Survivin启动子介导的RNA干涉喉鳞状细胞癌中eIF4E基因的实验研究
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摘要
目的真核翻译起始因子4E(eIF4E),是蛋白质合成起始阶段的重要调控因子。该起始因子在多种头颈部肿瘤细胞和组织中不仅高表达,而且与预后呈负相关;同时,eIF4E的过表达状态还与喉癌的发生、发展及其化疗耐药密切相关。首先构建survivin启动子调控增强型绿色荧光蛋白基因(EGFP)干涉表达载体,转染人头颈部鳞状细胞癌细胞系和脐静脉血管内皮细胞系,验证survivin启动子调控的RNA干涉系统具有肿瘤特异性和高转录活性。构建survivin启动子调控的以eIF4E为靶点的真核表达干涉载体,转染喉癌细胞系(Hep-2),筛选出eIF4E表达下调明显的稳定转染细胞,检测eIF4E表达下调对喉癌细胞的体内和体外增殖活性、细胞周期、细胞凋亡及其化疗敏感性的影响,从而为eIF4E分子成为喉癌治疗的新靶点提供实验性研究基础。
方法(1)针对增强型绿色荧光蛋白报告基因(EGFP)设计有效的siRNA干涉序列,退火后连接到上述载体H1RNA启动子下游形成载体pS-H1P-shEGFP,酶切鉴定。(2)采用PCR方法从构建好的pGL3-surP质粒载体中扩增获得survivin启动子基因,经测序后与Genbank中序列进行比较分析;将survivin启动子基因cDNA克隆到pSUPER.retro.puro干涉载体中替代H1RNA启动子并酶切鉴定,体外合成最小终止信号序列mpA插入到上述载体多克隆位点后面(pS-surP-shRNA-mpA)采用PCR方法从pEGFP-C1载体中扩增cmvP-EGFP-sv40polyA表达框,将该表达框插入到pSUPER.retro.puro中EcoRI和NheI限制性酶切位点之间,形成新的干涉和表达共同载体:pS-surP-shEGFP-mpA-cmvP-EGFP-sv40polyA,经酶切鉴定后分别转染鼻咽癌细胞系CNE-2,喉鳞癌细胞系Hep-2,甲状腺鳞癌细胞系SW579以及脐静脉血管内皮细胞系(ECV304)。转染72小时后,荧光显微镜下观察细胞发光情况;逆转录PCR(RT-PCR)和流氏细胞仪(FCM)分别检测EGFP的转录和荧光发光水平。(3)针对eIF4E cDNA序列设计和体外合成两条有效的siRNA干涉序列,并在BLAST中对靶序列进行同源性分析,退火后插入到pS-surP-shRNA-mpA载体中并酶切鉴定。通过脂质体2000转染喉癌细胞系(Hep-2)中,G418筛选两周后获得稳定转染的细胞。RT-PCR和蛋白印迹方法(Western blotting)分别检测eIF4E mRNA和蛋白表达水平,筛选抑制eIF4E表达最强的稳定转染Hep-2细胞用于进一步的实验研究。(4)蛋白印迹法检测血管内皮生长因子(VEGF)、成纤维细胞生长因子(FGF-2)、细胞周期素D1(cyclinD1)的表达变化;MTT法和克隆形成试验检测体外细胞增殖情况;流式细胞技术检测细胞周期变化及细胞凋亡情况;TUNEL染色法检测细胞凋亡变化;检测Caspase-3活性变化以及可能的凋亡机制;体内成瘤性和抑瘤性试验;体外及体内化疗敏感性试验(顺铂)。
结果(1)用PCR方法从pGL3-surP质粒载体中扩增出980bp的cDNA,测序证实为人survivin启动子基因;同时酶切鉴定表明survivin启动子基因片断(surP)和mpA中止信号片断成功插入到pSUPER.retro.puro干涉载体中形成新的干涉载体pS-surP-shRNA-mpA。(2)针对EGFP报告基因的真核表达和干涉共同载体构建成功。转染72小时后,RT-PCR结果显示EGFP在CNE-2、Hep-2及SW579细胞中转录表达水平明显低于在ECV304细胞中的转录表达水平;流式细胞仪检测显示EGFP在CNE-2、Hep-2及SW579细胞中蛋白荧光表达水平显著低于在ECV304细胞中的表达水平。(3)针对eIF4E基因的干涉表达载体构建成功。同未转染的Hep-2细胞相比,eIF4E的转录和蛋白表达水平在稳定表达eIF4E/shRNA2的Hep-2-s2细胞中分别抑制了68.5%和59.6%,而在稳定表达eIF4E/shRNA1的Hep-2-s1细胞中则没有明显变化,选择Hep-2-s2细胞用于下一步的试验研究。