沉默hTERT基因表达对不同p53状态卵巢癌细胞生长及凋亡的快速影响
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摘要
研究背景:端粒酶因可在细胞内通过其逆转录酶活性复制和延长染色体末端的端粒重复序列(TTAGGG)而广为人知。其可使细胞克服因每次有丝分裂而造成的端粒缩短,因此在细胞的永生化中扮演着重要的角色。人端粒酶活性的表达由端粒酶主要组成部分之一——端粒酶逆转录酶(human telomerase reverse transcriptase, hTERT)的转录水平所调控。由于hTERT仅在大部分恶性肿瘤组织中特异性表达,而正常组织中(造血干细胞,生殖细胞除外)一般无表达,故通过抑制hTERT表达从而抑制端粒酶活性可能成为肿瘤治疗的特异性靶点。靶向hTERT的基因治疗与目前认可的治疗方法相比,将具有更少的毒性副反应。
过去认为,单纯针对端粒酶的抑制剂由于端粒需要一定的时间才能缩短至可以诱发细胞衰老死亡的临界长度,故其治疗效果有一定的滞后性,且疗程的长短在很大程度上依赖于治疗起始时细胞端粒的长度以及细胞有丝分裂的周期长短。但与此不符的是,许多针对hTERT和端粒酶的研究都显示,一旦hTERT表达受抑,肿瘤细胞可以很快出现凋亡,且并不一定伴有端粒长度的明显改变。提示抑制端粒酶活性可能通过某种未知的机制,导致肿瘤细胞遵循另一条不依赖端粒长度损耗的“快速通道”而迅速走向死亡。由于抑制hTERT表达的抗肿瘤机制尚不明确,目前报道其在不同肿瘤细胞中的作用也各不相同,故有必要针对不同组织学类型的肿瘤进行具体的研究。
p53是在多条生长相关通路包括凋亡、细胞周期阻滞以及衰老中扮演关键角色的肿瘤抑制基因,已证实其可以通过抑制Sp1与hTERT启动子结合而下调hTERT的转录活性。但直到最近才有报道显示hTERT的过度表达或抑制也可以立即对p53的表达产生负性或正性的影响,且不伴有细胞端粒长度的改变,提示在hTERT与p53之间可能存在一定的反馈通路,hTERT可能通过某种端粒以外的效应作用于自身的细胞周期调节基因,而不需要依赖端粒长度的缩短。目前尚无研究显示由hTERT表达改变所引起的p53转录活性改变是否与导致细胞死亡的“快速通道”相关,但由于p53与凋亡的密切相关性,我们推测抑制hTERT表达所引起的细胞快速死亡可能与p53的表达改变有关。
研究目的:本研究的目的是检测hTERT基因表达沉默后能否引起多种卵巢癌细胞生长的快速抑制和死亡,并探讨这种现象与p53基因表达激活的相关性。我们旨在在含有不同p53状态的卵巢癌细胞中,探索抑制hTERT表达发挥效应的快速作用模式,并希望对此过程的更深刻理解能够为发现对hTERT抑制剂敏感或抵抗的原因提供新的线索,为卵巢癌的治疗提供新的思路。
研究方法:本研究构建了两个靶向hTERT的发夹状RNA干扰质粒pG1和pG2,和一个由与人类基因无同源性的随机序列组成的对照质粒pGCR,并将其在体外和体内分别转染入含不同p53状态的三株端粒酶阳性卵巢癌细胞中,包括含野生型p53的A2780细胞、含突变型p53的OVCAR3细胞和缺乏p53表达的SKOV3细胞。
在体外实验中,A2780, OVCAR3和SKOV3细胞分别进行了pG1, pG2质粒的转染,以pGCR质粒和未处理的细胞作为对照。hTERT的抑制效率分别通过实时荧光定量RT-PCR和免疫印迹进行了验证,转染后细胞的端粒酶活性则以TRAP法进行检测。采用CCK-8检测细胞的生长情况,流式细胞仪进行细胞周期分析。细胞凋亡情况则采用AnnexinⅤ-PE/7AAD双染和TUNEL法分别进行了检测,以免疫印迹检测p53和其下游基因p21的表达情况。
在动物实验中,将A2780和SKOV3细胞注射至裸鼠皮下以构建移植瘤模型。成瘤后随机分为3组,予以干扰质粒pG2、乱序质粒pGCR以及生理盐水分别瘤内注射,隔日一次共2周。每次注射前测量肿瘤大小,治疗完毕后完整剥离肿瘤称重并制成石蜡切片行HE染色进行形态学观察,采用TUNEL及电镜检测细胞凋亡情况,以免疫印迹法检测hTERT表达抑制情况,p53及p21的表达则采用免疫组化进行检测。研究结果:1.酶切及测序结果显示重组质粒pG1、pG2和pGCR构建成功。实时荧光定量RT-PCR、免疫印迹和TRAP分析显示靶向hTERT的发夹状RNA干扰质粒能够有效地抑制卵巢癌细胞中hTERT mRNA和蛋白的表达并抑制端粒酶活性。
2.体外实验中,沉默hTERT基因表达能够引起三株卵巢癌细胞快速的生长抑制,细胞周期分析显示抑制hTERT表达主要通过将细胞周期阻滞在S期而导致细胞生长减慢。其中两株细胞株,A2780和OVCAR3,在hTERT表达受抑后均很快出现了明显的凋亡,同时伴随着p53和p21表达的增高。相反的是,沉默hTERT基因在缺乏p53基因表达的SKOV3细胞中并未导致明显的凋亡,其p21基因表达也较对照组降低。
