【作者】 孔丽；

【导师】 于修平； 刘传聚；

【作者基本信息】 山东大学 ， 病原生物学， 2007， 博士

【摘要】 宫颈癌是女性生殖系统最常见的恶性肿瘤,在世界范围内发病率仅次子乳腺癌居第二位,每年约有超过50万新发病例,且引起较高的死亡率（1）。其中80%在发展中国家（2）,中国每年约有新发病例13.15万,占世界新发病例的约30%。近年来,年轻病例有逐年增加的趋势,发病年龄趋向年轻化,成为严重威胁中青年妇女健康的重大妇科肿瘤之一（3）。Southern转印杂交和PCR检测人乳头瘤病毒（human papillomavirus,HPV）检出阳性率达89-93%（4）,大量的研究资料表明HPV感染是诱发宫颈癌的首要启动因素（5,6）,因此,加强HPV致宫颈癌机制的研究是有效防治宫颈癌的前提。人乳头瘤病毒（Human papillomavirus,HPV）是双链DNA病毒,具有嗜上皮特性,能够感染皮肤与粘膜的上皮组织并形成增生性疾病（7）。高危型HPV（HPV16、18、33型等）与宫颈癌等恶性肿瘤的发生密切相关,低危型HPV（HPV 6、11型）可导致尖锐湿疣等良性病变（8）。HPV基因组为7.9kb的双链环状DNA,其早期区（E区）含有7个开放读码框（E1-E7 ORF）,编码与病毒复制、转录调控和细胞转化有关的早期蛋白。晚期区（L区）编码两种结构蛋白L1和L2,组成病毒的衣壳。HPV的转化基因主要包括病毒的早期基因E6和E7,该基因编码的蛋白产物与抑癌蛋白p53和pRb的相互作用是HPV致癌的主要机制,使细胞分化、增殖和凋亡紊乱,诱发肿瘤的形成（9）。一般认为,HPVE6与P⁵³结合,通过泛素依赖的蛋白水解酶将P⁵³降解,使P⁵³失去对细胞周期密切相关的P²¹（WAF1）、增殖细胞核抗原（PCNA）、细胞周期蛋白依赖激酶（CDK）的调节作用,E7蛋白干扰抑癌蛋白RB与转录因子E2F的结合,从而导致细胞增殖周期的紊乱,诱导细胞癌变（9-12）。随着研究的深入,不断发现一些矛盾的现象,在一些HPV阳性宫颈癌细胞中,P⁵³的DNA修复活性并未消失,提示HPV的E6蛋白不足以完全灭活P⁵³活性,突变的P⁵³也不能代替E6基因以维持HPV₁₆诱导的NIH3T3细胞的生长特性,因此HPVE₆致癌肯定存在非P⁵³依赖的途径。利用酵母双杂交表达系统发现一种细胞钙结合蛋白可以特异性与高危型HPVE6蛋白结合,即E6结合蛋白（E6 binding pmtem,E6BP）两者共同存在于内质网上,用FISH技术将E6BP基因定位于15q22.33-q24.1,即使P⁵³途径中E6结合并降解P⁵³也需E6相关蛋白,即E6BP参与,它是一种泛素激酶（9）,E6的一种突变体不能与P⁵³、E6BP、E6AP和端粒酶结合,但可以致细胞转化,表明细胞存在其它多种与HPVE6转化相关蛋白（13）,HPVE6还可以作用于Mcm7干扰细胞DNA复制（14）,作用于端粒酶影响染色体的结构（15,16）,作用于E6TP（17）影响信号转导,作用于C-Myc、Bax而抑制细胞凋亡（18-20）,E7也可通过非Rb途径影响细胞信号转导（21）。近来研究发现提示HPV还有新的靶分子和致癌途径,HPVE6和E7基因在增殖分化、转录调节、端粒酶活化和凋亡调节中涉及细胞内多个潜在靶目标,因此解码HPV原癌基因对细胞信号转导干扰的分子标志,并对其结构功能和表达调控方式进行研究是揭示宫颈癌发生机制的重要途径,目前该领域的研究还非常零碎和不完整,必须运用高通量的生物学技术从整体化的角度对该类问题进行研究。随着人类基因组测序工作的完成,生命科学的研究进入了后基因组时代,即功能基因组学时代,蛋白质组学（proteomics）是这一前沿研究的主要组成部分,从整体水平研究细胞内蛋白质的组成及其活动规律。由于蛋白质是生物细胞赖以生存的各种代谢和调控途径的主要执行者（22）,因此可更全面地认识蛋白质影响细胞生物学行为的方式和过程,为揭示HPV致癌机理提供了有效的技术体系。本研究运用基于固体pH梯度等电聚焦双向电泳（Two-dimensional gelelectrophoresis;2-DE）、肽质量指纹图谱鉴定技术（Peptide Massspectrometry analysis）和生物信息学（Bioinformatics）等蛋白质组学技术体系,鉴定了HPV16转化的人宫颈上皮永生化细胞系H8和人宫颈癌细胞系Caski的差异蛋白,RbAp48作为其中与肿瘤发生密切相关的差异表达蛋白质,引起了我们极大的兴趣并以此为研究目标展开了深入的功能研究。RbAP48,作为一种Rb（retinoblastoma-binding protein）结合蛋白（23）,是从涉及不同染色质集合、装配以及核小体修饰复合物,包括组蛋白乙酰转移酶（histonedeacetylase;HDAC）分离出的复合物中第一个被鉴定的高丰度蛋白（24,25）。本研究在国内外第一次系统全面的揭示了RbAP48与宫颈癌发生的密切关系,体内细胞水平和体外动物实验研究表明RbAP48是调控宫颈癌发病过程HPV转化活性的至关重要的分子,为全面深刻的认识HPV致癌机理从而更好的预防防治奠定基础。一、RbAp48在宫颈癌中的低表达利用双向电泳技术对正常永生化细胞H8及宫颈癌细胞Caski总蛋白的分离,并通过蛋白质组学技术分析,我们成功鉴定出20个表达有差异的蛋白质。其中RbAp48作为一个在肿瘤细胞中低表达的蛋白引起我们极大的兴趣。首先我们运用real-time PCR及western blot技术分别从RNA和蛋白水平上验证了其表达在H8中明显高于在Caski中的表达,然后又利用其他宫颈癌细胞系如SiHa、HeLa以及宫颈角质细胞HCK上也证实了RbAp48的表达在宫颈癌细胞中的普遍性。同时我们搜集临床上其他肿瘤组织如乳腺癌、卵巢癌、胃癌等相关的肿瘤及正常组织,试图发现RbAp48的表达在这些肿瘤中是否也有差异。Western blot分析显示在这些肿瘤组织中,RbAp48的表达没有明显差别,这证实了RbAp48的低表达在宫颈癌中的特异性。二、RbAp48的表达变化可以影响细胞的增殖分化及体外肿瘤形成利用RNAi（RNA干扰技术）构建能有效降低细胞内源性RbAp48表达的质粒pSUPER-RbAp48,将其转入H8建立稳定转染的细胞系H8+siRbAp48,同时将pSUPER空载体转染H8细胞作为对照组建立稳定细胞系。通过比较分析两种稳定细胞系的增殖分化等的差异,可以明显观察到转染pSUPER-RbAp48的H8细胞能够增加增殖细胞线粒体能量代谢过程中乳酸脱氢酶的释放,从而促进细胞能量代谢;能够促进细胞分裂增殖,细胞数目增加;增加细胞软琼脂克隆生长的数量;同时明显降低反映细胞衰老死亡指标的β-细胞半乳糖苷酶的活性。这些实验都表明降低细胞内源性的RbAp48的表达可以明显促进体外细胞水平的增殖分化并抑制细胞的死亡衰老。