RNA干扰沉默Aurora-A表达与激活PKR信号转导系统治疗宫颈癌的探索性研究
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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。
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.
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。
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.
引文
1. Di Paolo JA, Alvarez-Salas LM. Advances in the development of therapeutic nucleic acids against cervical cancer [J]. Expert Opin Biol Ther. 2004, 4(8): 1251-1257.
2. H.D.L.Birley. Human papillomaviruses, cervical cancer and the developing world. Annals of tropical medicine and parasitology, 1995, 89: 453-463.
3.卞美璐,刘树范.《子宫颈疾病的诊治》[M].科学技术文献出版社,2001年5月:68-73
4.许廷贵,工敬石,张惠珍. HPV的分子生物学特征[J].国外医学病毒学分册. 2004 11(3): 4
5.巴瑞琪,丁晓华.宫颈病变人乳头瘤病毒感染及型别分布的研究[J].华中科技大学学报(医学版). 2000,35( 6): 838-840
6.李键,薄莉,门新华.人乳头瘤病毒及行为因素与子宫颈癌的关系[J].中国性医学. 2005,14( 6): 12
7. Barnard P, McMillan NA. The human papillomavirus E7 oncoprotein abrogates signaling mediated by interferon-alpha. Virology. 1999 Jul 5;259(2):305-313.
8. Barnard P, Payne E, McMillan NA. The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-alpha. Virology. 2000 Nov 25;277(2):411-419.
9. Park JS, Kim EJ, Kwon HJ, Hwang ES, Namkoong SE, Um SJ. Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis. J Biol Chem. 2000 Mar 10; 275(10):6764-6769.
10. Um SJ, Rhyu JW, Kim EJ, Jeon KC, Hwang ES, Park JS. Abrogation of IRF-1 response by high-risk HPV E7 protein in vivo. Cancer Lett. 2002 May 28;179(2):205-212.
11. Perea SE, Massimi P, Banks L. Human papillomavirus type 16 E7 impairs the activation of the interferon regulatory factor-1. Int J Mol Med. 2000 Jun; 5(6): 661-6.
12. Nees M, Geoghegan JM, Hyman T, Frank S, Miller L, Woodworth CD. Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-kappaB-responsive genes in cervical keratinocytes. J Virol. 2001 May; 75(9): 4283-4296.
13. Pietenpol JA , Stewart ZA. Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis. Toxicology, 2002, 27 (1812182): 4752481.
14. Bartek J, Lukas J. Mammalian G1- and S- phase checkpoints in response to DNA damage. J. Curr Opin Cell Biol ,2001 ,13 (6) :738-747.
15. Narayanan BA, Geoffroy O, Willingham MC, et al. p53/p21 (WAF1P CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells. Cancer Lett, 1999, 136 (2) : 215-221.
16. Tan S, Wang TS. Analysis of fission yeast primase defines the checkpoint responses to aberrant S phase initiation. Mol Cell Biol, 2000, 20 (21): 7853-7866.
17. Foray N, Marot D, Randrianarison V, et al. Constitutive association of BRCA1 and c-Abl and its ATM-dependent disruption after irradiation. Mol Cell Biol, 2002, 22 (12): 4020-4032.
18. Yang J, Yu Y, Hamrick HE, et al. ATM, ATR and DNA-PK: initiators ofthe cellular genotoxic stress responses. Carcinogenesis, 2003, 24 (10): 1571-1580.
19. Duckworth BC, Weaver J S, Ruderman JV. G2 arrest in Xenopus oocytes depends on phosphorylation of cdc-5 by protein kinase A. Proc Natl Acad Sci USA, 2002, 99 (26): 16794-16799.
20. Qiu L, Burgess A, Fairlie DP, et al. Histone deacetylase inhibitors trigger a G2 checkpoint in normal cells that is defective in tumor cells. Mol Biol Cell, 2000, 11 (6): 2069-2083.
21. Lew DJ, Burke DJ. The spindle assembly and spindle position checkpoints. Annu Rev Genet, 2003, 37: 251-282.
22. Kraft C, Herzog F, Gieffers C, et al. Peters JM. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. EMBO J, 2003, 22 (24): 6598-6609.
23. Wentzensen N, Vinokurova S, von Knebel Doeberitz M. Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. Cancer Res. 2004 Jun 1;64(11):3878-3884.
24.张立妍,许妍,张爱荣.人乳头瘤病毒感染与宫颈癌的关系[J].山东医药.2005, 45(33): 68
25. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990 Apr 6;248(4951):76-79.
26. KatherA, Ferrara A,Nonn M , et al. Identification of a naturally processed HLA-A 0201 HPV 18E7 T cell epitope by tumor cell mediated in vitro vaccination [J]. Int J Cancer, 2003, 104: 345-353.
27. Classon M, Dyson N. p l07 and p130 versatile p roteinswith interestingpockets [J]. Exp Cell Res, 2001, 264: 135-137.
28. d′Adda di Fagagna F, Reaper PM, Clay2Farrace L, et al. A DNA damage checkpoint response in telomere-initiated senescence [J]. Nature, 2003, 426 (6963): 194-198.
29. Nanduri S, Rahman F, Willians B, et al . A dynamically tuned double-stranded RNA binding mechanism for the activation of antiviral kinase PKR [J].EMBO J,2000, 19 (20): 5567-5574.
30. Gupta V,Huang X,Patel RC.The carboxy-terminal,M3 motifs of PACT and TRBP have opposite effects on PKR activity [J]. Virology, 2003, 315(2): 253-291.
31. Francois C, Duverlie G, Rebouillat D, et al. Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis [J]. J Virol, 2000, 74(12): 5587-5596.
32. Langland JO, Jacobs BL. The role of the PKR-inhibitory genes,E3L and K3L,in determining vaccinia virus host range [J]. Virology, 2002, 299 (1):133-141.
33. Ward SV, Samuel CE. Regulation of the interferon-inducible PKR kinase gene: the KCS element is a constitutive promoter element that functions in concert with the interferon-stimulated response element [J]. Virology, 2002, 296 (1): 136-146.
34. Hebner C. M, Wilson R, Rader J, et al. Human papillomaviruses target the double-stranded RNA protein kinase pathway [J]. J GEN Virol, 2006, 87 (Pt 11): 3183-3193.
35.贾因棠,魏来,蒋栋等,双链RNA激活的蛋白激酶(PKR)的克隆表达及其对丙型肝炎病毒蛋白合成的抑制作用.中国生物化学与分子生物学报, 2006, 22(1)24~30.
36. Taylor DR, Shi ST, Romano PR, Barber GN, et al. Inhibition of the interferon-inducible protein kinase PKR by HCV E2protein. Science, 1999, 285:107-110.
37. Christy M. Hebner, Regina Wilson, Janet Rader, et al. Human papillomavirusestarget the double-stranded RNA protein kinase pathway. J Gen Virol, 2006, 87:3183-3193.
38. Ryman KD,White LJ,Johnston RE,et al. Effects of PKR/RNase L-dependent and alternative antiviral pathways on alphavirus replication and pathogenesis [J]. Viral Immunol, 2002, 15 (1):53-76.
39. Ben-Asouli Y, Banai Y, Pel-Or Y, et al.Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR [J]. Cell, 2002, 108 (2): 221-232.
40. Vattem KM, Staschke KA, Wek RC. Mechanism of activation of the double-stranded-RNA-dependent protein kinase, PKR : role of dimerization and cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (eIF2) [J]. Eur J Biochem, 2001, 268 (13):3674-3684.
41. Vattem KM,Staschke KA,Zhu S,et al.Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase,PKR,are important for regulating phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) [J]. Eur J Biochem, 2001, 268 (4): 1143-1153.
42. Gil J , Alcami J , Esteban M . Activation of NF-kappaB by the dsRNA-dependent protein kinase,PKR involves the I-kappaB kinase complex.Oncogene,2000,19:1369-1378.
43. Samuel CE. Antiviral actions of interferons. Clin Microbiol Rev. 2001 Oct;14(4):778-809, table of contents.
44. Sundar SS, Cornall RJ, Kehoe ST. Advances in the management of cervical cancer [J]. J Br Menopause Soc, 2005, 11(3): 91-95.
45. Nguyen, M., Song, S., Liem, A., et al. A mutant of human papillomavirus type 16 E6 deficient in bindingα-helix partners displays reduced oncogenic potential in vivo. J Vrol, 2002, 76:13039-13048.
46. Kazemi S, Papadopoulou S, Li S, et al. Control of alpha subunit of eukaryotic translation initiation factor 2 (eIF2 alpha) phosphorylation by the human papillomavirus type 18 E6 oncoprotein: implications for eIF2 alpha-dependent gene expression and cell death[J]. Mol Cell Biol, 2004, 24(8): 3415-3429.
47. Savinova O, Joshi B, Jagus R. Abnormal levels andminimal activity of the dsRNA-activated protein kinase, PKR, in breast carcinoma cells. Int J Biochem Cell Biol. 1999 Jan; 31(1):175-189.
48. Kim SH, Forman AP, Gunnery S. Mathews cells contain elevated levels Human breast cancer activity of the protein kinase PKR. Oncogene. 2000 Jun 22; 19 (27):3086-3094.
49. Shir Alexei, mbar Friedrich and Alexander Levitzki. Tumor specific activation of PKR as a non-toxic modality of cancer treatment. Semin Cancer Biol. 2003 Aug;13(4): 309-314.
50. Donze O., Jagus R., Koromilas A. E., Hershey J. W. and Sonenberg N. Abrogation of translation initiatibn factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J. 14 (1995): 3828-3834.
51. Kumar A, Yang YL and Flati V. Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1and NF- kappaB. EMBO J. 1997 Jan 15;16(2):406-416.
52. Giet R, Prigent C. Aurora/Ipl l p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. J Cell Sci. 1999; 112(Pt 21):3591-3601.
53. Bischoff JR, Plowman GD. The Aurora/Ipllp kinase family: regulators of chromosome segregation and cytokinesis. Trends Cell Biol. 1999; 9(11):454-459.
54. Kufer TA, Sillje HH, Korner R, Gruss OJ, Meraldi P, Nigg EA. Human TPX2 is required for targeting Aurora-A kinase to the spindle. J Cell Biol. 2002; 158(4):617-623.
55. Tanner MM, Tirkkonen M, Kallioniemi A, Collins C, Stokke T, Karhu R, Kowbel D, Shadravan F, Hintz M, Kuo WL. Increased copy number at 20q 13 in breast cancer: defining the critical region and exclusion of candidate genes. Cancer Res. 1994; 54(16):4257-4260.
56. Kallioniemi A, Kallioniemi OP, Piper J, Tanner M, Stokke T, Chen L, Smith HS, Pinkel D, Gray JW, Waldman FM. Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc Natl Acad Sci U S A. 1994; 91(6):2156-2160.
57. De Angelis PM, Clausen OP, Schjolberg A, Stokke T. Chromosomal gains and losses in primary colorectal carcinomas detected by CGH and their associations with tumour DNA ploidy, genotypes and phenotypes. Br J Cancer. 1999; 80(3-4):526-535.