3.成功构建A2780和SKOV3细胞裸鼠皮下移植瘤。与注射pGCR和生理盐水组相比,重组质粒pG2在体内能够显著减慢两种细胞移植瘤的生长,减小肿瘤体积并减轻肿瘤重量。在A2780细胞移植瘤中出现了大量的凋亡和坏死,同时p53和p21的表达也明显增强。但在SKOV3细胞移植瘤中仅观察到大片状的坏死,细胞凋亡不明显,p21的表达较对照组相比没有明显改变。未发现治疗后的裸鼠有明显的毒副反应。
结论:针对hTERT的发夹状RNA干扰质粒在体内外均能有效地和特异地沉默卵巢癌细胞hTERT基因的表达、降低端粒酶活性并迅速引起肿瘤细胞的生长抑制。但在含有不同p53状态的细胞系中,其引起细胞死亡的机制并不相同,抑制hTERT表达所立即引起的卵巢癌细胞凋亡与其引起抑癌基因p53及p21上调有关。我们再一次证实了在通过使端粒逐渐缩短而引起细胞衰老死亡的“经典途径”以外,抑制端粒酶活性还可以通过另一条并不依赖于细胞端粒长度改变的“快速通道”引起细胞的迅速死亡。这对以hTERT和端粒酶为靶点的抗肿瘤治疗具有重大意义。
Background : Telomerase is well-known as a cellular reverse transcriptase that synthesizes telomeric (TTAGGG) repeats at the ends of chromosomes. It permits cancer cells to overcome the progressive loss of telomere during each round of cell division thus plays a critical role in cell immortality. Telomerase expression is predominantly regulated at the transcriptional level of the human telomerase reverse transcriptase (hTERT), one of the main components of telomerase. Because of its preferential expression in malignant tumor and absence in most normal tissues, it has been widely accepted that hTERT is a potential target of a cancer therapy which would be more cancer specific and perhaps with fewer cytotoxic side effects, as compared with currently approved therapies.
It is believed that 'pure' telomerase inhibitors require a lag time before telomeres shorten to a critical length that could trigger senescence, and this process must be slow and depends on the initial telomere length and the cell division cycle. Interestingly, many present studies targeting hTERT and telomerase showed dramatic short-term effects leading to rapid cell death of the tumor cells,which was apparently not dependent on telomere loss, suggesting that some occult influence may present immediately in the absence of telomerase,which lead to a 'fast-track' resulting in rapid cell death without obvious telomere loss. As the anti-tumor mechanism of hTERT inhibition remains unclear, its effects are diverse in different tumor cell lines according to existing reports. It is necessary to test the anti-tumor effects of hTERT inhibition with different histological types.