(4)同未转染的Hep-2细胞相比,血管内皮生长因子、成纤维细胞生长因子及细胞周期素D1在Hep-2-s2细胞中的蛋白表达明显受到抑制;Hep-2-s2细胞的增殖活性明显降低,第7天抑制率达到最大(48.2±1.6%; P<0.01)。Hep-2-s2细胞的体外克隆形成能力明显降低(平均克隆形成数=342; P<0.01);G0/G1期Hep-2-s2细胞比例明显增加而S期细胞比例明显减少;流式细胞仪检测和TUNEL染色结果显示Hep-2-s2细胞凋亡率明显增加(18.3±1.7%; P<0.05);Caspase-3活性明显增加1.21倍;凋亡的产生可能和c-IAP1、c-IAP2、c-myc蛋白表达升高有关系而和Bcl-2家族蛋白表达没有关系; Hep-2-s2细胞的体内成瘤能力明显低于对照组(平均瘤体体积=233.5mm3);pS/surP-eIF4E/shRNA2处理组平均瘤体积(322.5±5.3mm3)显著低于其它处理组;Hep-2-s2细胞对顺铂的体外和体内敏感性明显增强。
结论(1) survivin启动子介导的RNA干涉系统能特异而高效的封闭肿瘤细胞内源基因的表达。(2) eIF4E基因表达的下调,能够诱导其下游靶基因的表达下调,体外和体内细胞增殖活性的降低;稳定表达eIF4E/shRNA2的细胞出现了G0/G1期阻滞,凋亡率显著增加;同时eIF4E基因表达的下调能够诱导增强喉癌细胞体内和体外对顺铂的化疗敏感性。(3)肿瘤特异性启动子survivin启动子和RNA干涉系统结合为喉癌靶向性治疗探索了一种新策略,同时靶向性封闭eIF4E基因表达则为喉癌治疗的新靶点开发和逆转化疗耐药开辟了新思路。
Objective Eukaryotic initiation factor eIF4E, an important regulator of translation, is overexpressed in a variety of human head and neck squamous cell carcinoma(HNSCC) cells and tissues, while the high levels of eIF4E expression is inversely correlated with prognosis. Morever, the overexpression of this gene is associated with tumorigenicity, development and chemoresistance of laryngeal carcinoma. To tesitify the tumor specificity and high efficacy of survivin promoter-mediated RNA interference system, survivin promoter-mediated RNAi expression vector targeting EGFP gene was transfected into various head and neck squamous cell carcinoma cell lines and normal blood vessel endothelium cell line, respectively. Secondly, survivin promoter-mediated siRNA eukaryotic expression vector targeting eIF4E was constructed and then transfected into laryngeal carcinoma cells(Hep-2). To explore the possibility of eIF4E as an therapeutic target for the treatment of laryngeal carcinomas, the effects of eIF4E downregulation on in vitro and in vivo proliferation, cell cycle, apoptosis and chemosensitivity of laryngeal carcinoma cells were evaluated.
