慢病毒载体介导的E2F-1基因沉默对人胃癌裸鼠移植瘤生长的影响及其机制的初步探讨
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摘要
1背景与目的
胃癌是人类常见的消化道肿瘤,严重的威胁着广西人民的健康。目前临床上主要利用手术、化疗和放疗等手段治疗胃癌,尽管经过多年的基础及临床研究,胃癌在早期诊断及化疗药物研究上取得重大进展,但效果不甚理想,基因治疗已成为肿瘤治疗研究的新领域。转录因子E2F-1基因作为E2F家族的一员,是细胞周期和细胞凋亡决定性调控因子之一。大量研究证实人类大多数肿瘤中E2F-1通路环诸多环节的失调与肿瘤发生有着密切的关系。我们在前期的研究中亦发现,E2F-1在胃癌的发生发展过程中是一个非常稳定、保守的基因,突变的发生率极低,而且E2F-1在胃癌组织中呈高表达,提示E2F-1在胃癌的发生发展过程中具有重要作用。通过构建E2F-1沉默的真核表达载体转染胃癌细胞MGC-803,我们发现转染后的胃癌细胞生长速度明显减慢,细胞凋亡明显增加,并促使MGC-803细胞停滞于M期,提示了E2F-1基因在体外具有癌基因的特性。为了进一步探讨E2F-1沉默后的体内抗肿瘤效应,本课题构建E2F-1基因RNA干扰(RNAi)慢病毒载体,并运用E2F-1的RNAi慢病毒载体进行了初步的动物实验,同时,应用半定量RT-PCR、Western blot和酵母双杂交等技术初步探讨其发生机制,以期为胃癌的基因治疗打下坚实的基础。
2方法
2.1根据前期的研究结果,选择干扰E2F-1基因效率最高的siRNA寡核苷酸序列,制备双链DNA oligo (E2F-1-shRNA),应用基因重组技术克隆到pLentiLox3.7(pLL3.7)'慢病毒表达载体中,XbaI和NotI进行双酶切和DNA测序鉴定重组克隆。在脂质体的介导下将慢病毒的混合包装载体质粒和包含E2F-1基因RNAi的重组慢病毒载体(pLL3.7-shRNA-E2F-1)共转染293T细胞,包装成病毒48小时后,收集病毒颗粒(Lv-shRNA-E2F-1),孔稀释法测定病毒滴度。另外,设计一条现有基因文库中基因均无同源性的非特异性siRNA序列作为阴性对照序列,同法构建阴性对照组慢病毒载体(pLL3.7-shRNA-NC),同法包装收集阴性对照组慢病毒颗粒(Lv-shRNA-NC),孔稀释法测定病毒滴度。
2.2取对数生长期的人胃癌细胞MGC-803常规消化,随机选取5只裸鼠,向其右腋皮下注射200μ1(1×106个细胞)单细胞悬液。待皮下移植瘤最长径长约1cm时,切取移植瘤,剪碎至1-2mm3,再移植回剩余裸鼠的右腋皮下,建立人胃癌裸鼠移植瘤模型。构建人胃癌裸鼠移植瘤模型成功后,将裸鼠编号,随机分为实验组(Lv-shRNA-E2F-1组)、阴性对照组(Lv-shRNA-NC组)和PBS组。隔天向各组分别注射相应的病毒颗粒100μl(5×108TU/ml),共6次,同时测量各组肿瘤的长径和短径以计算肿瘤的体积大小,并绘制其生长曲线；11天后颈椎脱臼处死裸鼠,称量肿瘤重量,取瘤体HE染色后观察其形态改变,应用免疫组化sp法、半定量逆转录-聚合酶链反应(RT-PCR)和蛋白印迹(Western Blot)分别检测肿瘤组织中E2F-1的mRNA和蛋白的表达,原位末端转移酶标记技术(TUNEL)检测皮下移植瘤细胞凋亡指数,探索基因治疗的效果。
2.3根据前期研究中基因芯片的结果,提取移植瘤组织的RNA及总蛋白,采用半定量RT-PCR检测移植瘤组织凋亡相关基因PTEN、Caspase-3、 Caspase-9和NF-κB mRNA的表达情况。采用Western blot检测胃癌细胞内PTEN、Caspase-3前体(pro-caspase-3)、Caspase-9前体(pro-caspase-9)、激活型Caspase-3(cleaved caspase-3)、激活型Caspase-9(cleaved caspase-9)和NF-κB的蛋白含量。
2.4根据前期预实验结果,设计引物,以pCMV-E2F-1-HA为模板,去掉E2F-1激活域,PCR扩增E2F-1截短体(309-1241bp),引入酶切位点EcoRI和BamHI,将E2F-1截短体连接至载体pGBKT7中,将构建成功的诱饵表达载体pGBKT7-E2F-1截短体运用化学转化法转化到酵母菌AH109中,采用选择性培养皿分析检测重组质粒的自激活活性和细胞毒性。再将pGBKT7-E2F-1和来源于人正常胃黏膜细胞的cDNA文库质粒顺序转化酵母AH109细胞中,通过营养缺陷型培养基进行阳性克隆的筛选。随后将所得的阳性克隆通过多次营养筛选、X-gal分析、酵母双杂交回转实验对获得的假定相互作用的可靠性加以验证。并对验证后的阳性克隆进行测序。最后借助数据库中的BLAST工具对候选阳性克隆的测序结果进行同源性分析,确定可能发生相互作用的蛋白。
3结果
3.1双酶切证实所设计的E2F-1及阴性对照组shRNA正确插入慢病毒载体,DNA测序证实插入的序列正确；293T细胞成功包装E2F-1-shRNA的重组慢病毒载体及其阴性对照组的重组慢病毒载体；收集的细胞培养上清液中,Lv-shRNA-E2F-1病毒的滴度为3×109TU/ml,Lv-shRNA-NC病毒的滴度为2×109TU/ml。