As a critical tumor suppressor implicated in several diverse growth-related pathways, including apoptosis, cell cycle arrest, and senescence, p53 is proved to down regulate the hTERT transcription, which is likely to be mediated by inhibition of the Sp1 binding to the hTERT promoter. Not until recently has it been found that the overexpression or repression of hTERT may have an immediate negative or positive effect on the transcription of p53 in some human cells before telomeric changes are detectable, suggesting that there may be a feedback loop between hTERT and p53, and hTERT may exert an extra-telomeric effect on its own regulatory gene components independent of telomere length. But whether the consequent change of p53 expression level is associated with the 'fast-track' of telomerase has not been elucidated yet. Due to the close relationship between p53 and apoptosis, we hypothesized that the possible rapid cell death caused by the knockdown of hTERT may be partially attributed to altered functional p53.
Objective:In this study, we constructed two recombinant plasmids of short hairpin RNA(shRNA) targeting hTERT named pG1 and pG2 respectively. Another clone, pGCR, which expresses a shRNA scrambled sequence, was also constructed as a control. Three telomerase-positive human epithelial ovarian cancer cell lines: wild type-p53(wt-p53) A2780, mutant type-p53(mt-p53) OVCAR3 and p53-null SKOV3 were transfected with pG1, pG2 and pGCR respectively both in vitro and in vivo, to test the possibility of rapid growth arrest and apoptosis induced by hTERT gene silence, and to determine whether this phenomenon is related to the activation of p53 expression. We sought to determine the mode of short-term effects induced by hTERT inhibitors in human ovarian carcinoma cells that harbor different status of p53, and we hoped an improved understanding of this process could provide insight into mechanisms of sensitivity and/or resistance to hTERT inhibitors, thus uncover new targets for ovarian cancer therapy.
Methods: A2780, OVCAR3 and SKOV3 were transfected with pG1, pG2 and pGCR in vitro respectively, and untreated cells were also used as one of controls. The efficiency of hTERT inhibition was confirmed with real-time RT-PCR and western blot, and telomerase activity was evaluated with TRAP assay. Cell proliferation was measured with CCK-8 assay, and cell circle analysis was performed by Flow cytometric. Apoptosis was detected with AnnexinⅤ-PE/7AAD and TUNEL assay, and expression of p53 and p21 were measured with western blot.
A2780 and SKOV3 cells were injected into hypodermic tissue to construct implanted-tumor model in nude mice. After xenograft tumor formed, recombinant plasmids of pG2, pGCR and saline were injected intratumorally every other day respectively for two weeks.The size of tumor was measured at the same time. Tumors were excised, weighted, sectioned and assessed for mophplogy by HE, and apoptosis was detected with TUNEL and electron microscope after treatment. The efficiency of hTERT inhibition was confirmed with western blot, and expression of p53 and p21 were measured with immunohistochemistry.
Results: 1. Restriction enzyme mapping and sequencing demonstrated that pG1, pG2 and pGCR were constructed successfully. Real-time RT-PCR, western blot and TRAP assay showed shRNA directed against hTERT could efficiently silence hTERT expression and inhibit telomerase activity in ovarian cancer cells.
2. knock-down of hTERT induced rapid growth reduction in vitro in all three cell lines of ovarian cancer, and cell cycle analysis showed that growth inhibited by hTERT silence is mediated by S-phase arrest. Two cell lines, A2780 and OVCAR3, with enhancive apoptosis under the influence of hTERT suppression, both presented accumulation of p53 and p21 proteins. In contrast, the knock down of hTERT in p53-null SKOV3 cells resulted in a rapid decreased expression of p21 and did not cause obvious cell apoptosis.