58. Courjal F, Cuny M, Rodriguez C, Louason G, Speiser P, Katsaros D, Tanner MM, Zeillinger R, Theillet C.DNA amplifications at 20q13 and MDM2 define distinct subsets of evolved breast and ovarian tumours. Br J Cancer. 1996; 74(21):1984-1989.
59. Farruggio DC, Townsley FM, Ruderman JV. Cdc20 associates with the kinase aurora2/Aik. Proc Natl Acad Sci U S A. 1999; 96(13):7306-7311.
60. Dutertre S., Descamps S., Prigent C. On the role of aurora-A in centrosome function. Oncogene, 2002, 21(40):6175-6183.
61. Sugimoto K, Urano T, Zushi H, moue K, Tasaka H, Tachibana M, Dotsu M. Molecular dynamics of Aurora-A kinase in living mitotic cells simultaneously visualized with histone H3 and nuclear membrane protein importinalpha. Cell Struct Funct. 2002; 27(6):457-467.
62. T. Wittmann, M. Wilm, E. Karsenti, I. Vernos, TPX2, a novel Xenopus MAP involved in spindle pole organization, J. Cell. Biol. 149 (2000) 1405–1418.
63. T.A. Kufer, H.H. Sillje, R. Korner, O.J. Gruss, P. Meraldi, E.A. Nigg, Human TPX2 is required for targeting Aurora-A kinase to the spindle, J. Cell. Biol. 158 (2002) 617–623.
64. R. Bayliss, T. Sardon, I. Vernos, E. Conti, Structural basis of Aurora-A activation by TPX2 at the mitotic spindle, Mol. Cell 12 (2003) 851–862.
65. P.A. Eyers, E. Erikson, L.G. Chen, J.L. Maller, A novel mechanism for activation of the protein kinase Aurora A, Curr. Biol. 13 (2003) 691–697.
66. A. Hutterer, D. Berdnik, F. Wirtz-Peitz, M. Zigman, A. Schleiffer, J.A. Knoblich, Mitotic activation of the kinase Aurora-A requires its binding partner Bora, Dev. Cell 11 (2006) 147–157.
67. Marumoto T, Zhang D, Saya H. Aurora-A - a guardian of poles. Nat Rev Cancer. 2005; 5( I ):42-50.
68. D.M. Glover, M.H. Leibowitz, D.A. McLean, H. Parry, Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles, Cell 81 (1995) 95–105.
69. E. Hannak, M. Kirkham, A.A. Hyman, K. Oegema, Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans, J. Cell. Biol. 155 (2001) 1109–1116.
70. Q. Liu, J.V. Ruderman, Aurora A, mitotic entry, and spindle bipolarity, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 5811–5816.
71. C. Roghi, R. Giet, R. Uzbekov, N. Morin, I. Chartrain, R. Le Guellec, A. Couturier, M. Doree, M. Philippe, C. Prigent, The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly, J. Cell. Sci. 111 (Pt 5) (1998) 557–572.
72. A. Khodjakov, R.W. Cole, B.R. Oakley, C.L. Rieder, Centrosome- independent mitotic spindle formation in vertebrates, Curr. Biol. 10 (2000) 59–67
73. H. Maiato, C.L. Rieder, A. Khodjakov, Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis, J. Cell. Biol. 167 (2004) 831–840.
74. Kufer TA, Nigg EA, SilljéHH. Regulation of Aurora-A kinase on the mitotic spindle.Chromosoma. 2003 Dec;112(4):159-163. Epub 2003 Nov 21.
75. Kimura M, Okano Y. Aurora kinases and cancer Gan To Kagaku Ryoho. 2005 Jan;32(1):1-5.
76. T. Hirota, N. Kunitoku, T. Sasayama, T. Marumoto, D. Zhang, M. Nitta, K. Hatakeyama, H. Saya, Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells, Cell 114 (2003) 585–598.
77. D.L. Satinover, D.L. Brautigan, P.T. Stukenberg, Aurora-A kinase andinhibitor-2 regulate the cyclin threshold for mitotic entry in Xenopus early embryonic cell cycles, Cell Cycle 5 (2006) 2268–2274.
78. M.A. van Vugt, A. Bras, R.H. Medema, Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells, Mol. Cell 15 (2004) 799–811.
79. M.A. van Vugt, A. Bras, R.H. Medema, Restarting the cell cycle when the checkpoint comes to a halt, Cancer Res. 65 (2005) 7037–7040.
80. Tomotoshi Marumoto, Shinobu Honda, Toshihiro Hara,et al. Aurora-A Kinase Maintains the Fidelity of Early and Late Mitotic Events in HeLa Cells. J. Biol. Chem., Vol. 278, Issue 51,51786-51795, December 19, 2003.
81. Bischoff JR, Anderson L, Zhu Y, Mossie K, Ng L, Souza B, Schryver B, Flanagan P, Clairvoyant Drosophila F, Ginther C, Chan CS, Novotny M, Slamon DJ, Plowman GD. A homologue of aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J. 1998; 17(11):3052-3065.
82. Zhou H, Kuang J, Zhong L, Kuo WL, Gray JW, Sahin A, Brinkley BR, Sen S. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet. 1998; 20(2):189-193.
83. Gritsko TM, Coppola D, Paciga JE, Yang L, Sun M, Shelley SA, Fiorica JV, Nicosia SV, Cheng JQ. Activation and overexpression of centrosome kinase BTAK/Aurora-A in human ovarian cancer. Clin Cancer Res. 2003; 9(4):1420-1426.
84. Sakakura C, Hagiwara A, Yasuoka R, Fujita Y, Nakanishi M, Masuda K, Shimomura K, Nakamura Y, Inazawa J, Abe T, Yamagishi H. Tumour-amplified kinase BTAK is amplified and overexpressed in gastriccancers with possible involvement in aneuploid formation. Br J Cancer. 2001; 84(6):824-831.
85. Jeng YM, Peng SY, Lin CY, Hsu HC. Overexpression and amplification of Aurora-A in hepatocellular carcinoma. Clin Cancer Res. 2004; 10(6):2065-2071.
86. Sen S, Zhou H, Zhang RD, Yoon DS, Vakar-Lopez F, Ito S, Jiang F, Johnston D, Grossman HB, Ruifrok AC, Katz RL, Brinkley W, Czerniak B. Amplification/overexpression of a mitotic kinase gene in human bladder cancer. J Natl Cancer Inst. 2002; 94(17):1320-1329.
87. Hamada M, Yakushijin Y, Ohtsuka M, Kakimoto M, Yasukawa M, Fujita S. Aurora2/BTAK/STK15 is involved in cell cycle checkpoint and cell survival of aggressive non-Hodgkin's lymphoma. Br J Haematol. 2003; 12l(3):439-447.
88. Li D, Zhu J, Firozi PF, Abbruzzese JL, Evans DB, Cleary K, Friess H, Sen S. Overexpression of oncogenic STK15/BTAK/Aurora A kinase in human pancreatic cancer. Clin Cancer Res. 2003; 9(3):991-997.
89. Li YH, Li FC, Zhao X, Zhao Z, Sun KL, Sun XH. Expression of STK15 gene and chromosomal instability in laryngeal carcinoma. Yi Chuan Xue Bao. 2002; 29(12):1048-1051.
90. Reichardt W, Jung V, Brunner C, Klein A, Wemmert S, Romeike BF, Zang KD, Urbschat S. The putative serine/threonine kinase gene STK15 on chromosome 20q13.2 is amplified in human gliomas. Oncol Rep. 2003; 10(5):1275-1279.
91. J.R. Bischoff, L. Anderson, Y. Zhu, K. Mossie, L. Ng, B. Souza, B. Schryver, P. Flanagan, F. Clairvoyant, C. Ginther, C.S. Chan, M. Novotny, D.J. Slamon, G.D. Plowman, A homologue of Drosophila aurora kinase isoncogenic and amplified in human colorectal cancers, Embo J. 17 (1998) 3052–3065.
92. H. Zhou, J. Kuang, L. Zhong, W.L. Kuo, J.W. Gray, A. Sahin, B.R. Brinkley, S. Sen, Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation, Nat. Genet. 20 (1998) 189–193.
93. P. Meraldi, R. Honda, E.A. Nigg, Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53-/- cells, Embo J. 21 (2002) 483–492.
94. S. Anand, S. Penrhyn-Lowe, A.R. Venkitaraman, AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol, Cancer Cells 3 (2003) 51–62.
95. Katayama H, Zhou H, Li Q, Tatsuka M, Sen S.Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle. J Biol Chem. 2001;276(49):46219-46224.
96. Fukasawa K, Choi T, Kwiyama R, Rulong S, Vande Woude GF. Abnorma centrosome amplification in the absence of p53. Science. 1996; 271(5256):1744-1747.
97. Lingle WL, Lutz WH, Ingle JN, Maihle NJ, Salisbury JL. Centrosome hypertrophy in human breast tumors: implications for genomic stability and cell polarity. Proc Natl Acad Sci U S A, 1998, 95(6):2950-2955.
98. Pihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenbeny P, Doxsey SJ. Centrosome defects and genetic instability in malignant tumors. Cancer Res. 1998; 58(17):3974-3985.
99. Marumoto T, Hirota T, Morisaki T, Kunitoku N, Zhang D, Ichikawa Y,Sasayama T, Kuninaka S, Mimori T, Tamaki N, Kimura M, Okano Y, Saya H. Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells. Genes Cells. 2002; 7(11):1173-1182.
100. Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk J, Suzuki F, Fujii S, Arlinghaus RB, Czerniak BA, Sen S. Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53. Nat Genet. 2004, 36(1):55-62.
101. Liu Q, Kaneko S, Yang L, Feldman RI, Nicosia SV, Chen J, Cheng JQ. Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215. J Bio1 Chem. 2004; 279(50):52175-52182.
102. Yang H, Ou CC, Feldman RI, Nicosia SV, ICruk PA, Cheng JQ. Aurora-A kinase regulates telomerase activity through c-Myc in human ovarian and breast epithelial cells. Cancer Res, 2004 ,64(2):463-467.
103. Zhang H, Jin Y, Chen X, et al. Papillomavirus type 16 E6/E7 and human telomerase reverse transcriptase in esophageal cell immortalization and early transformation. Cancer Lett 2007;245(1–2):184–94.
104. Mahadevan D, Bearss DJ, Vankayalapati H. Structure2based design of novel anti-cancer agents targeting aurora kinases. Curr Med Chem Anti2Canc Agents, 2003, 3 (1): 25234.
105. Hamada M , Yakushijin Y, Ohtsuka M , et al. Aurora2/BTAK/STK15 is involved in cell cycle checkpoint and cell survival of aggressive non2Hodgkin′s lymphoma. Br J Haematol, 2003, 121(3): 4392447.
106. Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 1990 Apr; 2(4):279-289.