将稳定转染pSUPER-RbAp48和pSUPER空载体的细胞分别皮下注射裸鼠,四周后裸鼠体内肿瘤形成的情况表明,由于RbAp48本身并不具备致瘤性,因此转染pSUPER空载体的细胞注射的裸鼠体内没有肿瘤形成:而稳定转染pSUPER-RbAp48细胞注射的6只裸鼠中有5只体内均长出肿瘤。为了进一步研究RbAp48在肿瘤形成过程中的功能,我们从另一方面增加RbAp48的表达,观察RbAp48的过表达对体内体外肿瘤发生的影响。我们分别转染了RbAp48的表达质粒及pcDNA 3.1到Caski细胞中建立稳定细胞系,进行细胞增殖实验分析。MTT分析、细胞生长曲线、以及软琼脂克隆形成试验均表明过表达RbAp48明显降低细胞的生长率,β-细胞半乳糖苷酶分析显示细胞衰老率增加。体外裸鼠肿瘤形成试验表明在宫颈癌细胞Caski中增加RbAp48的表达后,细胞的致瘤性明显降低,裸鼠体内形成的肿瘤明显缩小。这些都表明RbAp48作为一种新发现的调控人乳头瘤病毒HPV16阳性的宫颈癌转化活性的重要蛋白,能够抑制肿瘤细胞的生长,降低其致瘤性,结合最近研究报道RbAp48作为一种肿瘤放射敏感性蛋白,提示RbAp48可以作为一种HPV相关疾病的治疗靶位用于临床应用,为宫颈癌的预防和治疗提供一种新的方向和理论依据。三、RbAp48通过调节其他癌基因和抑癌基因的表达来影响肿瘤的发生为了进一步探讨RbAp48影响宫颈癌的机制,我们分别研究了其对肿瘤癌基因HPVE6、E7,c-myc,抑癌基因Rb,P53,以及Rb/E2F靶位基因的表达影响。通过将RbAp48利用siRNA干扰之后,我们发现HPVE6、E7以及c-myc基因的表达均上升,而抑癌基因Rb和p53的表达则明显下降。另外我们还检查了Rb/E2F靶位基因的表达情况包括Cyclin A,Cyclin E,Cyclin D1,proliferating cell nuclear antigen（PCNA）和p16INK4a,结果发现只有Cyclin D1的表达有所升高,也许是因为RbAp48通过影响Cyclin D1的表达来影响宫颈癌的分化。四、ECM1在软骨发育中抑制软骨发育的作用另外,我们通过酵母双杂交试验在研究COMP（Cartilage oligomericmatrix protein,软骨寡聚基质蛋白）蛋白这一基质蛋白在软骨发育中的作用时,用COMP蛋白的EGF片段做诱饵,筛选出来了ECM1（extracellular matrixprotein 1,细胞外基质蛋白1）这一细胞外基质蛋白。通过研究其与COMP的相互作用,我们明确了二者在体内外相互结合作用的模式,并发现ECM1具有抑制软骨发育的作用,更重要的是ECM1是PTHrP（Parathyriod hormone relatedpeptide,甲状旁腺激素相关蛋白）这一重要的软骨发育的抑制因子的重要下游分子,因为PTHrP能够明显诱导ECM1的表达,而且抑制了ECM1的表达之后,PTHrP也失去了抑制软骨发育的作用。更多还原

【Abstract】 Cervical cancer is one of the most common neoplastic diseases among women, with a combined worldwide incidence of approximately one-half million new cases annually and rates of morbidity and fatality second only to breast cancer(1).In addition,in recent years the average cervical cancer patient has become progressively younger(3).Over 99%of cervical carcinomas are positive for human papillomavirus (HPV)DNA(4),indicating that HPV infection is the most important cause for cervical cancer(5,6).Thus it is of great importance to elucidate the mechanism by which HPV induces cervical cancer from both the pathophysiological and the therapeutic standpoint.HPV is a double-stranded DNA virus(26)that affects skin and mucosa epithelia and induces hyperplasia,such as in benign hyperplastic verruca and malignant tumors. Although HPV infection appears to be the prerequisite for causing the great majority of cases of cervical cancer,the molecular events following HPV infection of cervical mucosa epithelial cells remain largely unknown.The transforming genes of HPV include the early genes E6 and E7,whose coding proteins bind tumor suppressor proteins p53 and Rb.These bindings results in an imbalance between proliferation and apoptosis,thereby inducing cervical cancer(9).Growing evidence suggests that in addition to p53 and Rb,various HPV E6- and E7-associated molecules and pathways are involved in cell proliferation,differentiation,transcription regulation,telomerase activation and apoptosis(14,15,17,21).Identification of novel target molecules participating in HPV-induced tumourgenesis will provide the foundation for better understanding