【作者】 张潍；

【导师】 辛晓燕； 王建；

【作者基本信息】 第四军医大学 ， 妇产科学， 2009， 博士

【摘要】 研究背景:宫颈癌是妇科高发恶性肿瘤,虽然宫颈癌的防治已取得很大成效,但近年来由于宫颈癌的发病率明显增高,目前,宫颈癌的治疗采用以手术和放射治疗为主,同时辅以化疗的综合治疗,因此不可避免的会给患者,尤其是尚未生育的育龄妇女带来巨大痛苦。如何更加安全、有效地早期预防宫颈癌发生;在常规手术的基础上,增加宫颈癌放、化疗敏感性,成为当前研究的热点与难点。近20年来,有关宫颈癌研究的最大进展即证明了HPV感染同宫颈癌发生关系密切。在所有宫颈癌病例中,明确由HPV16、18型引起的分别占总体的60%和72%,而99.7%的宫颈癌组织中存在HPV DNA。因此,从病因学讲,宫颈癌是可预防、可阻断、可治疗的。目前,国内外有关宫颈癌生物治疗的研究多从如何有效预防、治疗HPV感染的角度着手。高危型HPV16、18的E6、E7蛋白疫苗、L1病毒样颗粒疫苗已经问世,到2006年1月份为止,超过3000例患者参与的大规模II期临床实验已在国外相继完成,HPV疫苗对宫颈上皮内瘤样变(CIN)的预防及治疗有效率可达到95%～100%,显示了良好的治疗前景。但就目前而言,HPV疫苗在安全性、免疫源性、HLA相容性、经济性等方面也存在着一些不可回避的问题;另外,HPV疫苗的安全性评价得经过最少25年的临床观察才能给予最终定论。因此,以治疗HPV感染为切入点的针对宫颈癌的治疗的研究尚未取得突破性进展,仍需要进一步补充和完善。PKR是一种广泛存在于哺乳动物细胞中的丝氨酸/苏氨酸蛋白激酶,通常在细胞中以潜在和非激活状态存在,但可以被低浓度的双链RNA(dsRNA)如病毒复制过程中产生的dsRNA所激活,进而磷酸化其底物真核起始因子的α亚基(eIF-2α),磷酸化的eIF-2α抑制蛋白质合成,包括病毒蛋白的合成,诱导被感染细胞凋亡,在控制病毒复制中起着非常重要的作用。最近证实,PKR同时是一个重要的凋亡效应子,所以一些研究者开始关注是否PKR的调节是一种新的抗肿瘤策略。研究发现:PKR能被转录因子E2F-1直接诱导上调,也能通过与某些细胞因子直接的蛋白质-蛋白质相互作用而活化;另外,通过转染与肿瘤特异RNA序列互补的反义RNA而形成细胞内的dsRNA,能诱导PKR活化和选择性针对这些肿瘤细胞的凋亡。预示了PKR用于抗肿瘤治疗新靶点的美好前景。目前,关于宫颈癌中PKR、以及下游通路中重要功能蛋白eIF-2α的表达与活化水平还缺乏实质性的研究成果及文献报道。我们通过预实验发现:未受到HPV病毒感染的宫颈癌细胞中,PKR的表达与活化与其他肿瘤中的情况完全一致;在受到HPV感染的宫颈癌组织及细胞中,无活性PKR的表达与其他肿瘤中报道的情况相同,处于高表达水平,但其活性状态——磷酸化PKR却受到严重抑制。因此我们推断,①由于受到特殊病原体HPV的影响,PKR在宫颈癌组织中活化能力下降,进而限制其抵抗病毒感染、促进肿瘤细胞凋亡的作用;②如果能采用导入dsRNA的方法将其激活,打破与HPV病毒间的相对“平衡”,将产生强大的抗病毒与抗肿瘤效果。然而对肝炎等与病毒密切相关疾病研究发现,由于病毒表达不同蛋白成分对PKR的负调控作用,使得PKR对长链dsRNA的应答反应表现不一。结合预实验的发现,我们认为,如果选择其他的治疗靶位联合激活PKR途径的策略,势必将达到治疗宫颈癌更优的效果。同时,如果靶位选择得当,PKR诱导产生的细胞凋亡也能够具有相对的特异性。Aurora-A激酶在正常表达水平具有调节中心体、微管功能,从而对有丝分裂起关键作用。但在其发生过度表达时通常伴随肿瘤发生,并且表达水平与肿瘤的临床分期、细胞分化、转移等情况密切相关。但它与宫颈癌发生、发展的关系目前尚未见报道。在食道癌与HPV的研究中证实,HPV能够上调Aurora-A的表达;其他实验发现,Aurora-A在过度表达后通过抑制p53等蛋白功能,造成细胞周期检查点功能缺陷,诱导细胞发生永生化。而p53是PKR通路发挥抗肿瘤作用的重要途径;在国内外针对Aurora-A联合其他靶位用于肿瘤治疗的研究中,已有成功的先例。因此,我们推断:在与HPV病毒感染关系密切的宫颈癌中,Aurora-A极有可能出于过度表达状态;如果抑制Aurora-A过表达,保护p53的正常功能状态,将更好的发挥PKR通路的作用;同时,由于Aurora-A的过度表达仅发生于增殖速度明显异常的细胞,因此,针对Aurora-A的靶向治疗,具有一定的肿瘤特异性。研究目的:1.从细胞水平和组织水平明确PKR在宫颈不同病理状态下表达的差异与活性状态;2.了解宫颈正常与癌变情况下Aurora-A蛋白的表达水平,并结合病例分析,探讨其与宫颈癌的关系;3.探讨RNAi策略阻断抑制Aurora-A蛋白表达对宫颈治疗的可行性;4.探讨小片段RNA激活PKR通路的可能性与实用价值。研究方法:1.利用免疫组化、Western blotting、实时定量PCR等方法检测不同病理状态下宫颈组织,及不同宫颈细胞株中PKR通路中上游调节蛋白及下游主要作用产物的水平,研究在宫颈病变逐渐演进中PKR通路的表达水平与活性状态。2.利用免疫组化、Western blotting、实时定量PCR等方法检测正常宫颈组织与宫颈癌组织标本中Aurora-A蛋白的表达差异,并结合病例资料分析,明确Aurora-A与宫颈癌的关系。3.使用RNA干扰的手段对Hela细胞中Aurora-A基因实施特异性抑制,并通过Western blotting、实时定量PCR的方法检测其抑制效率。4.对已构建的Aurora-A的siRNA序列进行修饰,并再次转染细胞,观察其抑制效率及对PKR通路的激活能力。使用MTT方法比较常规设计的siRNA及修饰后的siRNA对Hela细胞增殖抑制的能力。研究结果:1.正常宫颈、CIN I、CIN II、CIN III、宫颈鳞状上皮癌、宫颈腺癌组织中均有PKR及其通路中多个蛋白的表达,表达存在不同程度差异,并且与HPV感染有关;2.在未感染HPV病毒的宫颈癌细胞株C33A中,PKR蛋白的表达与活化处于较高水平,最终体现在作用底物eIF-2α处于较高的活性状态;此发现与目前报道的PKR在其他肿瘤中的表达相一致;3.