hTERT非端粒酶依赖途径调控喉鳞癌细胞凋亡的研究
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摘要
目的：应用RNA干扰技术沉默人端粒酶逆转录酶(hTERT)前后,检测喉癌细胞及移植瘤组织中hTERT表达含量与端粒酶活性及前列腺凋亡反应因子(Par-4)表达相关性,探讨人端粒酶逆转录酶不依赖其催化活性的抗凋亡作用及其机制,为喉癌的基因治疗提供新的思路。
方法：构建靶向人端粒酶逆转录酶mRNA的质粒p-EGFP-shTERT,将质粒转染体外培养的喉癌Hep-2细胞,应用TRAP-PCR法检测检测转染后0h、12h、24h、48h四个时间点喉癌Hep-2细胞端粒酶活性,流式细胞术检测转染后四个时间点细胞凋亡程度,Western-blot检测hTERT蛋白及Par-4蛋白表达；建立喉癌移植瘤裸鼠动物模型,采用瘤体内多点注射方式将质粒p-EGFP-shTERT导入移植瘤内,采用量子点超敏荧光免疫技术检测质粒转染前以及转染后6d、10d、14d移植瘤组织内hTERT和Par-4两种蛋白表达情况,方差分析、卡方检验及Spearman相关性检验分析实验结果。
结果：质粒成功转染Hep-2细胞24h后端粒酶活性开始下降,48h后端粒酶活性被抑制；质粒转染12h后Hep-2细胞即有显著凋亡,随时间延长,凋亡率逐渐增高；hTERT蛋白表达随转染时间延长逐渐降低,Par-4蛋白表达逐渐增加；体内实验中发现,质粒转染前,hTERT蛋白在喉癌Hep-2细胞裸鼠移植瘤中呈高表达,而且胞核、胞浆内均有表达,但Par-4蛋白仅在胞浆表达,质粒转染14天后,hTERT表达降低,胞核内表达下调明显,而Par-4在胞核表达增多,Speraman等级相关分析发现,移植瘤中Par-4和hTERT在质粒p-EGFP-shTERT转染前后表达呈负相关(p<0.05,r=-0.908)。
结论：应用RNA干扰抑制hTERT表达过程中,我们发现质粒转染24h后,端粒酶活性仅有轻度降低,48小时后端粒酶活性才被抑制,然而质粒转染Hep-2细胞12h后,就有凋亡反应出现,24h后凋亡显著,因此推测,RNA干扰抑制hTERT诱导的凋亡反应并不依赖端粒酶活性的抑制。同样,hTERT介导的肿瘤细胞抗凋亡作用除端粒酶激活途径外,还存在其它非端粒酶途径；抑制hTERT蛋白表达的同时,我们还发现,前列腺凋亡反应因子(Par-4)表达上调,且质粒转染前后两种蛋白表达部位发生改变,提示Par-4可能参与hTERT不依赖其催化活性的抗凋亡途径,hTERT可能在胞浆中与Par-4相互作用,抑制了Par-4转入胞核,从而抑制了细胞凋亡。
Objective:To explore the mechanism of telomerase-independent anti-apoptosis of hTERT, investigated the relationship between the expression of human telomerase reverse transcriptase (hTERT) and telomerase activity or prostate apoptosis response4(Par-4) proteins in laryngocarcinoma cells after applying RNA interference to silencing the expression of hTERT gene in laryngeal carcinoma cells in vitro and in vivo, which supply an new method for the laryngocarcinoma gene therapy.
Methods:A plasmid termed pEGFP-shTERT was constructed. Hep-2cells were transfected with the plasmids. After0,12,24,48hours, cells were collected and detected with TRAP-PCR in telomerase activity, with flow cytometer in cell apoptosis, with and immunocytochemistry in expression of hTERT and Par-4protein and with Western-blot in expression of hTERT and Par-4protein; An animal model of laryngocarcinoma was constructed. The plasmids pEGFP-shTERT were transfected by multipoint injection. The expression of Par-4and hTERT after transfected of0,6,10,14days was detected by Quantum dots-based immunofluoresense technology in xenograft laryngocarcinoma from nude mice.
Results:The plasmids were successfully transfected after12h of treated with pEGFP-shTERT, the transfection rate achieved78.6%after24h. After24h of plasmids transfection, the telomerase activity of Hep-2cells started to decline but not be inhibited completely, while the apoptosis of Hep-2cells occurred before the inactivation of the telomerase after12h of plasmids transfection. The expression of hTERT was down-regulated and the expression of Par-4was increased after RNAi, and there are obvious dose-effect relationship (P<0.05); Meanwhile, the similar results were found in vivo study, furthermore before hTERT gene silencing, the hTERT protein were found both in nucleus and cytoplasm, while Par-4protein only expressed in cytoplasm. After14d of RNAi, the expression of hTERT protein decreased both in nucleus and cytoplasm, especially in nucleus, while the expression of Par-4protein obviously increased in nucleus.There was negative correlation between Par-4and hTERT expression in nucleus (P<0.05, r=-0.908).
Conclusion:After24h of plasmids transfection, the telomerase activity of Hep-2cells started to decline but not obviously, while the apoptosis of Hep-2cells occurred before the inactivation of the telomerase. After24h of plasmids transfection, there were16.5% Hep-2cells in apoptosis. So, we consider that hTERT may have anti-apoptosis effect independent of telomerase. There was negative correlation between Par-4and hTERT expression when silencing hTERT gene, which meant Par-4may took part in the hTERT mediated apoptosis independent of telomerase. hTERT may interact with Par-4in cytoplasm and inhibit the translocation of Par-4into nucleus, and then inhibit the Par-4induced apoptosis.