the mechanisms of cervical cancer.Proteomics has been shown to be a powerful approach to isolate the target molecules that are involved in various biological processes(22)and it also has been successfully used to identify proteins involving tumorigenesis(27-30).The transformation from human cervical mucosa epithelial cells to cervical cancer cells has been attributed to abnormal expression of oncogenes,tumor suppressor genes,growth factors,growth factor receptors,cell adhesion factors,and DNA repair genes as detected by northern blot,differential display PCR,and Cdna chips based on transcription level(31).Because mRNA expression level does not always correlate with protein level as a result of posttranslation regulation,and because proteins are the executor of genetic information,proteomics provides a powerful,straightforward tool for studying transformation of cells during carcinogenesis.In this study we utilized a combination of two-dimensional gel electrophoresis(2-DE),image analysis,mass spectrometry,and bioinformatics to quantify and characterize differentially expressed proteins between the HPV16 immortalized human cervical mucosa epithelial H8 cells and cervical cancer Caski cells and identified retinoblastoma-binding protein 4(RbAp48,also referred to as RbBP4)as one of the 18 differentially expressed proteins.RbAp48,initially identified as a retinoblastoma-binding protein(23),was characterized as a highly abundant component of various chromatin assembly, remodeling,and distinct nucleosome-modifying complexes,including the nuclear histone deacetylases(24,25).RbAp48 is best known as a component of large chromatin-associated complexes recruited by tumor suppressor Rb and most likely acts in concert with other proteins to modify histones and/or remodel nucleosomes(32, 33);furthermore,the Rb/RbAp48-associated histone deacetylase complex has been found to be involved in transcriptional repression of E2F responsive genes(34-36).It was reported that E2F-1 and RbAp48 are physically associated in the presence of Rb and histone deacetylase(37),suggesting that RbAp48 could be involved in the transcriptional repression of E2F-responsive genes,p55,the Drosophila ortholog of RbAp48,has been shown to be required for the repression of dE2F2/RBF-regulated genes(38).Fission yeast centromere protein Mis15,which bears a strong resemblance to human RbAp48,is part of the CENP-A recruitment pathway and forms an evolutionarily conserved complex that includes Mis6(39).RbAp48 was also isolated as one of three radiosensitive genes in a microarray analysis used for selecting radiosensitivity prediction molecules and RbAp48-overexpression-induced radiosensitization in HS-578T,MDA-MB-231,and MALME-3M cells,two breast cancer and a melanoma cell line,respectively,when compared with mock-transfected cell lines(40).Our studies provided first evidence linking RbAP48 to cervical cancer and both in