Methods (1) The survivin promoter gene was cloned from pGL3-surP vector by RT-PCR method, then sequenced and analyzed in Genbank. Then, the survivin promoter gene and in vitro-synthesized mpA sequence were inserted into pSUPER.retro.puro vector, respectively and confirmed by the digestion analysis of restriction endonuclease. (2) siRNA oligonucleotide sequence was designed and synthesized accoding to the EGFP cDNA sequence. The siRNA oligonucleotide was annealed and inserted into the downstream of survivin promoter. Then, the cmvP-EGFP-polyA expression frame was coloned from pEGFP-C1 vector by PCR and then inserted between EcoRI and NheI restricted enzyme sites of pSUPER.retro-pur vector. The reconstructed vector was named pS-surP-shEGFP-mpA-cmvP-EGFP-polyA. The vector was transfected into human nasopharyngeal carcinoma cell line(CNE-2), laryngeal squamous cell carcinoma cell line(Hep-2),thyroid squamous cell carcinoma cell line(SW579) and normal vascular endothelial cell(ECV304). 72h after transfection, the cells were photographed by using a fluorescence microscope. RT-PCR and flow cytometry assays were performed to detect the levels of EGFP mRNA and the levels of EGFP fluorescence using fluorescence-activated cell sorting. (3) Two siRNA olignucleotides were designed and in vitro synthesized, and the target sequence homology with other genes was analyzed by BLAST search. Then, those olignucleotides were annealed and inserted into pS-surP-shRNA-mpA vector, respectively. Followingly, the siRNA expression vectors were transfected into Hep-2 cells using Lipofectamine 2000. Stable cell lines were selected with G418 (600μg/mL)48h later after transfection, and individual clones were isolated and maintained in a medium containing G418(100μg/mL). RT-PCR and Western blotting assays were performed to detect the levels of eIF4E mRNA and protein expression, respectively. The stable transfectant showing downregulated-eIF4E expression of was chose for further assays. (4) Western blotting was performed to detect the changes of VEGF, FGF-2 and cyclinD1 protein expression in stable transfectants. MTT and colony formation assays were performed to evaluate effects of eIF4E/shRNA2 on the in vitro cell proliferation of Hep-2 cells. Flow cytomatery and TUNEL staining assays were performed to detect the changes of cell cycle and apoptosis. The activity of caspase-3 was determined and the possible mechanism of apoptosis was explored. Tumorigenicity and tumor inhibition assays were performed to detect in vivo proliferation. In vitro and in vivo chemotherapy assays were performed to evaluate the effect of eIF4E downregulation on the chemosensitivity of Hep-2 cells to cisplatin.
Results (1) The recombinant vector pS-surP-shRNA-mpA including 980bp survivin promoter gene and interrupted signal mpA were successfully constructed. (2) 72h after transfection, RT-PCR results showed that the levels of EGFP mRNA expression in CNE-2, Hep-2 and SW579 cells were significanty inhibited compared with that in ECV304 cells. (3) The levels of eIF4E mRNA and protein expression in Hep-2-s2 cells were decreased by 68.5% and 59.6%, respectively, but there were no obvious difference in Hep-2-s1. Hep-2-s2 cells were chose for further assays. (4) The levels of VEGF, FGF-2 and cyclinD1 protein expression were significantly inhibited in Hep-2-s2 cells. The proliferation of Hep-2-s2 cells was also obviously decreased and the highest inhibitory rate was 48.2±1.6% on day 7(P<0.01). The colony numbers of Hep-2-s2 cells were significantly reduced (averaged number=342, P<0.01). The percentage of Hep-2-s2 cells in the G0/G1 phase was increased and that in the S phase was decreased. The activity of Caspase-3 showed obvious enhancement. The high levels of c-IAP1, c-IAP2 and c-myc protein expression but not Bcl-2 family protein expression were involved in the process of apoptosis. The tumor volume developed from Hep-2-s2 cells(averaged size=233.5mm3)was obviously smaller than that developed from control cells. Moreover, pS-SP-eIF4E/shRNA2 could significantly inhibit the proliferation of in vivo tumor. eIF4E/shRNA2 also could lead to in vitro and in vivo chemosensitivity enhancement in Hep-2 cells.