3.2成功构建人胃癌裸鼠移植瘤模型,三组裸鼠成瘤情况为Lv-shRNA-E2F-1组9只、Lv-shRNA-NC组9只、PBS组8只；各组皮下移植瘤生长曲线表明：与PBS组和Lv-shRNA-NC组比较,Lv-shRNA-E2F-1组肿瘤生长速度明显减慢(P<0.05),PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)；移植瘤重量称量结果显示：Lv-shRNA-E2F-1组肿瘤重量(0.637±0.061g)显著小于Lv-shRNA-NC组(2.225±0.079g)和PBS组(2.334±0.087g),(P<0.05),PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)。HE染色后显微镜下确认切取肿瘤为胃癌皮下移植瘤组织；免疫组化E2F-1特殊染色后可见Lv-shRNA-NC组和PBS组的移植瘤细胞细胞核内出现较多的棕褐色颗粒,而Lv-shRNA-E2F-1组的肿瘤细胞细胞核内均未见或偶见棕褐色颗粒(P<0.05)；半定量RT-PCR和Western blot结果均显示Lv-shRNA-E2F-1组E2F-1mRNA及蛋白表达较Lv-shRNA-NC组和PBS组明显下调(P<0.05),PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)；TUNEL结果显示：Lv-shRNA-E2F-1组的凋亡指数(18.7±3.6%)明显高于Lv-shRNA-NC组(6.5±0.7%)和PBS组(5.8±0.3%)(P<0.05),PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)。
3.3在移植瘤组织中,半定量RT-PCR结果显示：与PBS组和Lv-shRNA-NC组比较,Lv-shRNA-E2F-1组的PTEN、Caspase-3、Caspase-9mRNA表达明显上调,NF-κB mRNA表达明显下调(P＜0.05), PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)；Western-blot结果显示：Lv-shRNA-E2F-1组PTEN、cleaved caspase-9、cleaved caspase-3的蛋白表达量显著高于Lv-shRNA-NC组和PBS组(P<0.05),pro-caspase-9、 pro-caspase-3、NF-κB的蛋白表达量显著低于Lv-shRNA-NC组和PBS组(P＜0.05), PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)。
3.4成功扩增了E2F-1截短体,并连接至载体pGBKT7中,成功构建E2F-1截短体的诱饵载体(pGBKT7-E2F-1),经测序比对结果正确。将pGBKT7-E2F-1转化到酵母菌AH109中,经检测无自激活活性和细胞毒性。AH109(pGBKT7-E2F-1)交配筛选人正常胃黏膜细胞的cDNA文库,在四缺培养基上进行筛选,经反复划线和X-gal检测后获得21个阳性克隆。将获得的阳性质粒一对一与诱饵质粒pGBKT7-E2F1重新共转化AH109酵母菌进行验证,获得阳性候选克隆20个,将阳性克隆测序后进行同源性分析鉴定,最终得到18个不同的相关蛋白,分别为：IRF7、CB、GUK1、ATPIF1、 RPSA、SERBP1、ASS1、MESDC2、WISP2、RCN1、CDC42EP1、EXOSC7、 EFEMP1、FSTL3、MT2A、LGALS1、SIVA1、未知基因。
4结论
4.1成功构建人E2F-1基因RNAi慢病毒载体,为下一步探讨E2F-1沉默对于胃癌生长的体内实验体内实验提供了理论基础和前期准备。为开展靶向E2F-1基因的胃癌基因治疗研究提供了必须的实验工具。
4.2瘤内注射靶向沉默E2F-1基因慢病毒颗粒Lv-shRNA-E2F-1可显著抑制人胃癌细胞MGC-803皮下移植瘤的生长速度,降低肿瘤重量,并明显下调移植瘤组织中E2F-1mRNA及蛋白表达水平。该慢病毒表达系统可能是胃癌治疗的一种有效工具。
4.3瘤内注射慢病毒颗粒Lv-shRNA-E2F-1抑制移植瘤组织中E2F-1的表达后,可引起PTEN的表达量上升,并激活Caspase-9和Caspase-3,降低NF-κB的表达。提示E2F-1沉默极有可能直接或间接通过PTEN/NF-κB信号传导通路,促进胃癌细胞凋亡,抑制胃癌的生长。
4.4成功构建了E2F-1截短体的诱饵载体,经检测无自激活活性和细胞毒性,应用酵母双杂交技术筛选出18个可与E2F-1相互作用的蛋白,结合前期的实验结果,为阐明E2F-1沉默抑制胃癌生长的作用机制提供了新的思路。
创新点
1目前,对于利用RNA干扰技术使E2F-1基因沉默后,探讨其对胃癌进展过程的影响的相关研究未见报道。本课题从体内实验及E2F-1的蛋白间相互作用出发,初步明确E2F-1基因沉默后对胃癌进展的影响,结合前期体外实验,有助于全面阐明E2F-1沉默在胃癌生长中的作用及其功能机制,为E2F-1作为胃癌治疗的一个新的治疗性和预后性靶基因提供理论和实验依据。
2酵母双杂交技术是研究新基因功能的有效手段,本研究拟采用此技术筛查与E2F-1相互作用的蛋白,这在国内外尚属首次。以寻找E2F-1的相互作用蛋白入手,有助于客观真实地阐明其作用的功能机制。
1. Background and Objective