vitro and in vivo studies demonstrated that RbAP48,previously unknown in cervical cancer,is a novel and critical mediator that control HPV16 transforming activity in cervical cancinogenesis1.Downregulation of RbAp48 and other components of RbAp48 complex in cervical cancer cells is specific for cervical cancer.Among all proteins identified,RbAp48 is of particular interest to us,since this protein is a highly abundant component of various chromatin assembly,remodeling, and distinct nucleosome-modifying complexes,in which it associates with tumor supressor retinoblastoma protein(Rb)and was recently isolated a radiosensitive marker genes in several cancer cell lines(40).To verify the altered expression between HPV16 immortalized cervical mucosa epithelial H8 cells and cervical cancer Caski cells,a Western blotting assay was performed with cell extracts prepared from these two cell lines.There was a robust reduction of RbAp48 protein in cervical cancer Caski cells compared to HPV16 immortalized cervical mucosa epithelial H8 cells. These findings were further verified in human native tissues,the similar expression pattern for RbAp48 was demonstrated in native tissues as that in cell lines.In addition, reduced expression of RbAp48 in cervical cancer was further confirmed with additional cervical cancer-derived cell lines,including Hela and SiHa cells when compared to the primary cervical keratinocytes(HCK)cells.To determine whether downregulation of RbAp48 is also true for other human cancers,we next examined the expression of RbAp48 in breast cancer,ovarian cancer and gastric cancer,and no significant differences of RbAp48 expression between cancers and corresponding control tissues were observed,indicating that reduced expression of RbAp48 might be specific for cervical cancer.2.Inhibition of RbAp48 expression via siRNA-mediated silencing results in significant stimulation of cell proliferation and colony formation and a reduction in cellular senescence in vitro as well as leading to tumor formation in nude mice, while RbAp48 overexpression inhibits cell proliferation in vitro and tumor growth in vivoTo determine whether a specific level of RbAp48 is required for preventing the transformation of HPV-infected cervical epithelial cells to cervical cancer cells,we first suppressed RbAp48 gene expression in H8 cells using an siRNA approach.We generated stable lines beating either pSUPER-RbAp48 or pSUPER vector based on H8 cells with a high level of endogenous RbAp48,and then investigated the effects of reduced RbAp48 on cell proliferation,colony formation,and cellular senescence. Both an MTT assay and cell number counting revealed that reduction in RbAp48 resulted in enhanced cell proliferation;in addition,reduced RbAp48 significantly increased colony formation,as revealed by a soft agar assay.Senescence-associatedβ-galactosidase is widely used as a biochemical marker for cellular