107. Romano N, Macino G. Quelling: transient inactivation of gene expressionin Neurospora crassa by transformation with homologous sequences. Mol Microbiol. 1992 Nov; 6(22):3343-3353.
108. Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell. 1995 May 19; 81(4):611-620.
109. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19; 391(6669):806-811.
110. Hannon GJ. RNA interference. Nature. 2002 Jul 11; 418(6894):244-251.
111. Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science. 1999 Oct 29; 286(5441):950-2.
112. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000 Mar 31;101(1):25-33.
113. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000 Mar 16; 404(6775):293-296.
114. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001 Jan 18; 409(6818):363-6.
115. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15; 15(2):188-200.
116. Wianny F and Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development [J]. Nat Cell Biol, 2000; 2(2): 70-75.
117. Elbashir S M, Harborth J, Lendeckel W, Yalcin A, Weber K, and Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001; 411(6836): 494-498.
118. Brummelkamp T R, Bernards R, and Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002; 296(5567): 550-553.
119. Song E, Lee S K, Wang J, Ince N, Ouyang N,min J, Chen J, Shankar P, and Lieberman J. RNA interference targeting Fas protects mice from fulminant hepatitis [J]. Nat Med, 2003; 9(3): 347-351.
120. Morris K V, Chan S W, Jacobsen S E, and Looney D J. Small interfering RNA-induced transcriptional gene silencing in human cells [J]. Science, 2004; 305(5688): 1289-1292.
121. Kawasaki H, Taira K, and Morris K V. siRNA Induced Transcriptional Gene Silencing in Mammalian Cells [J]. Cell Cycle, 2005; 4(3).
122. Kawasaki H and Taira K. Induction of DNA methylation and gene silencing by short interfering RNAs in human cells [J]. Nature, 2004; 431(7005): 211-217.
123. Bass BL. RNA interference. The short answer. Nature. 2001 May 24; 411(6836):428-9.
124. Matzke M, Matzke AJ, Kooter JM. RNA: guiding gene silencing. Science. 2001 Aug 10;293(5532):1080-3.
125. Bartel D P. MicroRNAs: genomics, biogenesis, mechanism, and function [J]. Cell, 2004; 116(2): 281-297.
126. Zhang H, Kolb F A, Jaskiewicz L, Westhof E, and Filipowicz W. Single processing center models for human Dicer and bacterial RNase III [J]. Cell, 2004; 118(1): 57-68.
127. Nykanen A, Haley B, and Zamore P D. ATP requirements and small interfering RNA structure in the RNA interference pathway [J]. Cell, 2001; 107(3): 309-321.
128. Hammond S M, Boettcher S, Caudy A A, Kobayashi R, and Hannon G J. Argonaute2, a link between genetic and biochemical analyses of RNAi [J]. Science, 2001; 293(5532): 1146-1150.
129. Pham J W, Pellino J L, Lee Y S, Carthew R W, and Sontheimer E J. A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila [J]. Cell, 2004; 117(1): 83-94.
130. Hutvagner G and Zamore P D. A microRNA in a multiple-turnover RNAi enzyme complex [J]. Science, 2002; 297(5589): 2056-2060.
131. Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M, and Dreyfuss G. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs [J]. Genes Dev, 2002; 16(6): 720-728.
132. Kim J, Krichevsky A, Grad Y, Hayes G D, Kosik K S, Church G M, and Ruvkun G. Identification of many microRNAs that copurify with polyribosorries in mammalian neurons [J]. Proc Natl Acad Sci U S A, 2004; 101(1): 360-365.
133. Liu J, Carmell M A, Rivas F V, Marsden C G, Thomson J M, Song J J, Hammond S M, Joshua-Tor L, and Hannon G J. Argonaute2 is the catalytic engine of mammalian RNAi [J]. Science, 2004; 305(5689): 1437-1441.
134. Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, and Tuschl T. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs [J]. Mol Cell, 2004; 15(2): 185-197.
135. Cerutti H. RNA interference: traveling in the cell and gaining function?Trends Genet. 2003 Jan; 19(1):39-46.
136. Marx J. Interfering with gene expression. Science. 2000 May 26;288(5470):1370-2.
137. Khvorova A, Reynolds A, and Jayasena S D. Functional siRNAs and miRNAs exhibit strand bias [J]. Cell, 2003; 115(2): 209-216.
138. Schwarz D S, Hutvagner G, Du T, Xu Z, Aronin N, and Zamore P D. Asymmetry in the assembly of the RNAi enzyme complex [J]. Cell, 2003; 115(2): 199-208.
139. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science. 2004 Sep 3;305(5689):1434-7.
140. Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal S I, and Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex [J]. Science, 2004; 303(5658): 672-676.
141. Tuschl T ,Zamore PD, Lehmann R, et al. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev, 1999, 13(24): 3191-3197.
142. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 2002; 16: 948-958.
143. Yu JY, Taylor J, DeRuiter SL, Vojtek AB, et al. Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors. Mol Ther 2003; 7: 228-236.
144. Kim VN. RNA interference in functional genomics and medicine. J orean Med Sci. 2003 Jun; l 8(3):309-318.
145. Nishikura K. A short primer on RNA: RNA-directed RNA polymerase actsas a key catalyst. cell, 2001, 107 (4):415-418.
146. Gan L, Anton KE, Masterson BA, et al. Specific interference with gene expression and gene function mediated by long double-stranded RNA in neural ce11s. J Neurosci Methods, 2002, 121(2): 151-157.
147. Wohlbold L, van der Kuip H, Miething C, Vornlocher H P, Knabbe C, Duyster J, and Aulitzky W E. Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571)[J]. Blood, 2003; 102(6): 2236-2239.
148. Scherr M, Battmer K, Winkler T, Heidenreich O, Ganser A, and Eder M. Specific inhibition of bcr-abl gene expression by small interfering RNA[J]. Blood, 2003; 101(4): 1566-1569.
149. Li M J, McMahon R, Snyder D S, Yee J K, and Rossi J J. Specific killing of Ph+ chronic myeloid leukemia cells by a lentiviral vector-delivered anti-bcr/abl small hairpin RNA[J]. Oligonucleotides, 2003; 13(5): 401-409.
150. Wilda M, Fuchs U, Wossmann W, and Borkhardt A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi) [J]. Oncogene, 2002; 21(37): 5716-5724.
151. Sumimoto H, Yamagata S, Shimizu A, Miyoshi H, Mizuguchi H, Hayakawa T, Miyagishi M, Taira K, and Kawakami Y. Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference. [J]. Gene Ther, 2005; 12(1): 95-100.
152. Duxbury M S, Matros E, Ito H, Zinner M J, Ashley S W, and Whang E E. Systemic siRNA-mediated gene silencing: a new approach to targeted therapy of cancer[J]. Ann Surg, 2004; 240(4): 667-674; discussion 675-666.
153. Lakka S S, Gondi C S, Yanamandra N, Olivero W C, Dinh D H, Gujrati M, and Rao J S. Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis[J]. Oncogene, 2004; 23(27): 4681-4689.
154. Zhang Y, Zhang Y F, Bryant J, Charles A, Boado R J, and Pardridge W M. Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer [J]. Clin Cancer Res, 2004; 10(11): 3667-3677.
155. Brummelkamp T R, Bernards R, and Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference [J]. Cancer Cell, 2002; 2(3): 243-247.
156. Stege A, Priebsch A, Nieth C, and Lage H. Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference [J]. Cancer Gene Ther, 2004; 11(11): 699-706.
157. Yague E, Higgins C F, and Raguz S. Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1 [J]. Gene Ther, 2004; 11(14): 1170-1174.
158. Shen H, Pan Y, Han Z, Hong L, Liu N, Han S, Yao L, Xie H, Zhaxi C, Shi Y, and Fan D. Reversal of multidrug resistance of gastric cancer cells by downregulation of TSG101 with TSG101siRNA [J]. Cancer Biol Ther, 2004; 3(6): 561-565.
159. Nieth C, Priebsch A, Stege A, and Lage H. Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi) [J]. FEBS Lett, 2003; 545(2-3): 144-150.
160. Miller V M, Gouvion C M, Davidson B L, and Paulson H L. TargetingAlzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles[J]. Nucleic Acids Res, 2004; 32(2): 661-668.
161. Barthel A, Herzig S, Muller H W, Harborth J, and Bornstein S R. RNA Interference-based Strategies for Metabolic Syndrome Treatment [J]. Horm Metab Res, 2005; 37(2): 59-62.
162. Lieberman J, Song E, Lee S K, and Shankar P. Interfering with disease: opportunities and roadblocks to harnessing RNA interference [J]. Trends Mol Med, 2003; 9(9): 397-403.
163. Gitlin L and Andino R. Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing [J]. J Virol, 2003, 77(13): 7159-7165.
164. Radhakrishnan S K, Layden T J, and Gartel A L. RNA interference as a new strategy against viral hepatitis [J]. Virology, 2004; 323(2): 173-181.
165. Shi Y, Yang de H, Xiong J, Jia J, Huang B, and Jin Y X. Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs [J]. Cell Res, 2005; 15(3): 193-200.
166. Li T, Zhang Y, Fu L, Yu C, Li X, Li Y, Zhang X, Rong Z, Wang Y, Ning H, Liang R, Chen W, Babiuk L A, and Chang Z. siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication [J]. Gene Ther, 2005 May; 12(9):751-61.
167. Wu C J, Huang H W, Liu C Y, Hong C F, and Chan Y L. Inhibition of SARS-CoV replication by siRNA [J]. Antiviral Res, 2005; 65(1): 45-48.
168. Zheng B J, Guan Y, Tang Q, Du C, Xie F Y, He M L, Chan K W, Wong K L, Lader E, Woodle M C, Lu P Y, Li B, and Zhong N. Prophylactic and therapeutic effects of small interfering RNA targeting SARS-coronavirus [J]. Antivir Ther, 2004; 9(3): 365-374.
169. Radhakrishnan S, Gordon J, Del Valle L, Cui J, and Khalili K. Intracellular approach for blocking JC virus gene expression by using RNA interference during viral infection [J]. J Virol, 2004; 78(13): 7264-7269.
170. Orba Y, Sawa H, Iwata H, Tanaka S, and Nagashima K. Inhibition of virus production in JC virus-infected cells by postinfection RNA interference [J]. J Virol, 2004; 78(13): 7270-7273.
171. Sorensen D R, Leirdal M, and Sioud M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice [J]. J Mol Biol, 2003; 327(4): 761-766.
172. Pusch O, Boden D, Silbermann R, Lee F, Tucker L, and Ramratnam B. Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA [J]. Nucleic Acids Res, 2003; 31(22): 6444-6449.
173. Jackson A L, Bartz S R, Schelter J, Kobayashi S V, Burchard J, Mao M, Li B, Cavet G, and Linsley P S. Expression profiling reveals off-target gene regulation by RNAi [J]. Nat Biotechnol, 2003; 21(6): 635-637.
174. Persengiev S P, Zhu X, and Green M R. Nonspecific, concentration- dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs) [J]. Rna, 2004; 10(1): 12-18.