3. Hypodermic tumor-implanted model were established successfully by 100% with A2780 and SKOV3 cells. As compared with pGCR and saline-treated mice, recombinant plasmid pG2 could significantly slow down tumor growth, decrease tumor volume and tumor weight in vivo in both two xenograft tumor. Massive apoptosis and necrosis presented in A2780-implanted tumor after treatment, with reinforcement of p53 and p21 expression. But only necrosis presented in SKOV3-implanted tumor, and the expression of p21 appeared to be unaffected. No obvious side effects were observed in treated mice.
Conclusions: Our data demonstrated that recombinant plasmids of shRNA could efficiently and specifically knock-down hTERT expression, decrease telomerase activity, and inhibit tumor cell growth immediately both in vitro and in vivo, but the mechanism of cell death is differ in ovarian cancer cells with different p53 state, and apoptosis induced immediately by inhibition of hTERT may be due, in part, to up-regulation of growth inhibitory genes p53 and p21. We confirmed once again that, besides the 'classical' mechanism by which inhibition of telomerase could lead to telomere shortening preceding cell death, an 'fast - track' not requiring telomere shortening also exist in cancer cells. This is meaningful for the anticancer therapy targeting telomerase and hTERT.
过去认为,单纯针对端粒酶的抑制剂由于端粒需要一定的时间才能缩短至可以诱发细胞衰老死亡的临界长度,故其治疗效果有一定的滞后性,且疗程的长短在很大程度上依赖于治疗起始时细胞端粒的长度以及细胞有丝分裂的周期长短。但与此不符的是,许多针对hTERT和端粒酶的研究都显示,一旦hTERT表达受抑,肿瘤细胞可以很快出现凋亡,且并不一定伴有端粒长度的明显改变。提示抑制端粒酶活性可能通过某种未知的机制,导致肿瘤细胞遵循另一条不依赖端粒长度损耗的“快速通道”而迅速走向死亡。由于抑制hTERT表达的抗肿瘤机制尚不明确,目前报道其在不同肿瘤细胞中的作用也各不相同,故有必要针对不同组织学类型的肿瘤进行具体的研究。
p53是在多条生长相关通路包括凋亡、细胞周期阻滞以及衰老中扮演关键角色的肿瘤抑制基因,已证实其可以通过抑制Sp1与hTERT启动子结合而下调hTERT的转录活性。但直到最近才有报道显示hTERT的过度表达或抑制也可以立即对p53的表达产生负性或正性的影响,且不伴有细胞端粒长度的改变,提示在hTERT与p53之间可能存在一定的反馈通路,hTERT可能通过某种端粒以外的效应作用于自身的细胞周期调节基因,而不需要依赖端粒长度的缩短。目前尚无研究显示由hTERT表达改变所引起的p53转录活性改变是否与导致细胞死亡的“快速通道”相关,但由于p53与凋亡的密切相关性,我们推测抑制hTERT表达所引起的细胞快速死亡可能与p53的表达改变有关。
研究目的:本研究的目的是检测hTERT基因表达沉默后能否引起多种卵巢癌细胞生长的快速抑制和死亡,并探讨这种现象与p53基因表达激活的相关性。我们旨在在含有不同p53状态的卵巢癌细胞中,探索抑制hTERT表达发挥效应的快速作用模式,并希望对此过程的更深刻理解能够为发现对hTERT抑制剂敏感或抵抗的原因提供新的线索,为卵巢癌的治疗提供新的思路。