senescence(41),and our staining results showed that knockdown of RbAp48 expression in H8 cells resulted in an approximately 30%inhibition of the cell senescence-like phonotype.To investigate the biological consequence of RbAp48 inactivation in tumorigenesis,we utilized nude mouse xenografts.Stable H8 cells bearing siRbAp48 and control cells were injected into 6-week-old female nude mice.As expected,none of the mice injected with the control cells developed tumors within 4 weeks of injection,whereas mice injected with H8 cells stably transfected with pSUPER-RbAp48 developed tumors of considerable size within the same time frame, demonstrating that RbAp48 is a critical modulator of the transforming action of HPV16 in cervical carcinogenesis.To further define the function of RbAp48 in cervical carcinogenesis and to determine whether it has the potential for use as a therapeutic target in cervical cancer treatment,we next generated stable cell lines overexpressing RbAp48 in Caski and HeLa cervical cancer cells.We then examined whether overexpression of RbAp48 inhibits cell proliferation and colony formation.MTT assay,cell number counting, and soft agar assay indicated this to be the case.In addition,significant higherβ-galactosidase activity was observed in the RbAp48 stable lines than in corresponding control and parent Caski or HeLa cell lines.These findings suggest that RbAp48 overexpression suppresses the transformation phenotype and reduces the tumorigenicity of cervical cancer cells in vivo.To test that possibility,we injected nude mice subcutaneously with stable cells overexpressing RbAp48 in Caski cells as well as control cells.Tumor growth was substantially inhibited in the RbAp48 group as compared with the control group,indicating that the level of RbAp48 is crucial for carcinogenesis in cervical cancer.3.To elucidate the molecular events underlying RbAp48-controlled transforming activity of HPV16 in cervical cancer,we examined the effect of RbAp48 on tumor suppressors p53 and Rb,apoptosis-related enzymes caspase-3 and caspase-8, oncogene c-myc,HPV E6/E7,and some Rb/E2F targets genes,including PCNA, cyclin E,cyclin A,and p16INK4a,Cyclin D1.We found that the levels of p53 and two other apoptosis-related enzymes caspase-3 and caspase-8 were dramatically reduced in cervical mucosa epithelial cells when RbAp48 was inhibited via siRNA approach.Altered expression of RbAp48 significantly affect the mRNA levels of E6 and E7 in the transfected cells.Furthermore,cyclin D1 was selectively induced, whereas other well-documented Rb/E2F targets genes,including PCNA,cyclin E, cyclin A,and pl6INK4a were not particularly affected by reduced RbAp48.It is conceivable that regulation of tumor suppressor p53,caspase enzymes and Rb/E2F targets genes,especially CylinD1,by RbAp48 contribute,at least in part,to the RbAp48-mediated transforming activity of HPV16 in cervical carcinogenesis.In