由于HPV病毒的影响,宫颈癌组织以及HPV(+)的宫颈癌细胞系中,PKR通路中的各种功能蛋白在宫颈癌中的表达与现已报道的大多数肿瘤中的表达有较大差异;实验中发现,HPV能够通过以下途径抑制PKR通路:在转录水平降低PKR mRNA的表达;提高PKR抑制物p58IPK的水平;降低PKR激活物PACT的表达,抑制PKR的活化;并通过直接和间接的途径抑制其作用底物eIF-2α的活化过程。4.在人类宫颈癌组织中存在Aurora-A过度表达的情况,细胞实验也证明,宫颈癌细胞株中Aurora-A表达明显升高;5.经过统计学分析,发现Aurora-A的过度表达与宫颈癌发生发展有密切关系,其中与肿瘤的FIGO分期、肿瘤细胞分化程度、癌肿浸润程度以及有无远处转移等因素呈正相关关系;而与年龄、肿瘤类型等情况无相关关系;6.成功构建Aurora-A的siRNA片段,并通过实时定量PCR证实抑制了Aurora-A的mRNA表达,蛋白印迹实验证实蛋白表达水平下降,综合说明构建的常规siRNA片段有明显抑制Aurora-A蛋白表达的效果;7. MTT实验中转染了Aurora-A siRNA的Hela细胞增殖受到明显抑制,证实Aurora-A能够用于宫颈癌治疗的潜力;8.成功构建了用于干涉Aurora-A含polyA尾的siRNA,将其转染Hela细胞后,实时定量PCR和蛋白印迹实验说明同样对Aurora-A蛋白具有抑制作用;9.通过蛋白印迹实验,证实修饰后的siRNA片段具有激活PKR系统的能力,但是作用较微弱;10. MTT实验中,转染了修饰后siRNA的Hela细胞增殖水平比转染常规siRNA细胞的更低,可能是被激活的PKR系统非特异的抑制细胞蛋白合成的结果。结论:1.通过对各个功能蛋白的分析,我们认为,PKR通路的调控与HPV病毒有密切关系;由于宫颈癌普遍合并HPV病毒的感染,因而表现出与目前已经报道的人类大多数肿瘤中PKR的高表达不一致;2.人类宫颈癌由于同时兼有肿瘤和特殊病原体感染的特性,使PKR通路表现出更复杂的多样性;因此,在与HPV密切相关的宫颈癌组织中,有丰富的PKR被严重抑制,处于无功能状态,如能打破这种相对平衡,将充分发挥PKR通路的抗病毒与抗肿瘤作用;3. Aurora-A蛋白过度表达于子宫颈癌中,并且其过度表达的状态与宫颈癌的预后密切相关;4. RNA干涉后造成Aurora-A蛋白表达下降,并抑制宫颈癌Hela细胞株的增殖,显示出干涉Aurora-A表达用于宫颈癌治疗的潜力;5.通过对小干扰RNA进行修饰的手段,可能会使siRNA获得对PKR的激活能力;联合激活PKR通路与抑制Aurora-A蛋白过度表达的方式具有更强的抑制细胞增殖的作用;6.在siRNA反义链3,端加入polyA时可以出现对PKR较弱的激活作用,但其机制与实用性尚待进一步研究。更多还原

【Abstract】 Background:Cervical cancer remains as one of the biggest fillers of women worldwide. Despite significant achievements in the treatment of cervical cancer, it is still a deadly disease that readily metastasize in the earlier period, hence newer therapeutical modalities are needed."High risk" genotypes of the human papillomavirus(HPV), most commonly HPV genotype 16/18, are the primary etiologic agents of cervical cancer. Indeed HPV DNA is detected in 99.7% of cervical carcinomas. Thus, cervical cancer and other HPV-associated malignancies might be prevented or treated by the induction of appropriate viral-antigen-specific immune responses. Two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation, therapeutic vaccines targeting E6 and E7 may provide the best opportunity to control HPV associated malignancies. Various candidate therapeutic HPV vaccines are currently being test in clinical trials, some trials are still ongoing. But, the results of the trials are not as desirable as expected.PKR, the protein kinase regulated by double-stranded RNA (dsRNA), is well established as an important component of the host response to viral infection. The N-terminal region of PKR includes a repeated domain that confers dsRNA-binding activity, and the C-terminal region possesses subdomains necessary for kinase catalytic activity. In addition to the autophosphorylation of PKR that