175. Saxena S, Jonsson Z O, and Dutta A. Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells [J]. J Biol Chem, 2003; 278(45): 44312-44319.
176. Pancoska P, Moravek Z, and Moll U M. Efficient RNA interference depends on global context of the target sequence: quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA [J]. Nucleic Acids Res, 2004; 32(4): 1469-1479.
177. Haley B and Zamore P D. Kinetic analysis of the RNAi enzyme complex [J]. Nat Struct Mol Biol, 2004; 11(7): 599-606.
178. Zamore P D. RNA interference: listening to the sound of silence [J]. Nat Struct Biol, 2001; 8(9): 746-750.
179. Bridge A J, Pebernard S, Ducraux A, Nicoulaz A L, and Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells [J]. Nat Genet, 2003; 34(3): 263-264.
180. Sledz C A, Holko M, de Veer M J, Silverman R H, and Williams B R. Activation of the interferon system by short-interfering RNAs[Jl. Nat Cell Biol, 2003; 5(9): 834-839.
181. Moss E G and Taylor J M. Small-interfering RNAs in the radar of the interferon system[J]. Nat Cell Biol, 2003; 5(9): 771-772.
182. Kim D H, Longo M, Han Y, Lundberg P, Cantin E, and Rossi J J. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase[J]. Nat Biotechnol, 2004; 22(3): 321-325.
183. Pebernard S and. Iggo R D. Determinants of interferon- stimulated gene induction by RNAi vectors[J]. Differentiation, 2004; 72(2-3): 103-111.
184. Diebold S S, Montoya M, Unger H, Alexopoulou L, Roy P, Haswell L E, Al-Shamkhani A, Flavell R, Borrow P, and Reis a Sousa C. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers [J]. Nature, 2003; 424(6946): 324-328.
185. Kariko K, Bhuyan P, Capodici J, and Weissman D. Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 [J]. J Immunol, 2004; 172(11): 6545-6549.
186. Lingor P, Michel U, Scholl U, Bahr M, and Kugler S. Transfection of"naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons [J]. Biochem Biophys Res Commun, 2004; 315(4): 1126-1133.
187.刘士德陈莲凤,原代培养的正常人宫颈鳞状上皮细胞超微结构.电子显微学报,1994.13(5).419-419
188. Huang X, Hutchins B, Patel RC. The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR). Biochem J, 2002, 366(1): 175-186.
189. Peters GA, Li S, Sen GC. Phosphorylation of specific serine residues in the PKR activation domain of PACT is essential for its ability to mediate apoptosis. J Biol Chem, 2006, 281(46): 35129-35136.
190. Gupta V, Patel RC. Proapoptotic protein PACT is expressed at high levels in colonic epithelial cells in mice. Am J Physiol Gastrointest Liver Physiol, 2002, 283(3): 801~808.
191. Patel CV, Handy I, Goldsmith T, Patel RC. PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR. J Biol Chem, 2000, 275(48): 37993-37998.
192. Kok KH, Ng MH, Ching YP, Jin DY. Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. J Biol Chem, 2007, 282(24):17649-17657.
193. Gale, M., Jr., S.-L. Tan, M. Wambach, and M. G. Katze. 1996. Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation. Mol. Cell. Biol. 16:4172-4181.
194. Goodman AG, Smith JA, Balachandran S, Perwitasari O, Proll SC, Thomas MJ, Korth MJ, Barber GN, Schiff LA, Katzemg. The Cellular Protein P58IPK Regulates Influenza Virus mRNA Translation and Replication through a PKR-Mediated Mechanism. J Virol. 2007 Mar;81(5):2221-30.
195. Yan W, Frank CL, Korth MJ, Sopher BL, Novoa I, Ron D, Katzemg. Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK. Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):15920-5.
196. Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe ? Nat Rev Cancer, 2004, 4(12): 966-977.
197. Khan MM, Nomura T, Chiba T, et al. The fusion oncoprotein PML- RARalpha induces endoplasmic reticulum(ER)-associated degradation of N-CoR and ER stress. J Biol Chem, 2004, 279(12): 11814-11824.
198. Kitamura Y, Miyamura A, Takata K. Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in thapsigargin-induced apoptosis in human neuroblastoma SH-SY5Y cells. J Pharmacol Sci, 2003, 92 (3): 228-236.
199. Thomlinson RH, GrayLH. The histological structure of some human lung cancers and the possible imp lications for radiotherapy[J]. Br J Cancer, 1955, 9 (4): 539-549.
200. FelsDR, Koumenis C. The PERK/e-IF2alpha /ATF4 module of the UPR in hypoxia resistance and tumor growth [J]. Cancer Biol Ther, 2006, 5 (7): 723-728.
201. Tatsuo Hata, Toru Furukawa, Makoto Sunamura, Shinichi Egawa,Fuyuhiko Motoi, Noriyuki Ohmura, Tomotoshi Marumoto, Hideyuki Saya and Akira Horii RNA Interference Targeting Aurora Kinase A Suppresses Tumor Growth and Enhances the Taxane Chemosensitivity in Human Pancreatic Cancer Cells. Cancer Research 65, 2899-2905
202. Klein A, Flügel D, Kietzmann T. Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1. Mol Biol Cell. 2008 Sep;19(9):3667-75.
203. Tao Y, Zhang P, Frascogna V, Lecluse Y, Auperin A, Bourhis J, Deutsch E.Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells. Br J Cancer. 2007 Dec 17;97(12):1664-72.
204. Dutertre S, Cazales M, Quaranta M, Froment C, Trabut V, Dozier C, Mirey G, BouchéJP, Theis-Febvre N, Schmitt E, Monsarrat B, Prigent C, Ducommun B.Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition. J Cell Sci. 2004 May 15;117(Pt 12):2523-31.
205. Song SJ, Song MS, Kim SJ, Kim SY, Kwon SH, Kim JG, Calvisi DF, Kang D, Lim DS. Aurora A Regulates Prometaphase Progression by Inhibiting the Ability of RASSF1A to Suppress APC-Cdc20 Activity. Cancer Res. 2009 Mar 15;69(6):2314-23. Epub 2009 Mar 10.
206. Hata T, Furukawa T, Sunamura M, Egawa S, Motoi F, Ohmura N, Marumoto T, Saya H, Horii A. RNA interference targeting aurora kinase a suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells. Cancer Res. 2005 Apr 1;65(7):2899-905.
207. Kufer, T. A., Sillje, H. H., Korner, R., Gruss, O. J., Meraldi, P., and Nigg, E. A.(2002). Human TPX2 is required for targeting Aurora-A kinase to thespindle.J. Cell Biol. 158, 617–623.
208. Matsumoto S, Miyagishi M, Akashi H, Nagai R, Taira K.Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library. J Biol Chem. 2005 Jul 8;280(27):25687-96.
209. Cardin E, Larose L.Nck-1 interacts with PKR and modulates its activation by dsRNA. Biochem Biophys Res Commun. 2008 Dec 5;377(1):231-5.
210. Cabanski M, Steinmüller M, Marsh LM, Surdziel E, Seeger W, Lohmeyer J. PKR regulates TLR2/TLR4-dependent signaling in murine alveolar macrophages. Am J Respir Cell Mol Biol. 2008 Jan;38(1):26-31.
211. von Holzen U, Pataer A, Raju U, Bocangel D, Vorburger SA, Liu Y, Lu X, Roth JA, Aggarwal BB, Barber GN, Keyomarsi K, Hunt KK, Swisher SG. The double-stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation.
212. Scharer CD, Laycock N, Osunkoya AO, Logani S, McDonald JF, Benigno BB, Moreno CS. Aurora kinase inhibitors synergize with paclitaxel to induce apoptosis in ovarian cancer cells. J Transl Med. 2008 Dec 11;6:79.
213. Carpinelli P, Moll J. Aurora kinases and their inhibitors: more than one target and one drug. Adv Exp Med Biol. 2008;610:54-73.
214. Girdler F, Sessa F, Patercoli S, Villa F, Musacchio A, Taylor S. Molecular basis of drug resistance in aurora kinases. Chem Biol. 2008 Jun;15(6):552-62.
215. Gontarewicz A, Balabanov S, Keller G, Panse J, Schafhausen P, Bokemeyer C, Fiedler W, Moll J, Brümmendorf TH. PHA-680626 exhibits anti-proliferative and pro-apoptotic activity on Imatinib-resistantchronic myeloid leukemia cell lines and primary CD34+ cells by inhibition of both Bcr-Abl tyrosine kinase and Aurora kinases. Leuk Res. 2008 Dec;32(12):1857-65.
216. Grant KM. Targeting the cell cycle in the pursuit of novel chemotherapies against parasitic protozoa. Curr Pharm Des. 2008;14(9):917-24.
217. Akahane D, Tauchi T, Okabe S, Nunoda K, Ohyashiki K. PHA-680626 exhibits anti-proliferative and pro-apoptotic activity on Imatinib-resistant chronic myeloid leukemia cell lines and primary CD34+ cells by inhibition of both Bcr-Abl tyrosine kinase and Aurora kinases. Leuk Res. 2008 Dec;32(12):1857-65.
218. Gontarewicz A, Balabanov S, Keller G, Panse J, Schafhausen P, Bokemeyer C, Fiedler W, Moll J, Brümmendorf TH.PHA-680626 exhibits anti-proliferative and pro-apoptotic activity on Imatinib-resistant chronic myeloid leukemia cell lines and primary CD34+ cells by inhibition of both Bcr-Abl tyrosine kinase and Aurora kinases.Leuk Res. 2008 Dec;32(12):1857-65.
219. Nallagatla SR, Bevilacqua PC. Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner. RNA. 2008 Jun;14(6):1201-13.
220. Zheng X, Bevilacqua PC. Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails. RNA. 2004 Dec;10(12):1934-45.
221. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol. 2003 Sep;5(9):834-9.
222. Puthenveetil S, Whitby L, Ren J, Kelnar K, Krebs JF, Beal PA. Controllingactivation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification. Nucleic Acids Res. 2006;34(17):4900-11.
223. Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.Nat Biotechnol. 2005 Apr;23(4):457-62.
224. McAllister CS, Samuel CE.The RNA-activated protein kinase enhances the induction of interferon-beta and apoptosis mediated by cytoplasmic RNA sensors.J Biol Chem. 2009 Jan 16;284(3):1644-51.
225. Jie Li, Guang Yang, Shaohua Li, Guojun Cao, Qiang Zhao, Xuemei Liu,ming Fan, Beifen Shen, Ningsheng Shao. 3’-Poly(A) tail enhances siRNA activity against exogenous reporter genes in MCF-7 cells. Journal of RNAi and Gene Silencing (2006), 2(2), 195-204
226. Kojima K, Konopleva M, Tsao T, Nakakuma H, Andreeff M. Concomitant inhibition of Mdm2-p53 interaction and Aurora kinases activates the p53-dependent postmitotic checkpoints and synergistically induces p53-mediated mitochondrial apoptosis along with reduced endoreduplication in acute myelogenous leukemia. Blood. 2008 Oct 1;112(7):2886-95.