conclusion,RbAp48 was isolated in a global analysis of protein expression profiling based on 2-dimensional gel electrophoresis with mass spectrometry and identified as critical modulator of the transforming activity of HPV16 in cervical cancer in both vitro and in vivo experiments.Identification of RbAp48 as a novel critical mediator in cervical cancer as well as the elucidation of the molecular events involved provide insights into the processes regulating HPV16-induced carcinogenesis and may highlight a new research direction in understanding pathological mechanisms for cervical cancer.Considering its potent inhibition of tumor growth,RbAp48 also has great potential to be employed as a therapeutic target for treating HPV-related disorders,including cervical cancer.4.In a functional genetic screen for proteins associating with cartilage oligomeric matrix protein,we identified a novel extracellular matrix protein,ECM1(extracellular matrix protein 1),a molecule that had previously been linked to lipoid proteinosis and lichen sclerosus,a common chronic inflammatory condition.We verified this novel interaction using in vitro pulldown and in vivo coimmunoprecipitation assays and demonstrated that these two proteins colocalized on the cellular surface of primary human chondrocytes.Using RT-PCR and western blotting assays,we showed that both mRNA and protein levels of ECM1 were upregulated during differentiation of chondrocytes.ECM1 significantly inhibited mRNA levels of both early and later genes critical for chondrogenesis such as Sox9,collagen typeⅡ,and collagen X and repression of ECM1 via the siRNA silencing markedly enhanced the expression of Collagen X in the course of chondrogenesis.To determine the potential mechanisms by which ECM1 negatively regulates chondrogenesis,we studied the effects of ECM1 on PTHrP(an essential negative regulator for later chondrogenesis)and IHH(a positive regulator of chondrogenesis).ECM1 induced PTHrP 5- to 18-fold and inhibited IHH during chondrogenesis.We further showed that PTHrP induced mRNA levels of ECM1 in chondrocytes.Importantly,knocking down ECM1 mRNA levels via the siRNA silencing or blocking ECM1 protein activity via anti-ECM1 antibodies completely abolished the effects of PTHrP on chondrogenesis in vitro.Using an immunohistochemistry assay,we showed that ECM1 was expressed throughout the chondrocyte zone of growth plates and articular cartilage and that ECM was no longer detectable in PTHrP null growth plates at day E18.5 in vivo.Last,we demonstrated that both mRNA and protein levels of ECM1 were upregulated in arthritis using microarray,real-time PCR and western blotting assays.Our findings demonstrate for the first time that ECM1,a direct downstream molecule of PTHrP in cartilage,is a novel negative regulator of chondrocyte differentiation and suggest that ECM1 may also plays an important role in the pathology of arthritis.更多还原