occurs during the dsRNA-mediated autoactivation process, the best-characterized PKR substrate is theαsubunit of protein synthesis eukaryotic initiation factor 2 (eIF-2α). PKR catalyzes the phosphorylation of eIF-2αon serine 51, a modification that alters the translation pattern within cells and leads to an inhibition of protein synthesis under stress conditions, including virus infection . PKR also modulates nuclear factorκB (NF-κB)-mediated signal transduction processes in response to dsRNA.Studies have provided evidence that PKR plays an important role in a variety of physiologic processes, including cell proliferation and death, in addition to the role that PKR plays in the antiviral actions of interferons. But the expression level of PKR in cervical caner have not been described.Aurora-A is a centrosome-associated gene,the amplification or high expression of Aurora-A cause centrosomal anomalies,anuploidy formation and cell transformation .The expression level of Aurora-A in most normal tissues remains low,but the overexpression of Aurora-A is a common event in tumor.Recently,the studies on Aurora-A expression in pancreatic tumor,esophageal cancer,gastric cancer and some other tumors were more, and the RNA interference targeting aurora kinase could suppresses tumor growth in some kinds of human cancer cells. But the study in cevical neoplasm were little.Objective:1. To investigate the expression of PKR in different tissues and cells from normal cervix or cervical cancer;2. To observe the expression level of Aurora-A in cervical tissues and cells, and investigate whether Aurora-A expression correlates with clinicopathologic factors and prognosis of cervical carcinoma patients;3. To observe the bionomics effect in Hela cell by inhibition expression of Aurora-A with RNAi targeting;4. To investigate the feasibility of activation PKR by modified siRNA.Methods:1. Differential expression and activition of PKR was examined by RT-PCR, Western blotting and Immunohistochemistry analysis in different cervical tissues and cell lines;2. Differential expression of Aurora-A was examined by RT-PCR, Western blotting and Immunohistochemistry analysis in different cervical tissues and cell lines; to analyze whether Aurora-A expression correlates with clinicopathologic factors and prognosis of cervical carcinoma patients with statistics;3. Inhibition the expression of Aurora-A with RNAi targeting, and the reproduction of Hela cell were tested by MTT method; and the inhibition effect of siRNA was detected by the RT-PCR, Western-blotting;4. To modify the the siRNA oligonucleotide with