研究方法:本研究构建了两个靶向hTERT的发夹状RNA干扰质粒pG1和pG2,和一个由与人类基因无同源性的随机序列组成的对照质粒pGCR,并将其在体外和体内分别转染入含不同p53状态的三株端粒酶阳性卵巢癌细胞中,包括含野生型p53的A2780细胞、含突变型p53的OVCAR3细胞和缺乏p53表达的SKOV3细胞。
在体外实验中,A2780, OVCAR3和SKOV3细胞分别进行了pG1, pG2质粒的转染,以pGCR质粒和未处理的细胞作为对照。hTERT的抑制效率分别通过实时荧光定量RT-PCR和免疫印迹进行了验证,转染后细胞的端粒酶活性则以TRAP法进行检测。采用CCK-8检测细胞的生长情况,流式细胞仪进行细胞周期分析。细胞凋亡情况则采用AnnexinⅤ-PE/7AAD双染和TUNEL法分别进行了检测,以免疫印迹检测p53和其下游基因p21的表达情况。
在动物实验中,将A2780和SKOV3细胞注射至裸鼠皮下以构建移植瘤模型。成瘤后随机分为3组,予以干扰质粒pG2、乱序质粒pGCR以及生理盐水分别瘤内注射,隔日一次共2周。每次注射前测量肿瘤大小,治疗完毕后完整剥离肿瘤称重并制成石蜡切片行HE染色进行形态学观察,采用TUNEL及电镜检测细胞凋亡情况,以免疫印迹法检测hTERT表达抑制情况,p53及p21的表达则采用免疫组化进行检测。研究结果:1.酶切及测序结果显示重组质粒pG1、pG2和pGCR构建成功。实时荧光定量RT-PCR、免疫印迹和TRAP分析显示靶向hTERT的发夹状RNA干扰质粒能够有效地抑制卵巢癌细胞中hTERT mRNA和蛋白的表达并抑制端粒酶活性。
2.体外实验中,沉默hTERT基因表达能够引起三株卵巢癌细胞快速的生长抑制,细胞周期分析显示抑制hTERT表达主要通过将细胞周期阻滞在S期而导致细胞生长减慢。其中两株细胞株,A2780和OVCAR3,在hTERT表达受抑后均很快出现了明显的凋亡,同时伴随着p53和p21表达的增高。相反的是,沉默hTERT基因在缺乏p53基因表达的SKOV3细胞中并未导致明显的凋亡,其p21基因表达也较对照组降低。
3.成功构建A2780和SKOV3细胞裸鼠皮下移植瘤。与注射pGCR和生理盐水组相比,重组质粒pG2在体内能够显著减慢两种细胞移植瘤的生长,减小肿瘤体积并减轻肿瘤重量。在A2780细胞移植瘤中出现了大量的凋亡和坏死,同时p53和p21的表达也明显增强。但在SKOV3细胞移植瘤中仅观察到大片状的坏死,细胞凋亡不明显,p21的表达较对照组相比没有明显改变。未发现治疗后的裸鼠有明显的毒副反应。
结论:针对hTERT的发夹状RNA干扰质粒在体内外均能有效地和特异地沉默卵巢癌细胞hTERT基因的表达、降低端粒酶活性并迅速引起肿瘤细胞的生长抑制。但在含有不同p53状态的细胞系中,其引起细胞死亡的机制并不相同,抑制hTERT表达所立即引起的卵巢癌细胞凋亡与其引起抑癌基因p53及p21上调有关。我们再一次证实了在通过使端粒逐渐缩短而引起细胞衰老死亡的“经典途径”以外,抑制端粒酶活性还可以通过另一条并不依赖于细胞端粒长度改变的“快速通道”引起细胞的迅速死亡。这对以hTERT和端粒酶为靶点的抗肿瘤治疗具有重大意义。
Background : Telomerase is well-known as a cellular reverse transcriptase that synthesizes telomeric (TTAGGG) repeats at the ends of chromosomes. It permits cancer cells to overcome the progressive loss of telomere during each round of cell division thus plays a critical role in cell immortality. Telomerase expression is predominantly regulated at the transcriptional level of the human telomerase reverse transcriptase (hTERT), one of the main components of telomerase. Because of its preferential expression in malignant tumor and absence in most normal tissues, it has been widely accepted that hTERT is a potential target of a cancer therapy which would be more cancer specific and perhaps with fewer cytotoxic side effects, as compared with currently approved therapies.
