lower than the normal and control cells. Cell cycle analysis by flow cytometry showed that the percentage of G0-G1 phase cells of E2-EPF low expression clone was increased and the percentage of S, G2-M phase (particularly S phase) cells was decreased. Using the classic transwell cell invasion assay, reduced cell invasion ability of E2-EPF low expression clone was observed. These results showed that E2-EPF knock down has significant negative effects on the cell growth, cell cycle and invasion.Effects of E2-EPF knock down on cell sensitivity to radiotherapy and chemotherapy Radiotherapy of OGy,4 Gy,7 Gy,10 Gy dosage were given to cells and MTT cell proliferation assay kit was used to evaluate the cell number. Results showed that the relative survival cell number of the E2-EPF low expression clone was not significant different from the normal and control cells. Annexin-V、PI kit combined with flow cytometer were used to detect the apoptosis level of each dosage, a similar results of the apoptosis and necrosis cell number were showed.Different kinds of chemotherapeutic drugs (Cisplatin, Doxorubicin, Paclitaxel, Doxetaxel, Topotecan, Etoposide)were added to the cell culture media to observe the effect of E2-EPF knock down on the sensitivity of cells to these agents. MTT assay was used to evaluate the relative cell number. Results showed that E2-EPF knock down increased the cell sensitivity to the topoisomerase 1 inhibitor (Topotecan) andⅡ(Etoposide and Doxorubicin) and no effect on the sensitivity to Cisplatin, Paclitaxel, Doxetaxel. These results indicate that down regulation of E2-EPF can increase the cell sensitivity to topoisomerase inhibitors and can be used in combination to improve therapeutic effect of chemotherapy.E2-EPF knock down showed decreased tumorigenesis abilityCertain no. of tumor cells were s.c. inoculated into nude mice to monitor the tumor development. Weight of tumors from E2-EPF low expression clone cells was about 1/4 of the control cells. These results indicate that E2-EPF play an important role in tumor development in vivo. Targeting E2-EPF pathway may be an effective biotherapy method of late stage cervical cancer.In conclusion:E2-EPF is overexpressed in cervical tumor specimens and its expression level positively correlated with the clinical stage. Down regulation of E2-EPF by RNAi results in a decreased expression level of HIF-1α. E2-EPF knockdown decreases the growth, clone formation, cell invasion and tumorigenesis of SIHA cell line and increases the chemosensitivity to topoisomerase inhibitors. E2-EPF plays an important role in the tumorigenesis and development. Targeting of E2-EPF pathway may be a new therapeutic way of cervical tumor treatment.更多还原