Gastric cancer is a common human gastrointestinal cancer which is a serious threat to the health of the people of Guangxi. Currently, the primary treatment for gastric cancer is surgery, chemotherapy, radiotherapy and so on. Despite diagnosis and chemotherapy drugs have made significant progress, the therapeutic effects of gastric cancer remains poorly. Gene therapy has become a new tool in the field of treatment and research in the cancer. As one of the most important transcription factors during the cell cycle and apoptosis, E2F-1is a well-characterized member of the E2F gene family. A lot of studies have demonstrated that disorders of E2F-1signaling pathways will lead to the cancer. In our previous studies, we found that E2F-1was a very stable and conservative gene, and it was overexpressed in gastric cancer tissues. This research suggests that E2F-1may play an important role in the occurrence and development of gastric cancer. Moreover, we constructed a silencing eukaryotic vector targeting E2F-1, and transfected into human gastric cancer cell line MGC-803. The result showed that growth rate of gastric cancer cells was slower than control group, and apoptosis rate of cells increased significantly. We also found that more gastric cancer cells were stoped at M phase when E2F-1was suppressed. It can be concluded that E2F-1gene served as an oncogene in vitro. In order to further investigate anti-tumor effects of silencing E2F-1gene in vivo, lentiviral vector of RNA interference (RNAi) of the E2F-1gene will be constructed and used in preliminary animal experiments. At the same time, we will use semi-quantitative reverse transcription PCR (RT-PCR), Western blot and yeast two-hybrid system to reveal its mechanism.
2Methods
2.1According to our previous research, the double-stranded DNA oligo (E2F-1-shRNA) which can cause most RNA interference (RNAi) effect to E2F-1was made. The shRNA sequences were annealed and linked with linearized into the pLentiLox3.7lentiviral vector (pLL3.7). The recombinants were named as pLL3.7-shRNA-E2F-1, and were identified by double restriction digestion with XbaI/NotI and DNA sequencing. Moreover, the recombinant lentivirus (Lv-shRNA-E2F-1) was harvested from293T cells when the pLL3.7-shRNA-E2F-1co-transfected with lentiviral packing materials into them after48h. The virus particles were collected. Virus titer was determined by hole dilution method. In addition, the negative control sequences which had no significant homology to any human gene sequences were constructed. This complementary hairpin DNA oligo were synthesized, annealed, and ligated into linearized pLL3.7vector, and virus particles were collected by criteria method mentioned above. The negative control recombinants were named as pLL3.7-shRNA-NC. The negative control virus particles were named as Lv-shRNA-NC.