It is believed that 'pure' telomerase inhibitors require a lag time before telomeres shorten to a critical length that could trigger senescence, and this process must be slow and depends on the initial telomere length and the cell division cycle. Interestingly, many present studies targeting hTERT and telomerase showed dramatic short-term effects leading to rapid cell death of the tumor cells,which was apparently not dependent on telomere loss, suggesting that some occult influence may present immediately in the absence of telomerase,which lead to a 'fast-track' resulting in rapid cell death without obvious telomere loss. As the anti-tumor mechanism of hTERT inhibition remains unclear, its effects are diverse in different tumor cell lines according to existing reports. It is necessary to test the anti-tumor effects of hTERT inhibition with different histological types.
As a critical tumor suppressor implicated in several diverse growth-related pathways, including apoptosis, cell cycle arrest, and senescence, p53 is proved to down regulate the hTERT transcription, which is likely to be mediated by inhibition of the Sp1 binding to the hTERT promoter. Not until recently has it been found that the overexpression or repression of hTERT may have an immediate negative or positive effect on the transcription of p53 in some human cells before telomeric changes are detectable, suggesting that there may be a feedback loop between hTERT and p53, and hTERT may exert an extra-telomeric effect on its own regulatory gene components independent of telomere length. But whether the consequent change of p53 expression level is associated with the 'fast-track' of telomerase has not been elucidated yet. Due to the close relationship between p53 and apoptosis, we hypothesized that the possible rapid cell death caused by the knockdown of hTERT may be partially attributed to altered functional p53.
Objective:In this study, we constructed two recombinant plasmids of short hairpin RNA(shRNA) targeting hTERT named pG1 and pG2 respectively. Another clone, pGCR, which expresses a shRNA scrambled sequence, was also constructed as a control. Three telomerase-positive human epithelial ovarian cancer cell lines: wild type-p53(wt-p53) A2780, mutant type-p53(mt-p53) OVCAR3 and p53-null SKOV3 were transfected with pG1, pG2 and pGCR respectively both in vitro and in vivo, to test the possibility of rapid growth arrest and apoptosis induced by hTERT gene silence, and to determine whether this phenomenon is related to the activation of p53 expression. We sought to determine the mode of short-term effects induced by hTERT inhibitors in human ovarian carcinoma cells that harbor different status of p53, and we hoped an improved understanding of this process could provide insight into mechanisms of sensitivity and/or resistance to hTERT inhibitors, thus uncover new targets for ovarian cancer therapy.
Methods: A2780, OVCAR3 and SKOV3 were transfected with pG1, pG2 and pGCR in vitro respectively, and untreated cells were also used as one of controls. The efficiency of hTERT inhibition was confirmed with real-time RT-PCR and western blot, and telomerase activity was evaluated with TRAP assay. Cell proliferation was measured with CCK-8 assay, and cell circle analysis was performed by Flow cytometric. Apoptosis was detected with AnnexinⅤ-PE/7AAD and TUNEL assay, and expression of p53 and p21 were measured with western blot.
A2780 and SKOV3 cells were injected into hypodermic tissue to construct implanted-tumor model in nude mice. After xenograft tumor formed, recombinant plasmids of pG2, pGCR and saline were injected intratumorally every other day respectively for two weeks.The size of tumor was measured at the same time. Tumors were excised, weighted, sectioned and assessed for mophplogy by HE, and apoptosis was detected with TUNEL and electron microscope after treatment. The efficiency of hTERT inhibition was confirmed with western blot, and expression of p53 and p21 were measured with immunohistochemistry.
Results: 1. Restriction enzyme mapping and sequencing demonstrated that pG1, pG2 and pGCR were constructed successfully. Real-time RT-PCR, western blot and TRAP assay showed shRNA directed against hTERT could efficiently silence hTERT expression and inhibit telomerase activity in ovarian cancer cells.