single-stranded 3’-poly(A) tails at antisense strands, further more, evaluate its effect on downregulation the expression of Aurora-A and activation of PKR.Result:1. The expression and activation of several proteins in PKR-way are in different level and concerned with HPV infection in normal cervix, CIN I, CIN II, CIN III and cervical cancer;2. The expression level of PACT, the important reactivator of PKR, is direct correlated with the progress in cervical pathological changes, and whether the cancer cell be infected by HPV;3. The expression level of p58IPK, the inhibition factor of PKR, is in same condition as PACT;4. PKR over expressed in cancer tissues, and the expression level is associate with HPV;5. The expression level of p-PKR is upregulated in CIN I~II, but downregulated in cancer tissue with HPV infection. High-risk HPV could depress the activation of PKR;6. The expression and activation level of eIF-2α, the most important substrate of PKR, are similar with the level of PKR in cervical tissue;7. Aurora-A is over expressed in human cervical cancer, just like the condition in other cancers;8. The overexpression of Aurora-A is associated with FIGO stage, tumor differentiation, parametrial invasion and lymphnode or hematogenous metastasis, but not other clinicopathological factors.9. Successfully construct the siRNA of Aurora-A, which been proved could suppress Aurora-A overexpression in Hela cell; 10. By MTT analysis, Aurora-A siRNA has been proved could reduce proliferation of Hela cell;11. Although very weak, modified siRNA shows the ability to activate PKR by Western blotting, and the mechanism of the ability needs more explore.Conclusion:1. The expression and activation level of PKR are associated with HPV infection. PKR over expresses in tumor cell lines without HPV infection;2. It is shown that PKR over expressed and phosphorylated in HPV(-) cell line(C33A), just as in other cancers;3. In cervical tissue and HPV(+) cell lines, the expression and activation of PKR are suppressed by HPV in several ways: such as up regulation the level of p58IPK, reducing transcription of PKR mRNA and suppressing the phosphorylation of eIF-2α;4. Aurora-A over expresses in cervical cancer, and the level of expression correlate with the prognosis of cervical cancer patients;5. Aurora-A siRNA could reduce the expression of Aurora-A and the proliferation in Hela cell, which indicate the feasibility of Aurora-A siRNA being used into treating cervical cancer;6. The siRNA, modified with poly A tail, could enhance silencing effect by activating PKR, but the mechanism is still unclear.更多还原