Conclusions (1) Survivin promoter-mediated RNA interference system could silence endogenous gene expression with tumor specificity and high efficacy. (2) The downregulation of eIF4E expression could reduce the levels of downstream target gene expression and lead to proliferation inhibition both in vitro and in vivo. The Hep-2 cells stably expressing eIF4E/shRNA2 showed cell arrest in the G0/G1 phase and obvious apoptosis induction. The downregulation of this gene could also enhance chemosensitivity of Hep-2 cells to cisplatin. (3) Combination use of survivin promoter and RNA interference provides a novel strategy for the targeted therapy of human laryngeal carcinoma. Specific silencing of eIF4E gene provides a new approach for exploiting novel therapeutic target and reversing chemoresistance phenotype of laryngeal carcinoma.
方法(1)针对增强型绿色荧光蛋白报告基因(EGFP)设计有效的siRNA干涉序列,退火后连接到上述载体H1RNA启动子下游形成载体pS-H1P-shEGFP,酶切鉴定。(2)采用PCR方法从构建好的pGL3-surP质粒载体中扩增获得survivin启动子基因,经测序后与Genbank中序列进行比较分析;将survivin启动子基因cDNA克隆到pSUPER.retro.puro干涉载体中替代H1RNA启动子并酶切鉴定,体外合成最小终止信号序列mpA插入到上述载体多克隆位点后面(pS-surP-shRNA-mpA)采用PCR方法从pEGFP-C1载体中扩增cmvP-EGFP-sv40polyA表达框,将该表达框插入到pSUPER.retro.puro中EcoRI和NheI限制性酶切位点之间,形成新的干涉和表达共同载体:pS-surP-shEGFP-mpA-cmvP-EGFP-sv40polyA,经酶切鉴定后分别转染鼻咽癌细胞系CNE-2,喉鳞癌细胞系Hep-2,甲状腺鳞癌细胞系SW579以及脐静脉血管内皮细胞系(ECV304)。转染72小时后,荧光显微镜下观察细胞发光情况;逆转录PCR(RT-PCR)和流氏细胞仪(FCM)分别检测EGFP的转录和荧光发光水平。(3)针对eIF4E cDNA序列设计和体外合成两条有效的siRNA干涉序列,并在BLAST中对靶序列进行同源性分析,退火后插入到pS-surP-shRNA-mpA载体中并酶切鉴定。通过脂质体2000转染喉癌细胞系(Hep-2)中,G418筛选两周后获得稳定转染的细胞。RT-PCR和蛋白印迹方法(Western blotting)分别检测eIF4E mRNA和蛋白表达水平,筛选抑制eIF4E表达最强的稳定转染Hep-2细胞用于进一步的实验研究。(4)蛋白印迹法检测血管内皮生长因子(VEGF)、成纤维细胞生长因子(FGF-2)、细胞周期素D1(cyclinD1)的表达变化;MTT法和克隆形成试验检测体外细胞增殖情况;流式细胞技术检测细胞周期变化及细胞凋亡情况;TUNEL染色法检测细胞凋亡变化;检测Caspase-3活性变化以及可能的凋亡机制;体内成瘤性和抑瘤性试验;体外及体内化疗敏感性试验(顺铂)。