2. H.D.L.Birley. Human papillomaviruses, cervical cancer and the developing world. Annals of tropical medicine and parasitology, 1995, 89: 453-463.
3.卞美璐,刘树范.《子宫颈疾病的诊治》[M].科学技术文献出版社,2001年5月:68-73
4.许廷贵,工敬石,张惠珍. HPV的分子生物学特征[J].国外医学病毒学分册. 2004 11(3): 4
5.巴瑞琪,丁晓华.宫颈病变人乳头瘤病毒感染及型别分布的研究[J].华中科技大学学报(医学版). 2000,35( 6): 838-840
6.李键,薄莉,门新华.人乳头瘤病毒及行为因素与子宫颈癌的关系[J].中国性医学. 2005,14( 6): 12
7. Barnard P, McMillan NA. The human papillomavirus E7 oncoprotein abrogates signaling mediated by interferon-alpha. Virology. 1999 Jul 5;259(2):305-313.
8. Barnard P, Payne E, McMillan NA. The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-alpha. Virology. 2000 Nov 25;277(2):411-419.
9. Park JS, Kim EJ, Kwon HJ, Hwang ES, Namkoong SE, Um SJ. Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis. J Biol Chem. 2000 Mar 10; 275(10):6764-6769.
10. Um SJ, Rhyu JW, Kim EJ, Jeon KC, Hwang ES, Park JS. Abrogation of IRF-1 response by high-risk HPV E7 protein in vivo. Cancer Lett. 2002 May 28;179(2):205-212.
11. Perea SE, Massimi P, Banks L. Human papillomavirus type 16 E7 impairs the activation of the interferon regulatory factor-1. Int J Mol Med. 2000 Jun; 5(6): 661-6.
12. Nees M, Geoghegan JM, Hyman T, Frank S, Miller L, Woodworth CD. Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-kappaB-responsive genes in cervical keratinocytes. J Virol. 2001 May; 75(9): 4283-4296.
13. Pietenpol JA , Stewart ZA. Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis. Toxicology, 2002, 27 (1812182): 4752481.
14. Bartek J, Lukas J. Mammalian G1- and S- phase checkpoints in response to DNA damage. J. Curr Opin Cell Biol ,2001 ,13 (6) :738-747.
15. Narayanan BA, Geoffroy O, Willingham MC, et al. p53/p21 (WAF1P CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells. Cancer Lett, 1999, 136 (2) : 215-221.
16. Tan S, Wang TS. Analysis of fission yeast primase defines the checkpoint responses to aberrant S phase initiation. Mol Cell Biol, 2000, 20 (21): 7853-7866.
17. Foray N, Marot D, Randrianarison V, et al. Constitutive association of BRCA1 and c-Abl and its ATM-dependent disruption after irradiation. Mol Cell Biol, 2002, 22 (12): 4020-4032.
18. Yang J, Yu Y, Hamrick HE, et al. ATM, ATR and DNA-PK: initiators ofthe cellular genotoxic stress responses. Carcinogenesis, 2003, 24 (10): 1571-1580.
19. Duckworth BC, Weaver J S, Ruderman JV. G2 arrest in Xenopus oocytes depends on phosphorylation of cdc-5 by protein kinase A. Proc Natl Acad Sci USA, 2002, 99 (26): 16794-16799.
20. Qiu L, Burgess A, Fairlie DP, et al. Histone deacetylase inhibitors trigger a G2 checkpoint in normal cells that is defective in tumor cells. Mol Biol Cell, 2000, 11 (6): 2069-2083.
21. Lew DJ, Burke DJ. The spindle assembly and spindle position checkpoints. Annu Rev Genet, 2003, 37: 251-282.
22. Kraft C, Herzog F, Gieffers C, et al. Peters JM. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. EMBO J, 2003, 22 (24): 6598-6609.
23. Wentzensen N, Vinokurova S, von Knebel Doeberitz M. Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. Cancer Res. 2004 Jun 1;64(11):3878-3884.
24.张立妍,许妍,张爱荣.人乳头瘤病毒感染与宫颈癌的关系[J].山东医药.2005, 45(33): 68
25. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990 Apr 6;248(4951):76-79.
26. KatherA, Ferrara A,Nonn M , et al. Identification of a naturally processed HLA-A 0201 HPV 18E7 T cell epitope by tumor cell mediated in vitro vaccination [J]. Int J Cancer, 2003, 104: 345-353.
27. Classon M, Dyson N. p l07 and p130 versatile p roteinswith interestingpockets [J]. Exp Cell Res, 2001, 264: 135-137.
28. d′Adda di Fagagna F, Reaper PM, Clay2Farrace L, et al. A DNA damage checkpoint response in telomere-initiated senescence [J]. Nature, 2003, 426 (6963): 194-198.
29. Nanduri S, Rahman F, Willians B, et al . A dynamically tuned double-stranded RNA binding mechanism for the activation of antiviral kinase PKR [J].EMBO J,2000, 19 (20): 5567-5574.
30. Gupta V,Huang X,Patel RC.The carboxy-terminal,M3 motifs of PACT and TRBP have opposite effects on PKR activity [J]. Virology, 2003, 315(2): 253-291.
31. Francois C, Duverlie G, Rebouillat D, et al. Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis [J]. J Virol, 2000, 74(12): 5587-5596.
32. Langland JO, Jacobs BL. The role of the PKR-inhibitory genes,E3L and K3L,in determining vaccinia virus host range [J]. Virology, 2002, 299 (1):133-141.
33. Ward SV, Samuel CE. Regulation of the interferon-inducible PKR kinase gene: the KCS element is a constitutive promoter element that functions in concert with the interferon-stimulated response element [J]. Virology, 2002, 296 (1): 136-146.
34. Hebner C. M, Wilson R, Rader J, et al. Human papillomaviruses target the double-stranded RNA protein kinase pathway [J]. J GEN Virol, 2006, 87 (Pt 11): 3183-3193.
35.贾因棠,魏来,蒋栋等,双链RNA激活的蛋白激酶(PKR)的克隆表达及其对丙型肝炎病毒蛋白合成的抑制作用.中国生物化学与分子生物学报, 2006, 22(1)24~30.
36. Taylor DR, Shi ST, Romano PR, Barber GN, et al. Inhibition of the interferon-inducible protein kinase PKR by HCV E2protein. Science, 1999, 285:107-110.
37. Christy M. Hebner, Regina Wilson, Janet Rader, et al. Human papillomavirusestarget the double-stranded RNA protein kinase pathway. J Gen Virol, 2006, 87:3183-3193.
38. Ryman KD,White LJ,Johnston RE,et al. Effects of PKR/RNase L-dependent and alternative antiviral pathways on alphavirus replication and pathogenesis [J]. Viral Immunol, 2002, 15 (1):53-76.
39. Ben-Asouli Y, Banai Y, Pel-Or Y, et al.Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR [J]. Cell, 2002, 108 (2): 221-232.
40. Vattem KM, Staschke KA, Wek RC. Mechanism of activation of the double-stranded-RNA-dependent protein kinase, PKR : role of dimerization and cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (eIF2) [J]. Eur J Biochem, 2001, 268 (13):3674-3684.
41. Vattem KM,Staschke KA,Zhu S,et al.Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase,PKR,are important for regulating phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) [J]. Eur J Biochem, 2001, 268 (4): 1143-1153.
42. Gil J , Alcami J , Esteban M . Activation of NF-kappaB by the dsRNA-dependent protein kinase,PKR involves the I-kappaB kinase complex.Oncogene,2000,19:1369-1378.
43. Samuel CE. Antiviral actions of interferons. Clin Microbiol Rev. 2001 Oct;14(4):778-809, table of contents.
44. Sundar SS, Cornall RJ, Kehoe ST. Advances in the management of cervical cancer [J]. J Br Menopause Soc, 2005, 11(3): 91-95.
45. Nguyen, M., Song, S., Liem, A., et al. A mutant of human papillomavirus type 16 E6 deficient in bindingα-helix partners displays reduced oncogenic potential in vivo. J Vrol, 2002, 76:13039-13048.
46. Kazemi S, Papadopoulou S, Li S, et al. Control of alpha subunit of eukaryotic translation initiation factor 2 (eIF2 alpha) phosphorylation by the human papillomavirus type 18 E6 oncoprotein: implications for eIF2 alpha-dependent gene expression and cell death[J]. Mol Cell Biol, 2004, 24(8): 3415-3429.
47. Savinova O, Joshi B, Jagus R. Abnormal levels andminimal activity of the dsRNA-activated protein kinase, PKR, in breast carcinoma cells. Int J Biochem Cell Biol. 1999 Jan; 31(1):175-189.
48. Kim SH, Forman AP, Gunnery S. Mathews cells contain elevated levels Human breast cancer activity of the protein kinase PKR. Oncogene. 2000 Jun 22; 19 (27):3086-3094.
49. Shir Alexei, mbar Friedrich and Alexander Levitzki. Tumor specific activation of PKR as a non-toxic modality of cancer treatment. Semin Cancer Biol. 2003 Aug;13(4): 309-314.
50. Donze O., Jagus R., Koromilas A. E., Hershey J. W. and Sonenberg N. Abrogation of translation initiatibn factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J. 14 (1995): 3828-3834.
51. Kumar A, Yang YL and Flati V. Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1and NF- kappaB. EMBO J. 1997 Jan 15;16(2):406-416.
52. Giet R, Prigent C. Aurora/Ipl l p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. J Cell Sci. 1999; 112(Pt 21):3591-3601.
53. Bischoff JR, Plowman GD. The Aurora/Ipllp kinase family: regulators of chromosome segregation and cytokinesis. Trends Cell Biol. 1999; 9(11):454-459.
54. Kufer TA, Sillje HH, Korner R, Gruss OJ, Meraldi P, Nigg EA. Human TPX2 is required for targeting Aurora-A kinase to the spindle. J Cell Biol. 2002; 158(4):617-623.
55. Tanner MM, Tirkkonen M, Kallioniemi A, Collins C, Stokke T, Karhu R, Kowbel D, Shadravan F, Hintz M, Kuo WL. Increased copy number at 20q 13 in breast cancer: defining the critical region and exclusion of candidate genes. Cancer Res. 1994; 54(16):4257-4260.
56. Kallioniemi A, Kallioniemi OP, Piper J, Tanner M, Stokke T, Chen L, Smith HS, Pinkel D, Gray JW, Waldman FM. Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc Natl Acad Sci U S A. 1994; 91(6):2156-2160.
57. De Angelis PM, Clausen OP, Schjolberg A, Stokke T. Chromosomal gains and losses in primary colorectal carcinomas detected by CGH and their associations with tumour DNA ploidy, genotypes and phenotypes. Br J Cancer. 1999; 80(3-4):526-535.