2. knock-down of hTERT induced rapid growth reduction in vitro in all three cell lines of ovarian cancer, and cell cycle analysis showed that growth inhibited by hTERT silence is mediated by S-phase arrest. Two cell lines, A2780 and OVCAR3, with enhancive apoptosis under the influence of hTERT suppression, both presented accumulation of p53 and p21 proteins. In contrast, the knock down of hTERT in p53-null SKOV3 cells resulted in a rapid decreased expression of p21 and did not cause obvious cell apoptosis.
3. Hypodermic tumor-implanted model were established successfully by 100% with A2780 and SKOV3 cells. As compared with pGCR and saline-treated mice, recombinant plasmid pG2 could significantly slow down tumor growth, decrease tumor volume and tumor weight in vivo in both two xenograft tumor. Massive apoptosis and necrosis presented in A2780-implanted tumor after treatment, with reinforcement of p53 and p21 expression. But only necrosis presented in SKOV3-implanted tumor, and the expression of p21 appeared to be unaffected. No obvious side effects were observed in treated mice.
Conclusions: Our data demonstrated that recombinant plasmids of shRNA could efficiently and specifically knock-down hTERT expression, decrease telomerase activity, and inhibit tumor cell growth immediately both in vitro and in vivo, but the mechanism of cell death is differ in ovarian cancer cells with different p53 state, and apoptosis induced immediately by inhibition of hTERT may be due, in part, to up-regulation of growth inhibitory genes p53 and p21. We confirmed once again that, besides the 'classical' mechanism by which inhibition of telomerase could lead to telomere shortening preceding cell death, an 'fast - track' not requiring telomere shortening also exist in cancer cells. This is meaningful for the anticancer therapy targeting telomerase and hTERT.
引文
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[3]. Fakhoury, J.; Nimmo, G.A.; Autexier, C. Harnessing telomerase in cancer therapeutics. Anticancer Agents Med Chem. 2007,7(4):475-483.
[4]. Tuschl T, Borkhardt A. small interfering RNAs: a revolutionary tool for the analysis of gene function and gene therapy[J]. Mol Interv. 2002, 2(3): 158-167.
[5]. Boutla A, Delidakis C, Livadaras I, et al. Variations of the 3’protruding ends in synthetic short interfering RNA(siRNA) tested by microinjection in Drosophila embryos[J]. Oligonucleotides. 2003,13(5):295-301.
[6]. Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009,10(2):94-108.
[7]. Huang C, Li M, Chen C, Yao Q. Small interfering RNA therapy in cancer: mechanism, potential targets, and clinical applications. Expert Opin Ther Targets. 2008,12(5):637-645.
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[9]. Singer O, Verma IM. Applications of lentiviral vectors for shRNA delivery and transgenesis. Curr Gene Ther. 2008,8(6):483-488.
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[12]. Angela R. Rational siRNA design for RNA interference[J]. Nature biotechnology. 2004,22(3):326-330.
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[16]. Yuebo, G.; Yiqun, M.; Jeffrey, J. Telomere maintenance in telomerase-positive human ovarian SKOV-3 cells cannot be retarded by complete inhibition of telomerase. FEBS Lett. 2002,527(1-3):10-14.
[17]. Lian Z, Fujimoto J, Yokoyama Y, et al. Herbal complex suppresses telomerase activity in chemo-endocrine resistant cancer cell lines. Eur J Gynaecol Oncol. 2001,22(5):347-349.
[1]. Chen, S.M.; Tao, Z.Z.; Hua, Q.Q.; Xiao, B.K.; Xu, Y.; Wang, Y.; Deng, Y.Q. Inhibition of telomerase activity in cancer cells using short hairpin RNA expression vectors. Cancer Investig. 2007,25(8),691-698.
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[2]. Chen, S.M.; Tao, Z.Z.; Hua, Q.Q.; Xiao, B.K.; Xu, Y.; Wang, Y.; Deng, Y.Q. Inhibition of telomerase activity in cancer cells using short hairpin RNA expression vectors. Cancer Investig. 2007,25(8):691-698.
[3]. Fakhoury, J.; Nimmo, G.A.; Autexier, C. Harnessing telomerase in cancer therapeutics. Anticancer Agents Med Chem. 2007,7(4):475-483.