方法：构建靶向人端粒酶逆转录酶mRNA的质粒p-EGFP-shTERT,将质粒转染体外培养的喉癌Hep-2细胞,应用TRAP-PCR法检测检测转染后0h、12h、24h、48h四个时间点喉癌Hep-2细胞端粒酶活性,流式细胞术检测转染后四个时间点细胞凋亡程度,Western-blot检测hTERT蛋白及Par-4蛋白表达；建立喉癌移植瘤裸鼠动物模型,采用瘤体内多点注射方式将质粒p-EGFP-shTERT导入移植瘤内,采用量子点超敏荧光免疫技术检测质粒转染前以及转染后6d、10d、14d移植瘤组织内hTERT和Par-4两种蛋白表达情况,方差分析、卡方检验及Spearman相关性检验分析实验结果。
结果：质粒成功转染Hep-2细胞24h后端粒酶活性开始下降,48h后端粒酶活性被抑制；质粒转染12h后Hep-2细胞即有显著凋亡,随时间延长,凋亡率逐渐增高；hTERT蛋白表达随转染时间延长逐渐降低,Par-4蛋白表达逐渐增加；体内实验中发现,质粒转染前,hTERT蛋白在喉癌Hep-2细胞裸鼠移植瘤中呈高表达,而且胞核、胞浆内均有表达,但Par-4蛋白仅在胞浆表达,质粒转染14天后,hTERT表达降低,胞核内表达下调明显,而Par-4在胞核表达增多,Speraman等级相关分析发现,移植瘤中Par-4和hTERT在质粒p-EGFP-shTERT转染前后表达呈负相关(p<0.05,r=-0.908)。
结论：应用RNA干扰抑制hTERT表达过程中,我们发现质粒转染24h后,端粒酶活性仅有轻度降低,48小时后端粒酶活性才被抑制,然而质粒转染Hep-2细胞12h后,就有凋亡反应出现,24h后凋亡显著,因此推测,RNA干扰抑制hTERT诱导的凋亡反应并不依赖端粒酶活性的抑制。同样,hTERT介导的肿瘤细胞抗凋亡作用除端粒酶激活途径外,还存在其它非端粒酶途径；抑制hTERT蛋白表达的同时,我们还发现,前列腺凋亡反应因子(Par-4)表达上调,且质粒转染前后两种蛋白表达部位发生改变,提示Par-4可能参与hTERT不依赖其催化活性的抗凋亡途径,hTERT可能在胞浆中与Par-4相互作用,抑制了Par-4转入胞核,从而抑制了细胞凋亡。
Objective:To explore the mechanism of telomerase-independent anti-apoptosis of hTERT, investigated the relationship between the expression of human telomerase reverse transcriptase (hTERT) and telomerase activity or prostate apoptosis response4(Par-4) proteins in laryngocarcinoma cells after applying RNA interference to silencing the expression of hTERT gene in laryngeal carcinoma cells in vitro and in vivo, which supply an new method for the laryngocarcinoma gene therapy.
Methods:A plasmid termed pEGFP-shTERT was constructed. Hep-2cells were transfected with the plasmids. After0,12,24,48hours, cells were collected and detected with TRAP-PCR in telomerase activity, with flow cytometer in cell apoptosis, with and immunocytochemistry in expression of hTERT and Par-4protein and with Western-blot in expression of hTERT and Par-4protein; An animal model of laryngocarcinoma was constructed. The plasmids pEGFP-shTERT were transfected by multipoint injection. The expression of Par-4and hTERT after transfected of0,6,10,14days was detected by Quantum dots-based immunofluoresense technology in xenograft laryngocarcinoma from nude mice.
Results:The plasmids were successfully transfected after12h of treated with pEGFP-shTERT, the transfection rate achieved78.6%after24h. After24h of plasmids transfection, the telomerase activity of Hep-2cells started to decline but not be inhibited completely, while the apoptosis of Hep-2cells occurred before the inactivation of the telomerase after12h of plasmids transfection. The expression of hTERT was down-regulated and the expression of Par-4was increased after RNAi, and there are obvious dose-effect relationship (P<0.05); Meanwhile, the similar results were found in vivo study, furthermore before hTERT gene silencing, the hTERT protein were found both in nucleus and cytoplasm, while Par-4protein only expressed in cytoplasm. After14d of RNAi, the expression of hTERT protein decreased both in nucleus and cytoplasm, especially in nucleus, while the expression of Par-4protein obviously increased in nucleus.There was negative correlation between Par-4and hTERT expression in nucleus (P<0.05, r=-0.908).
Conclusion:After24h of plasmids transfection, the telomerase activity of Hep-2cells started to decline but not obviously, while the apoptosis of Hep-2cells occurred before the inactivation of the telomerase. After24h of plasmids transfection, there were16.5% Hep-2cells in apoptosis. So, we consider that hTERT may have anti-apoptosis effect independent of telomerase. There was negative correlation between Par-4and hTERT expression when silencing hTERT gene, which meant Par-4may took part in the hTERT mediated apoptosis independent of telomerase. hTERT may interact with Par-4in cytoplasm and inhibit the translocation of Par-4into nucleus, and then inhibit the Par-4induced apoptosis.
引文
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[17]Lourdes M. Aleman, John Doench and Philip A. Sharp. Comparison of siRNA-induced off-target RNA and protein effects. RNA (2007),13:385-395
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[24]Kumiko Ui-Tei, Yuki Naito, et al. Functional dissection of siRNA sequence by systematic DNA substitution. Nucleic Acids Research,2008,36(7):2136-2138