58. Courjal F, Cuny M, Rodriguez C, Louason G, Speiser P, Katsaros D, Tanner MM, Zeillinger R, Theillet C.DNA amplifications at 20q13 and MDM2 define distinct subsets of evolved breast and ovarian tumours. Br J Cancer. 1996; 74(21):1984-1989.
59. Farruggio DC, Townsley FM, Ruderman JV. Cdc20 associates with the kinase aurora2/Aik. Proc Natl Acad Sci U S A. 1999; 96(13):7306-7311.
60. Dutertre S., Descamps S., Prigent C. On the role of aurora-A in centrosome function. Oncogene, 2002, 21(40):6175-6183.
61. Sugimoto K, Urano T, Zushi H, moue K, Tasaka H, Tachibana M, Dotsu M. Molecular dynamics of Aurora-A kinase in living mitotic cells simultaneously visualized with histone H3 and nuclear membrane protein importinalpha. Cell Struct Funct. 2002; 27(6):457-467.
62. T. Wittmann, M. Wilm, E. Karsenti, I. Vernos, TPX2, a novel Xenopus MAP involved in spindle pole organization, J. Cell. Biol. 149 (2000) 1405–1418.
63. T.A. Kufer, H.H. Sillje, R. Korner, O.J. Gruss, P. Meraldi, E.A. Nigg, Human TPX2 is required for targeting Aurora-A kinase to the spindle, J. Cell. Biol. 158 (2002) 617–623.
64. R. Bayliss, T. Sardon, I. Vernos, E. Conti, Structural basis of Aurora-A activation by TPX2 at the mitotic spindle, Mol. Cell 12 (2003) 851–862.
65. P.A. Eyers, E. Erikson, L.G. Chen, J.L. Maller, A novel mechanism for activation of the protein kinase Aurora A, Curr. Biol. 13 (2003) 691–697.
66. A. Hutterer, D. Berdnik, F. Wirtz-Peitz, M. Zigman, A. Schleiffer, J.A. Knoblich, Mitotic activation of the kinase Aurora-A requires its binding partner Bora, Dev. Cell 11 (2006) 147–157.
67. Marumoto T, Zhang D, Saya H. Aurora-A - a guardian of poles. Nat Rev Cancer. 2005; 5( I ):42-50.
68. D.M. Glover, M.H. Leibowitz, D.A. McLean, H. Parry, Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles, Cell 81 (1995) 95–105.
69. E. Hannak, M. Kirkham, A.A. Hyman, K. Oegema, Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans, J. Cell. Biol. 155 (2001) 1109–1116.
70. Q. Liu, J.V. Ruderman, Aurora A, mitotic entry, and spindle bipolarity, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 5811–5816.
71. C. Roghi, R. Giet, R. Uzbekov, N. Morin, I. Chartrain, R. Le Guellec, A. Couturier, M. Doree, M. Philippe, C. Prigent, The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly, J. Cell. Sci. 111 (Pt 5) (1998) 557–572.
72. A. Khodjakov, R.W. Cole, B.R. Oakley, C.L. Rieder, Centrosome- independent mitotic spindle formation in vertebrates, Curr. Biol. 10 (2000) 59–67
73. H. Maiato, C.L. Rieder, A. Khodjakov, Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis, J. Cell. Biol. 167 (2004) 831–840.
74. Kufer TA, Nigg EA, SilljéHH. Regulation of Aurora-A kinase on the mitotic spindle.Chromosoma. 2003 Dec;112(4):159-163. Epub 2003 Nov 21.
75. Kimura M, Okano Y. Aurora kinases and cancer Gan To Kagaku Ryoho. 2005 Jan;32(1):1-5.
76. T. Hirota, N. Kunitoku, T. Sasayama, T. Marumoto, D. Zhang, M. Nitta, K. Hatakeyama, H. Saya, Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells, Cell 114 (2003) 585–598.
77. D.L. Satinover, D.L. Brautigan, P.T. Stukenberg, Aurora-A kinase andinhibitor-2 regulate the cyclin threshold for mitotic entry in Xenopus early embryonic cell cycles, Cell Cycle 5 (2006) 2268–2274.
78. M.A. van Vugt, A. Bras, R.H. Medema, Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells, Mol. Cell 15 (2004) 799–811.
79. M.A. van Vugt, A. Bras, R.H. Medema, Restarting the cell cycle when the checkpoint comes to a halt, Cancer Res. 65 (2005) 7037–7040.
80. Tomotoshi Marumoto, Shinobu Honda, Toshihiro Hara,et al. Aurora-A Kinase Maintains the Fidelity of Early and Late Mitotic Events in HeLa Cells. J. Biol. Chem., Vol. 278, Issue 51,51786-51795, December 19, 2003.
81. Bischoff JR, Anderson L, Zhu Y, Mossie K, Ng L, Souza B, Schryver B, Flanagan P, Clairvoyant Drosophila F, Ginther C, Chan CS, Novotny M, Slamon DJ, Plowman GD. A homologue of aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J. 1998; 17(11):3052-3065.
82. Zhou H, Kuang J, Zhong L, Kuo WL, Gray JW, Sahin A, Brinkley BR, Sen S. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet. 1998; 20(2):189-193.
83. Gritsko TM, Coppola D, Paciga JE, Yang L, Sun M, Shelley SA, Fiorica JV, Nicosia SV, Cheng JQ. Activation and overexpression of centrosome kinase BTAK/Aurora-A in human ovarian cancer. Clin Cancer Res. 2003; 9(4):1420-1426.
84. Sakakura C, Hagiwara A, Yasuoka R, Fujita Y, Nakanishi M, Masuda K, Shimomura K, Nakamura Y, Inazawa J, Abe T, Yamagishi H. Tumour-amplified kinase BTAK is amplified and overexpressed in gastriccancers with possible involvement in aneuploid formation. Br J Cancer. 2001; 84(6):824-831.
85. Jeng YM, Peng SY, Lin CY, Hsu HC. Overexpression and amplification of Aurora-A in hepatocellular carcinoma. Clin Cancer Res. 2004; 10(6):2065-2071.
86. Sen S, Zhou H, Zhang RD, Yoon DS, Vakar-Lopez F, Ito S, Jiang F, Johnston D, Grossman HB, Ruifrok AC, Katz RL, Brinkley W, Czerniak B. Amplification/overexpression of a mitotic kinase gene in human bladder cancer. J Natl Cancer Inst. 2002; 94(17):1320-1329.
87. Hamada M, Yakushijin Y, Ohtsuka M, Kakimoto M, Yasukawa M, Fujita S. Aurora2/BTAK/STK15 is involved in cell cycle checkpoint and cell survival of aggressive non-Hodgkin's lymphoma. Br J Haematol. 2003; 12l(3):439-447.
88. Li D, Zhu J, Firozi PF, Abbruzzese JL, Evans DB, Cleary K, Friess H, Sen S. Overexpression of oncogenic STK15/BTAK/Aurora A kinase in human pancreatic cancer. Clin Cancer Res. 2003; 9(3):991-997.
89. Li YH, Li FC, Zhao X, Zhao Z, Sun KL, Sun XH. Expression of STK15 gene and chromosomal instability in laryngeal carcinoma. Yi Chuan Xue Bao. 2002; 29(12):1048-1051.
90. Reichardt W, Jung V, Brunner C, Klein A, Wemmert S, Romeike BF, Zang KD, Urbschat S. The putative serine/threonine kinase gene STK15 on chromosome 20q13.2 is amplified in human gliomas. Oncol Rep. 2003; 10(5):1275-1279.
91. J.R. Bischoff, L. Anderson, Y. Zhu, K. Mossie, L. Ng, B. Souza, B. Schryver, P. Flanagan, F. Clairvoyant, C. Ginther, C.S. Chan, M. Novotny, D.J. Slamon, G.D. Plowman, A homologue of Drosophila aurora kinase isoncogenic and amplified in human colorectal cancers, Embo J. 17 (1998) 3052–3065.
92. H. Zhou, J. Kuang, L. Zhong, W.L. Kuo, J.W. Gray, A. Sahin, B.R. Brinkley, S. Sen, Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation, Nat. Genet. 20 (1998) 189–193.
93. P. Meraldi, R. Honda, E.A. Nigg, Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53-/- cells, Embo J. 21 (2002) 483–492.
94. S. Anand, S. Penrhyn-Lowe, A.R. Venkitaraman, AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol, Cancer Cells 3 (2003) 51–62.
95. Katayama H, Zhou H, Li Q, Tatsuka M, Sen S.Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle. J Biol Chem. 2001;276(49):46219-46224.
96. Fukasawa K, Choi T, Kwiyama R, Rulong S, Vande Woude GF. Abnorma centrosome amplification in the absence of p53. Science. 1996; 271(5256):1744-1747.
97. Lingle WL, Lutz WH, Ingle JN, Maihle NJ, Salisbury JL. Centrosome hypertrophy in human breast tumors: implications for genomic stability and cell polarity. Proc Natl Acad Sci U S A, 1998, 95(6):2950-2955.
98. Pihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenbeny P, Doxsey SJ. Centrosome defects and genetic instability in malignant tumors. Cancer Res. 1998; 58(17):3974-3985.
99. Marumoto T, Hirota T, Morisaki T, Kunitoku N, Zhang D, Ichikawa Y,Sasayama T, Kuninaka S, Mimori T, Tamaki N, Kimura M, Okano Y, Saya H. Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells. Genes Cells. 2002; 7(11):1173-1182.
100. Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk J, Suzuki F, Fujii S, Arlinghaus RB, Czerniak BA, Sen S. Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53. Nat Genet. 2004, 36(1):55-62.
101. Liu Q, Kaneko S, Yang L, Feldman RI, Nicosia SV, Chen J, Cheng JQ. Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215. J Bio1 Chem. 2004; 279(50):52175-52182.
102. Yang H, Ou CC, Feldman RI, Nicosia SV, ICruk PA, Cheng JQ. Aurora-A kinase regulates telomerase activity through c-Myc in human ovarian and breast epithelial cells. Cancer Res, 2004 ,64(2):463-467.
103. Zhang H, Jin Y, Chen X, et al. Papillomavirus type 16 E6/E7 and human telomerase reverse transcriptase in esophageal cell immortalization and early transformation. Cancer Lett 2007;245(1–2):184–94.
104. Mahadevan D, Bearss DJ, Vankayalapati H. Structure2based design of novel anti-cancer agents targeting aurora kinases. Curr Med Chem Anti2Canc Agents, 2003, 3 (1): 25234.
105. Hamada M , Yakushijin Y, Ohtsuka M , et al. Aurora2/BTAK/STK15 is involved in cell cycle checkpoint and cell survival of aggressive non2Hodgkin′s lymphoma. Br J Haematol, 2003, 121(3): 4392447.
106. Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 1990 Apr; 2(4):279-289.
107. Romano N, Macino G. Quelling: transient inactivation of gene expressionin Neurospora crassa by transformation with homologous sequences. Mol Microbiol. 1992 Nov; 6(22):3343-3353.
108. Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell. 1995 May 19; 81(4):611-620.
109. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19; 391(6669):806-811.
110. Hannon GJ. RNA interference. Nature. 2002 Jul 11; 418(6894):244-251.
111. Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science. 1999 Oct 29; 286(5441):950-2.
112. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000 Mar 31;101(1):25-33.
113. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000 Mar 16; 404(6775):293-296.
114. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001 Jan 18; 409(6818):363-6.
115. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15; 15(2):188-200.
116. Wianny F and Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development [J]. Nat Cell Biol, 2000; 2(2): 70-75.
117. Elbashir S M, Harborth J, Lendeckel W, Yalcin A, Weber K, and Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001; 411(6836): 494-498.
118. Brummelkamp T R, Bernards R, and Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002; 296(5567): 550-553.
119. Song E, Lee S K, Wang J, Ince N, Ouyang N,min J, Chen J, Shankar P, and Lieberman J. RNA interference targeting Fas protects mice from fulminant hepatitis [J]. Nat Med, 2003; 9(3): 347-351.
120. Morris K V, Chan S W, Jacobsen S E, and Looney D J. Small interfering RNA-induced transcriptional gene silencing in human cells [J]. Science, 2004; 305(5688): 1289-1292.
121. Kawasaki H, Taira K, and Morris K V. siRNA Induced Transcriptional Gene Silencing in Mammalian Cells [J]. Cell Cycle, 2005; 4(3).
122. Kawasaki H and Taira K. Induction of DNA methylation and gene silencing by short interfering RNAs in human cells [J]. Nature, 2004; 431(7005): 211-217.
123. Bass BL. RNA interference. The short answer. Nature. 2001 May 24; 411(6836):428-9.
124. Matzke M, Matzke AJ, Kooter JM. RNA: guiding gene silencing. Science. 2001 Aug 10;293(5532):1080-3.
125. Bartel D P. MicroRNAs: genomics, biogenesis, mechanism, and function [J]. Cell, 2004; 116(2): 281-297.
126. Zhang H, Kolb F A, Jaskiewicz L, Westhof E, and Filipowicz W. Single processing center models for human Dicer and bacterial RNase III [J]. Cell, 2004; 118(1): 57-68.
127. Nykanen A, Haley B, and Zamore P D. ATP requirements and small interfering RNA structure in the RNA interference pathway [J]. Cell, 2001; 107(3): 309-321.
128. Hammond S M, Boettcher S, Caudy A A, Kobayashi R, and Hannon G J. Argonaute2, a link between genetic and biochemical analyses of RNAi [J]. Science, 2001; 293(5532): 1146-1150.
129. Pham J W, Pellino J L, Lee Y S, Carthew R W, and Sontheimer E J. A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila [J]. Cell, 2004; 117(1): 83-94.
130. Hutvagner G and Zamore P D. A microRNA in a multiple-turnover RNAi enzyme complex [J]. Science, 2002; 297(5589): 2056-2060.
131. Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M, and Dreyfuss G. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs [J]. Genes Dev, 2002; 16(6): 720-728.
132. Kim J, Krichevsky A, Grad Y, Hayes G D, Kosik K S, Church G M, and Ruvkun G. Identification of many microRNAs that copurify with polyribosorries in mammalian neurons [J]. Proc Natl Acad Sci U S A, 2004; 101(1): 360-365.
133. Liu J, Carmell M A, Rivas F V, Marsden C G, Thomson J M, Song J J, Hammond S M, Joshua-Tor L, and Hannon G J. Argonaute2 is the catalytic engine of mammalian RNAi [J]. Science, 2004; 305(5689): 1437-1441.
134. Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, and Tuschl T. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs [J]. Mol Cell, 2004; 15(2): 185-197.
135. Cerutti H. RNA interference: traveling in the cell and gaining function?Trends Genet. 2003 Jan; 19(1):39-46.
136. Marx J. Interfering with gene expression. Science. 2000 May 26;288(5470):1370-2.
137. Khvorova A, Reynolds A, and Jayasena S D. Functional siRNAs and miRNAs exhibit strand bias [J]. Cell, 2003; 115(2): 209-216.
138. Schwarz D S, Hutvagner G, Du T, Xu Z, Aronin N, and Zamore P D. Asymmetry in the assembly of the RNAi enzyme complex [J]. Cell, 2003; 115(2): 199-208.
139. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science. 2004 Sep 3;305(5689):1434-7.
140. Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal S I, and Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex [J]. Science, 2004; 303(5658): 672-676.
141. Tuschl T ,Zamore PD, Lehmann R, et al. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev, 1999, 13(24): 3191-3197.
142. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 2002; 16: 948-958.
143. Yu JY, Taylor J, DeRuiter SL, Vojtek AB, et al. Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors. Mol Ther 2003; 7: 228-236.
144. Kim VN. RNA interference in functional genomics and medicine. J orean Med Sci. 2003 Jun; l 8(3):309-318.
145. Nishikura K. A short primer on RNA: RNA-directed RNA polymerase actsas a key catalyst. cell, 2001, 107 (4):415-418.
146. Gan L, Anton KE, Masterson BA, et al. Specific interference with gene expression and gene function mediated by long double-stranded RNA in neural ce11s. J Neurosci Methods, 2002, 121(2): 151-157.
147. Wohlbold L, van der Kuip H, Miething C, Vornlocher H P, Knabbe C, Duyster J, and Aulitzky W E. Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571)[J]. Blood, 2003; 102(6): 2236-2239.
148. Scherr M, Battmer K, Winkler T, Heidenreich O, Ganser A, and Eder M. Specific inhibition of bcr-abl gene expression by small interfering RNA[J]. Blood, 2003; 101(4): 1566-1569.
149. Li M J, McMahon R, Snyder D S, Yee J K, and Rossi J J. Specific killing of Ph+ chronic myeloid leukemia cells by a lentiviral vector-delivered anti-bcr/abl small hairpin RNA[J]. Oligonucleotides, 2003; 13(5): 401-409.
150. Wilda M, Fuchs U, Wossmann W, and Borkhardt A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi) [J]. Oncogene, 2002; 21(37): 5716-5724.
151. Sumimoto H, Yamagata S, Shimizu A, Miyoshi H, Mizuguchi H, Hayakawa T, Miyagishi M, Taira K, and Kawakami Y. Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference. [J]. Gene Ther, 2005; 12(1): 95-100.
152. Duxbury M S, Matros E, Ito H, Zinner M J, Ashley S W, and Whang E E. Systemic siRNA-mediated gene silencing: a new approach to targeted therapy of cancer[J]. Ann Surg, 2004; 240(4): 667-674; discussion 675-666.
153. Lakka S S, Gondi C S, Yanamandra N, Olivero W C, Dinh D H, Gujrati M, and Rao J S. Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis[J]. Oncogene, 2004; 23(27): 4681-4689.
154. Zhang Y, Zhang Y F, Bryant J, Charles A, Boado R J, and Pardridge W M. Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer [J]. Clin Cancer Res, 2004; 10(11): 3667-3677.
155. Brummelkamp T R, Bernards R, and Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference [J]. Cancer Cell, 2002; 2(3): 243-247.
156. Stege A, Priebsch A, Nieth C, and Lage H. Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference [J]. Cancer Gene Ther, 2004; 11(11): 699-706.
157. Yague E, Higgins C F, and Raguz S. Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1 [J]. Gene Ther, 2004; 11(14): 1170-1174.
158. Shen H, Pan Y, Han Z, Hong L, Liu N, Han S, Yao L, Xie H, Zhaxi C, Shi Y, and Fan D. Reversal of multidrug resistance of gastric cancer cells by downregulation of TSG101 with TSG101siRNA [J]. Cancer Biol Ther, 2004; 3(6): 561-565.
159. Nieth C, Priebsch A, Stege A, and Lage H. Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi) [J]. FEBS Lett, 2003; 545(2-3): 144-150.
160. Miller V M, Gouvion C M, Davidson B L, and Paulson H L. TargetingAlzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles[J]. Nucleic Acids Res, 2004; 32(2): 661-668.
161. Barthel A, Herzig S, Muller H W, Harborth J, and Bornstein S R. RNA Interference-based Strategies for Metabolic Syndrome Treatment [J]. Horm Metab Res, 2005; 37(2): 59-62.
162. Lieberman J, Song E, Lee S K, and Shankar P. Interfering with disease: opportunities and roadblocks to harnessing RNA interference [J]. Trends Mol Med, 2003; 9(9): 397-403.
163. Gitlin L and Andino R. Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing [J]. J Virol, 2003, 77(13): 7159-7165.
164. Radhakrishnan S K, Layden T J, and Gartel A L. RNA interference as a new strategy against viral hepatitis [J]. Virology, 2004; 323(2): 173-181.
165. Shi Y, Yang de H, Xiong J, Jia J, Huang B, and Jin Y X. Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs [J]. Cell Res, 2005; 15(3): 193-200.
166. Li T, Zhang Y, Fu L, Yu C, Li X, Li Y, Zhang X, Rong Z, Wang Y, Ning H, Liang R, Chen W, Babiuk L A, and Chang Z. siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication [J]. Gene Ther, 2005 May; 12(9):751-61.
167. Wu C J, Huang H W, Liu C Y, Hong C F, and Chan Y L. Inhibition of SARS-CoV replication by siRNA [J]. Antiviral Res, 2005; 65(1): 45-48.
168. Zheng B J, Guan Y, Tang Q, Du C, Xie F Y, He M L, Chan K W, Wong K L, Lader E, Woodle M C, Lu P Y, Li B, and Zhong N. Prophylactic and therapeutic effects of small interfering RNA targeting SARS-coronavirus [J]. Antivir Ther, 2004; 9(3): 365-374.
169. Radhakrishnan S, Gordon J, Del Valle L, Cui J, and Khalili K. Intracellular approach for blocking JC virus gene expression by using RNA interference during viral infection [J]. J Virol, 2004; 78(13): 7264-7269.
170. Orba Y, Sawa H, Iwata H, Tanaka S, and Nagashima K. Inhibition of virus production in JC virus-infected cells by postinfection RNA interference [J]. J Virol, 2004; 78(13): 7270-7273.
171. Sorensen D R, Leirdal M, and Sioud M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice [J]. J Mol Biol, 2003; 327(4): 761-766.
172. Pusch O, Boden D, Silbermann R, Lee F, Tucker L, and Ramratnam B. Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA [J]. Nucleic Acids Res, 2003; 31(22): 6444-6449.
173. Jackson A L, Bartz S R, Schelter J, Kobayashi S V, Burchard J, Mao M, Li B, Cavet G, and Linsley P S. Expression profiling reveals off-target gene regulation by RNAi [J]. Nat Biotechnol, 2003; 21(6): 635-637.
174. Persengiev S P, Zhu X, and Green M R. Nonspecific, concentration- dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs) [J]. Rna, 2004; 10(1): 12-18.
175. Saxena S, Jonsson Z O, and Dutta A. Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells [J]. J Biol Chem, 2003; 278(45): 44312-44319.