结果(1)用PCR方法从pGL3-surP质粒载体中扩增出980bp的cDNA,测序证实为人survivin启动子基因;同时酶切鉴定表明survivin启动子基因片断(surP)和mpA中止信号片断成功插入到pSUPER.retro.puro干涉载体中形成新的干涉载体pS-surP-shRNA-mpA。(2)针对EGFP报告基因的真核表达和干涉共同载体构建成功。转染72小时后,RT-PCR结果显示EGFP在CNE-2、Hep-2及SW579细胞中转录表达水平明显低于在ECV304细胞中的转录表达水平;流式细胞仪检测显示EGFP在CNE-2、Hep-2及SW579细胞中蛋白荧光表达水平显著低于在ECV304细胞中的表达水平。(3)针对eIF4E基因的干涉表达载体构建成功。同未转染的Hep-2细胞相比,eIF4E的转录和蛋白表达水平在稳定表达eIF4E/shRNA2的Hep-2-s2细胞中分别抑制了68.5%和59.6%,而在稳定表达eIF4E/shRNA1的Hep-2-s1细胞中则没有明显变化,选择Hep-2-s2细胞用于下一步的试验研究。(4)同未转染的Hep-2细胞相比,血管内皮生长因子、成纤维细胞生长因子及细胞周期素D1在Hep-2-s2细胞中的蛋白表达明显受到抑制;Hep-2-s2细胞的增殖活性明显降低,第7天抑制率达到最大(48.2±1.6%; P<0.01)。Hep-2-s2细胞的体外克隆形成能力明显降低(平均克隆形成数=342; P<0.01);G0/G1期Hep-2-s2细胞比例明显增加而S期细胞比例明显减少;流式细胞仪检测和TUNEL染色结果显示Hep-2-s2细胞凋亡率明显增加(18.3±1.7%; P<0.05);Caspase-3活性明显增加1.21倍;凋亡的产生可能和c-IAP1、c-IAP2、c-myc蛋白表达升高有关系而和Bcl-2家族蛋白表达没有关系; Hep-2-s2细胞的体内成瘤能力明显低于对照组(平均瘤体体积=233.5mm3);pS/surP-eIF4E/shRNA2处理组平均瘤体积(322.5±5.3mm3)显著低于其它处理组;Hep-2-s2细胞对顺铂的体外和体内敏感性明显增强。
结论(1) survivin启动子介导的RNA干涉系统能特异而高效的封闭肿瘤细胞内源基因的表达。(2) eIF4E基因表达的下调,能够诱导其下游靶基因的表达下调,体外和体内细胞增殖活性的降低;稳定表达eIF4E/shRNA2的细胞出现了G0/G1期阻滞,凋亡率显著增加;同时eIF4E基因表达的下调能够诱导增强喉癌细胞体内和体外对顺铂的化疗敏感性。(3)肿瘤特异性启动子survivin启动子和RNA干涉系统结合为喉癌靶向性治疗探索了一种新策略,同时靶向性封闭eIF4E基因表达则为喉癌治疗的新靶点开发和逆转化疗耐药开辟了新思路。
Objective Eukaryotic initiation factor eIF4E, an important regulator of translation, is overexpressed in a variety of human head and neck squamous cell carcinoma(HNSCC) cells and tissues, while the high levels of eIF4E expression is inversely correlated with prognosis. Morever, the overexpression of this gene is associated with tumorigenicity, development and chemoresistance of laryngeal carcinoma. To tesitify the tumor specificity and high efficacy of survivin promoter-mediated RNA interference system, survivin promoter-mediated RNAi expression vector targeting EGFP gene was transfected into various head and neck squamous cell carcinoma cell lines and normal blood vessel endothelium cell line, respectively. Secondly, survivin promoter-mediated siRNA eukaryotic expression vector targeting eIF4E was constructed and then transfected into laryngeal carcinoma cells(Hep-2). To explore the possibility of eIF4E as an therapeutic target for the treatment of laryngeal carcinomas, the effects of eIF4E downregulation on in vitro and in vivo proliferation, cell cycle, apoptosis and chemosensitivity of laryngeal carcinoma cells were evaluated.