[4]. Tuschl T, Borkhardt A. small interfering RNAs: a revolutionary tool for the analysis of gene function and gene therapy[J]. Mol Interv. 2002, 2(3): 158-167.
[5]. Boutla A, Delidakis C, Livadaras I, et al. Variations of the 3’protruding ends in synthetic short interfering RNA(siRNA) tested by microinjection in Drosophila embryos[J]. Oligonucleotides. 2003,13(5):295-301.
[6]. Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009,10(2):94-108.
[7]. Huang C, Li M, Chen C, Yao Q. Small interfering RNA therapy in cancer: mechanism, potential targets, and clinical applications. Expert Opin Ther Targets. 2008,12(5):637-645.
[8]. Carter PJ, Samulski RJ. Adeno-associated viral vectors as gene delivery vehicles. Int J Mol Med. 2000,6(1):17-27.
[9]. Singer O, Verma IM. Applications of lentiviral vectors for shRNA delivery and transgenesis. Curr Gene Ther. 2008,8(6):483-488.
[10]. Chen Y, Huang L. Tumor-targeted delivery of siRNA by non-viral vector: safe and effective cancer therapy. Expert Opin Drug Deliv. 2008,5(12):1301-1311.
[11]. Elbashir SM, Harborth J, Lendckel W, et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature. 2001,4119(6836): 494-498.
[12]. Angela R. Rational siRNA design for RNA interference[J]. Nature biotechnology. 2004,22(3):326-330.
[13]. Duxbury MS, Whang EE. RNA interference: a practical approach. J Surg Res. 2004, 117(2):339-44.
[14]. Li W, Cha L. Predicting siRNA efficiency. Cell Mol Life Sci. 2007,64(14): 1785-1792.
[15]. Sun PM, Wei LH, Luo MY, et al. The telomerase activity and expression of hTERT gene can serve as indicators in the anti-cancer treatment of human ovarian cancer. Eur J Obstet Gynecol Reprod Biol. 2007,130(2):249-257.
[16]. Yuebo, G.; Yiqun, M.; Jeffrey, J. Telomere maintenance in telomerase-positive human ovarian SKOV-3 cells cannot be retarded by complete inhibition of telomerase. FEBS Lett. 2002,527(1-3):10-14.
[17]. Lian Z, Fujimoto J, Yokoyama Y, et al. Herbal complex suppresses telomerase activity in chemo-endocrine resistant cancer cell lines. Eur J Gynaecol Oncol. 2001,22(5):347-349.
[1]. Chen, S.M.; Tao, Z.Z.; Hua, Q.Q.; Xiao, B.K.; Xu, Y.; Wang, Y.; Deng, Y.Q. Inhibition of telomerase activity in cancer cells using short hairpin RNA expression vectors. Cancer Investig. 2007,25(8),691-698.
[2]. Kraemer, K.; Fuessel, S.; Schmidt, U.; Kotzsch, M.; Schwenzer, B.; Wirth, M.P.; Meye, A. Antisense-mediated hTERT inhibition specifically reduces the growth of human bladder cancer cells. Clin. Cancer Res. 2003,9(10),3794–3800.
[3]. Ludwig, A.; Saretzki, G.; Holm, P.S.; Tiemann, F.; Lorenz, M.; Emrich, T.; Harley, C.B.; vonZglinicki, T. Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase. Cancer Res. 2001,61(7),3053–3061.
[4]. Xu, D.; Wang, Q.; Gruber, A.; Bjorkholm, M.; Chen, Z.G..; Zaid, A.; Selivanova, G.; Peterson, C.; Wiman, K.G.; Pisa, P. Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells. Oncogene.2000,19(45),5123–5133.
[5]. Kanaya, T.; Kyo, S.; Hamada, K.; Takakura, M.; Kitagawa, Y.; Harada, H.; Inoue, M. Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription. Clin. Cancer Res. 2000,6(4),1239–1247.
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