2.2Human gastric cancer cells MGC-803in logarithmic growth phase were obtained by centrifugation and stored. Five BALB/C nude mice were inplanted subcutaneously in flanks with200μl (1×106cells per nude) MGC-803cells to produce the tumor. When the maximum diameter of the tumor was longer than1cm, we pulled out the tumor, and cut it into several small pieces (1-2mm3) which were tansplanted into the rest of nude mouse subcutaneously to establish the nude mouse gastric cell carcinoma model. They were randomly divided into experimental group (Lv-shRNA-E2F-1group), negative control group (Lv-shRNA-NC group) and PBS group. The corresponding agents were injected100μl per time (5×108TU/ml) for a consecutive of6times on every two days, and measured the tumor volumes at the same time. The animals were sacrificed at11days, and the subcutaneous tumors were analyzed. Firstly, the tumor growth-curve was drawn, and the weight of tumor was measured. Secondly, the morphological changes of tumor cells were observed by the optical microscope. Thirdly, the expression of E2F-1in tumor was detected by immunohistochemistry, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting respectively. The apoptosis of tumor was measured by in situ end labeling technique (TUNEL).
2.3In our previous studies, we found that PTEN expression was increased when the E2F-1was downregulated in MGC-803cells in virto by using the gene chip assay. Therefore, to investigate the mechanisms of E2F-1-shRNA induction of transplant tumor cell apoptosis, we observed the expression of some apoptosis-associated genes by semi-quantitative RT-PCR and western blot, such as PTEN, caspase-3, caspase-9and NF-κB in this research.
2.4According to the results of our pilot experiments, we decide to remove the E2F-1activatied domain, and construct a bait vector of truncated E2F-1gene (309-1241bp) for yeast two-hybrid system. Above all, PCR primers were designed, and the gene sequence of truncated E2F-1was amplified from the PCMV-E2F-1-HA which was conserved in our lab by using PCR. Secondly, the truncated fragment was cloned into pGBKT7, and resulting recombinant plasmid was named as pGBKT7-E2F-1. This recombinant plasmid was transfered into AH109, and the autonomic activation and cytotoxicity of recombinants was determined by selective petri dish method. After that, The cDNA library of human gastric mucosa which was also conserved in our lab was screened with pGBKT7-E2F1as a bait plasmid by yeast two-hybrid system, and His+/Leu+/Trp+/Ade+/LacZ+positive yeast clones were obtained. In addtion, the inserted fragments of identified positive clones were confirmed and sequenced by using yeast turning test and BLAST tools.
3Results
3.1The small hairpin RNA sequences (shRNA) were successfully inserted into pLentiLox3.7vector by double restriction digestion, and the sequences were identified by DNA sequencing. The shRNA of E2F-1gene of the recombinant lentiviral vector was successfully packed into293T cells. The recombinant lentivirus was harvested from293T cells, and the titer of the virus of Lv-shRNA-E2F-1and Lv-shRNA-NC was3×109TU/ml and2×109TU/ml respectively.
3.2The model of human gastric cancer in nude mice was successfully established by subcutaneous transplantation. The formation rate of transplanted tumour in Lv-shRNA-E2F-1, Lv-shRNA-NC and PBS group was90%,90%and80%respectively. The tumor growth-curve showed that the tumor growth rate in the Lv-shRNA-E2F-1treatment group was much slower than that in the Lv-shRNA-NC and PBS group (P<0.05).The weight of transplant tumor in Lv-shRNA-E2F-1group (0.637±0.061g) was lighter than Lv-shRNA-NC (2.225±0.079g) and PBS group (2.334±0.087g). The tissues got from the nude mice haven been diagnosed that was malignant tumor under microscope after HE stain. Immunohistochemistry results showed that there were a few of brown granules in Lv-shRNA-NC and PBS group, while they were almost not found in the Lv-shRNA-E2F-1group (P＜0.05). Compared with Lv-shRNA-NC and PBS group, the expression level of E2F-1mRNA and protein were decreased in the Lv-shRNA-E2F-1group (P＜0.05). The apoptotic rate in the Lv-shRNA-E2F-1treatment group was18.7±3.6%, significantly higher than that in the other two groups (6.5±0.7%for Lv-shRNA-NC and5.8±0.3%for PBS, P＜0.05). There was no difference to find between the Lv-shRNA-NC and PBS group in all the experiment mention above (P＞0.05).