Methods (1) The survivin promoter gene was cloned from pGL3-surP vector by RT-PCR method, then sequenced and analyzed in Genbank. Then, the survivin promoter gene and in vitro-synthesized mpA sequence were inserted into pSUPER.retro.puro vector, respectively and confirmed by the digestion analysis of restriction endonuclease. (2) siRNA oligonucleotide sequence was designed and synthesized accoding to the EGFP cDNA sequence. The siRNA oligonucleotide was annealed and inserted into the downstream of survivin promoter. Then, the cmvP-EGFP-polyA expression frame was coloned from pEGFP-C1 vector by PCR and then inserted between EcoRI and NheI restricted enzyme sites of pSUPER.retro-pur vector. The reconstructed vector was named pS-surP-shEGFP-mpA-cmvP-EGFP-polyA. The vector was transfected into human nasopharyngeal carcinoma cell line(CNE-2), laryngeal squamous cell carcinoma cell line(Hep-2),thyroid squamous cell carcinoma cell line(SW579) and normal vascular endothelial cell(ECV304). 72h after transfection, the cells were photographed by using a fluorescence microscope. RT-PCR and flow cytometry assays were performed to detect the levels of EGFP mRNA and the levels of EGFP fluorescence using fluorescence-activated cell sorting. (3) Two siRNA olignucleotides were designed and in vitro synthesized, and the target sequence homology with other genes was analyzed by BLAST search. Then, those olignucleotides were annealed and inserted into pS-surP-shRNA-mpA vector, respectively. Followingly, the siRNA expression vectors were transfected into Hep-2 cells using Lipofectamine 2000. Stable cell lines were selected with G418 (600μg/mL)48h later after transfection, and individual clones were isolated and maintained in a medium containing G418(100μg/mL). RT-PCR and Western blotting assays were performed to detect the levels of eIF4E mRNA and protein expression, respectively. The stable transfectant showing downregulated-eIF4E expression of was chose for further assays. (4) Western blotting was performed to detect the changes of VEGF, FGF-2 and cyclinD1 protein expression in stable transfectants. MTT and colony formation assays were performed to evaluate effects of eIF4E/shRNA2 on the in vitro cell proliferation of Hep-2 cells. Flow cytomatery and TUNEL staining assays were performed to detect the changes of cell cycle and apoptosis. The activity of caspase-3 was determined and the possible mechanism of apoptosis was explored. Tumorigenicity and tumor inhibition assays were performed to detect in vivo proliferation. In vitro and in vivo chemotherapy assays were performed to evaluate the effect of eIF4E downregulation on the chemosensitivity of Hep-2 cells to cisplatin.
Results (1) The recombinant vector pS-surP-shRNA-mpA including 980bp survivin promoter gene and interrupted signal mpA were successfully constructed. (2) 72h after transfection, RT-PCR results showed that the levels of EGFP mRNA expression in CNE-2, Hep-2 and SW579 cells were significanty inhibited compared with that in ECV304 cells. (3) The levels of eIF4E mRNA and protein expression in Hep-2-s2 cells were decreased by 68.5% and 59.6%, respectively, but there were no obvious difference in Hep-2-s1. Hep-2-s2 cells were chose for further assays. (4) The levels of VEGF, FGF-2 and cyclinD1 protein expression were significantly inhibited in Hep-2-s2 cells. The proliferation of Hep-2-s2 cells was also obviously decreased and the highest inhibitory rate was 48.2±1.6% on day 7(P<0.01). The colony numbers of Hep-2-s2 cells were significantly reduced (averaged number=342, P<0.01). The percentage of Hep-2-s2 cells in the G0/G1 phase was increased and that in the S phase was decreased. The activity of Caspase-3 showed obvious enhancement. The high levels of c-IAP1, c-IAP2 and c-myc protein expression but not Bcl-2 family protein expression were involved in the process of apoptosis. The tumor volume developed from Hep-2-s2 cells(averaged size=233.5mm3)was obviously smaller than that developed from control cells. Moreover, pS-SP-eIF4E/shRNA2 could significantly inhibit the proliferation of in vivo tumor. eIF4E/shRNA2 also could lead to in vitro and in vivo chemosensitivity enhancement in Hep-2 cells.
Conclusions (1) Survivin promoter-mediated RNA interference system could silence endogenous gene expression with tumor specificity and high efficacy. (2) The downregulation of eIF4E expression could reduce the levels of downstream target gene expression and lead to proliferation inhibition both in vitro and in vivo. The Hep-2 cells stably expressing eIF4E/shRNA2 showed cell arrest in the G0/G1 phase and obvious apoptosis induction. The downregulation of this gene could also enhance chemosensitivity of Hep-2 cells to cisplatin. (3) Combination use of survivin promoter and RNA interference provides a novel strategy for the targeted therapy of human laryngeal carcinoma. Specific silencing of eIF4E gene provides a new approach for exploiting novel therapeutic target and reversing chemoresistance phenotype of laryngeal carcinoma.
引文
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