176. Pancoska P, Moravek Z, and Moll U M. Efficient RNA interference depends on global context of the target sequence: quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA [J]. Nucleic Acids Res, 2004; 32(4): 1469-1479.
177. Haley B and Zamore P D. Kinetic analysis of the RNAi enzyme complex [J]. Nat Struct Mol Biol, 2004; 11(7): 599-606.
178. Zamore P D. RNA interference: listening to the sound of silence [J]. Nat Struct Biol, 2001; 8(9): 746-750.
179. Bridge A J, Pebernard S, Ducraux A, Nicoulaz A L, and Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells [J]. Nat Genet, 2003; 34(3): 263-264.
180. Sledz C A, Holko M, de Veer M J, Silverman R H, and Williams B R. Activation of the interferon system by short-interfering RNAs[Jl. Nat Cell Biol, 2003; 5(9): 834-839.
181. Moss E G and Taylor J M. Small-interfering RNAs in the radar of the interferon system[J]. Nat Cell Biol, 2003; 5(9): 771-772.
182. Kim D H, Longo M, Han Y, Lundberg P, Cantin E, and Rossi J J. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase[J]. Nat Biotechnol, 2004; 22(3): 321-325.
183. Pebernard S and. Iggo R D. Determinants of interferon- stimulated gene induction by RNAi vectors[J]. Differentiation, 2004; 72(2-3): 103-111.
184. Diebold S S, Montoya M, Unger H, Alexopoulou L, Roy P, Haswell L E, Al-Shamkhani A, Flavell R, Borrow P, and Reis a Sousa C. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers [J]. Nature, 2003; 424(6946): 324-328.
185. Kariko K, Bhuyan P, Capodici J, and Weissman D. Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 [J]. J Immunol, 2004; 172(11): 6545-6549.
186. Lingor P, Michel U, Scholl U, Bahr M, and Kugler S. Transfection of"naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons [J]. Biochem Biophys Res Commun, 2004; 315(4): 1126-1133.
187.刘士德陈莲凤,原代培养的正常人宫颈鳞状上皮细胞超微结构.电子显微学报,1994.13(5).419-419
188. Huang X, Hutchins B, Patel RC. The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR). Biochem J, 2002, 366(1): 175-186.
189. Peters GA, Li S, Sen GC. Phosphorylation of specific serine residues in the PKR activation domain of PACT is essential for its ability to mediate apoptosis. J Biol Chem, 2006, 281(46): 35129-35136.
190. Gupta V, Patel RC. Proapoptotic protein PACT is expressed at high levels in colonic epithelial cells in mice. Am J Physiol Gastrointest Liver Physiol, 2002, 283(3): 801~808.
191. Patel CV, Handy I, Goldsmith T, Patel RC. PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR. J Biol Chem, 2000, 275(48): 37993-37998.
192. Kok KH, Ng MH, Ching YP, Jin DY. Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. J Biol Chem, 2007, 282(24):17649-17657.
193. Gale, M., Jr., S.-L. Tan, M. Wambach, and M. G. Katze. 1996. Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation. Mol. Cell. Biol. 16:4172-4181.
194. Goodman AG, Smith JA, Balachandran S, Perwitasari O, Proll SC, Thomas MJ, Korth MJ, Barber GN, Schiff LA, Katzemg. The Cellular Protein P58IPK Regulates Influenza Virus mRNA Translation and Replication through a PKR-Mediated Mechanism. J Virol. 2007 Mar;81(5):2221-30.
195. Yan W, Frank CL, Korth MJ, Sopher BL, Novoa I, Ron D, Katzemg. Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK. Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):15920-5.
196. Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe ? Nat Rev Cancer, 2004, 4(12): 966-977.
197. Khan MM, Nomura T, Chiba T, et al. The fusion oncoprotein PML- RARalpha induces endoplasmic reticulum(ER)-associated degradation of N-CoR and ER stress. J Biol Chem, 2004, 279(12): 11814-11824.
198. Kitamura Y, Miyamura A, Takata K. Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in thapsigargin-induced apoptosis in human neuroblastoma SH-SY5Y cells. J Pharmacol Sci, 2003, 92 (3): 228-236.
199. Thomlinson RH, GrayLH. The histological structure of some human lung cancers and the possible imp lications for radiotherapy[J]. Br J Cancer, 1955, 9 (4): 539-549.
200. FelsDR, Koumenis C. The PERK/e-IF2alpha /ATF4 module of the UPR in hypoxia resistance and tumor growth [J]. Cancer Biol Ther, 2006, 5 (7): 723-728.
201. Tatsuo Hata, Toru Furukawa, Makoto Sunamura, Shinichi Egawa,Fuyuhiko Motoi, Noriyuki Ohmura, Tomotoshi Marumoto, Hideyuki Saya and Akira Horii RNA Interference Targeting Aurora Kinase A Suppresses Tumor Growth and Enhances the Taxane Chemosensitivity in Human Pancreatic Cancer Cells. Cancer Research 65, 2899-2905
202. Klein A, Flügel D, Kietzmann T. Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1. Mol Biol Cell. 2008 Sep;19(9):3667-75.
203. Tao Y, Zhang P, Frascogna V, Lecluse Y, Auperin A, Bourhis J, Deutsch E.Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells. Br J Cancer. 2007 Dec 17;97(12):1664-72.
204. Dutertre S, Cazales M, Quaranta M, Froment C, Trabut V, Dozier C, Mirey G, BouchéJP, Theis-Febvre N, Schmitt E, Monsarrat B, Prigent C, Ducommun B.Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition. J Cell Sci. 2004 May 15;117(Pt 12):2523-31.
205. Song SJ, Song MS, Kim SJ, Kim SY, Kwon SH, Kim JG, Calvisi DF, Kang D, Lim DS. Aurora A Regulates Prometaphase Progression by Inhibiting the Ability of RASSF1A to Suppress APC-Cdc20 Activity. Cancer Res. 2009 Mar 15;69(6):2314-23. Epub 2009 Mar 10.
206. Hata T, Furukawa T, Sunamura M, Egawa S, Motoi F, Ohmura N, Marumoto T, Saya H, Horii A. RNA interference targeting aurora kinase a suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells. Cancer Res. 2005 Apr 1;65(7):2899-905.
207. Kufer, T. A., Sillje, H. H., Korner, R., Gruss, O. J., Meraldi, P., and Nigg, E. A.(2002). Human TPX2 is required for targeting Aurora-A kinase to thespindle.J. Cell Biol. 158, 617–623.
208. Matsumoto S, Miyagishi M, Akashi H, Nagai R, Taira K.Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library. J Biol Chem. 2005 Jul 8;280(27):25687-96.
209. Cardin E, Larose L.Nck-1 interacts with PKR and modulates its activation by dsRNA. Biochem Biophys Res Commun. 2008 Dec 5;377(1):231-5.
210. Cabanski M, Steinmüller M, Marsh LM, Surdziel E, Seeger W, Lohmeyer J. PKR regulates TLR2/TLR4-dependent signaling in murine alveolar macrophages. Am J Respir Cell Mol Biol. 2008 Jan;38(1):26-31.
211. von Holzen U, Pataer A, Raju U, Bocangel D, Vorburger SA, Liu Y, Lu X, Roth JA, Aggarwal BB, Barber GN, Keyomarsi K, Hunt KK, Swisher SG. The double-stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation.
212. Scharer CD, Laycock N, Osunkoya AO, Logani S, McDonald JF, Benigno BB, Moreno CS. Aurora kinase inhibitors synergize with paclitaxel to induce apoptosis in ovarian cancer cells. J Transl Med. 2008 Dec 11;6:79.
213. Carpinelli P, Moll J. Aurora kinases and their inhibitors: more than one target and one drug. Adv Exp Med Biol. 2008;610:54-73.
214. Girdler F, Sessa F, Patercoli S, Villa F, Musacchio A, Taylor S. Molecular basis of drug resistance in aurora kinases. Chem Biol. 2008 Jun;15(6):552-62.
215. Gontarewicz A, Balabanov S, Keller G, Panse J, Schafhausen P, Bokemeyer C, Fiedler W, Moll J, Brümmendorf TH. PHA-680626 exhibits anti-proliferative and pro-apoptotic activity on Imatinib-resistantchronic myeloid leukemia cell lines and primary CD34+ cells by inhibition of both Bcr-Abl tyrosine kinase and Aurora kinases. Leuk Res. 2008 Dec;32(12):1857-65.
216. Grant KM. Targeting the cell cycle in the pursuit of novel chemotherapies against parasitic protozoa. Curr Pharm Des. 2008;14(9):917-24.
217. Akahane D, Tauchi T, Okabe S, Nunoda K, Ohyashiki K. PHA-680626 exhibits anti-proliferative and pro-apoptotic activity on Imatinib-resistant chronic myeloid leukemia cell lines and primary CD34+ cells by inhibition of both Bcr-Abl tyrosine kinase and Aurora kinases. Leuk Res. 2008 Dec;32(12):1857-65.
218. Gontarewicz A, Balabanov S, Keller G, Panse J, Schafhausen P, Bokemeyer C, Fiedler W, Moll J, Brümmendorf TH.PHA-680626 exhibits anti-proliferative and pro-apoptotic activity on Imatinib-resistant chronic myeloid leukemia cell lines and primary CD34+ cells by inhibition of both Bcr-Abl tyrosine kinase and Aurora kinases.Leuk Res. 2008 Dec;32(12):1857-65.
219. Nallagatla SR, Bevilacqua PC. Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner. RNA. 2008 Jun;14(6):1201-13.
220. Zheng X, Bevilacqua PC. Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails. RNA. 2004 Dec;10(12):1934-45.
221. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol. 2003 Sep;5(9):834-9.
222. Puthenveetil S, Whitby L, Ren J, Kelnar K, Krebs JF, Beal PA. Controllingactivation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification. Nucleic Acids Res. 2006;34(17):4900-11.
223. Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.Nat Biotechnol. 2005 Apr;23(4):457-62.
224. McAllister CS, Samuel CE.The RNA-activated protein kinase enhances the induction of interferon-beta and apoptosis mediated by cytoplasmic RNA sensors.J Biol Chem. 2009 Jan 16;284(3):1644-51.
225. Jie Li, Guang Yang, Shaohua Li, Guojun Cao, Qiang Zhao, Xuemei Liu,ming Fan, Beifen Shen, Ningsheng Shao. 3’-Poly(A) tail enhances siRNA activity against exogenous reporter genes in MCF-7 cells. Journal of RNAi and Gene Silencing (2006), 2(2), 195-204
226. Kojima K, Konopleva M, Tsao T, Nakakuma H, Andreeff M. Concomitant inhibition of Mdm2-p53 interaction and Aurora kinases activates the p53-dependent postmitotic checkpoints and synergistically induces p53-mediated mitochondrial apoptosis along with reduced endoreduplication in acute myelogenous leukemia. Blood. 2008 Oct 1;112(7):2886-95.
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