3.3To investigate the mechanisms of E2F-1-shRNA induction of transplant tumor cell apoptosis, we used semi-quantitative RT-PCR and western blot to examine the mRNA and protein expression of PTEN, Caspase-3, Caspase-9and NF-κB. Densitometry showed that PTEN, Caspase-3and Caspase-9mRNA expression in the Lv-shRNA-E2F-1group was higher while NF-κB was lower than that of the Lv-shRNA-NC and PBS groups (P＜0.05), and no difference was found between Lv-shRNA-NC and PBS groups (P＞0.05). In addition, E2F-1-shRNA induced cleavage of pro-caspase-3(35kDa) and pro-caspase-9(47kDa) into other multiple, cleaved, maturation products, but only the17kDa form of cleaved caspase-3and37kDa form of cleaved caspase-9were observered in tumor tissue. Densitometry showed that PTEN, p17cleaved caspase-3and p37cleaved caspase-9protein expression in the Lv-shRNA-E2F-1group was higher while NF-κB, pro-caspase-3and pro-caspase-9expression was lower than that in the Lv-shRNA-NC and PBS groups (P<0.05), and no difference was found between the Lv-shRNA-NC and PBS groups (P>0.05).
3.4The bait vector pGBKT7-E2F-1was constructed successfully. The result of selective petri dish method showed pGBKT7-E2F-1could not autonomously activate the reporter gene, and it did not have cytotoxicity on AH109yeasts. After the cDNA library of human gastric mucosa was screened with pGBKT7-E2F1, we could obtain20His+/Leu+/Trp+/Ade+/LacZ+positive yeast clones.18different candidate gene sequences were confirmed by yeast turning test and BLAST analysis in NCBI, including IRF7, CB, GUK1, ATPIF1, RPSA, SERBP1, ASS1, MESDC2, WISP2, RCN1, CDC42EP1, EXOSC7, EFEMP1, FSTL3, MT2A, LGALS1, SIVA1and one unknown gene.
4Conclusions
4.1Lentiviral shRNA expression vector targeting E2F-1gene has been successfully constructed, which provides a stable vector for further study of the relationship between gastric cancer and E2F-1gene.
4.2Intra-tumor injection of retrovirus Lv-shRNA-E2F-1has significantly inhibitory effect on the growth of human gastric subcutaneous tumor of Nude Mice. This lentiviral expression vetor would be an effective tool for gastric cancer treatment.
4.3E2F-1-shRNA could increase PTEN, Caspase-3and Caspase-9expression while NF-κB expression was decreased. This result indicate that blocking E2F-1expression probably upregulates PTEN expression directly or indirectly, decreases NF-κB expression, activates Caspase-3and Caspase-9via PTEN/NF-Kb signaling pathway, and inhibits the growth of MGC-803cells in vivo.
4.4The bait vector pGBKT7-E2F-1was constructed successfully. There was not self-activation activity and cytotoxicity.18different proteins interacting with E2F-1have been successfully screened from the cDNA library of human gastric mucosa by yeast two-hybrid system. At the same time, the prophase work related, we could illuminate mechanism How E2F-1silencing inhibited the growth of gastric cancer.
Innovation
1. So far, the effect of RNA interference targeting E2F-1on gastric cancer is still not clear. In present study, we investigated how E2F-1-shRNA affected the human gastric cancer cells growth in vivo by using lentivirus-mediated RNA interference system. Moreover, this study also for the first time searched the protein interacting with E2F-1by yeast two-hybrid system analysis. Combining with our previous research, the mechanism how E2F-1silencing inhibited the growth of gastric cancer could be illuminated. This study lays the foundation for treatment of gastric cancer through downregulation of E2F-1expression.
2. Yeast two-hybrid sysstem is an effective tool to explore the gene function. This study for the first time searched the protein interacting with E2F-1at home and abroad by using yeast two-hybrid system, and helped clarify the role of E2F-1in gastric cancer.
胃癌是人类常见的消化道肿瘤,严重的威胁着广西人民的健康。目前临床上主要利用手术、化疗和放疗等手段治疗胃癌,尽管经过多年的基础及临床研究,胃癌在早期诊断及化疗药物研究上取得重大进展,但效果不甚理想,基因治疗已成为肿瘤治疗研究的新领域。转录因子E2F-1基因作为E2F家族的一员,是细胞周期和细胞凋亡决定性调控因子之一。大量研究证实人类大多数肿瘤中E2F-1通路环诸多环节的失调与肿瘤发生有着密切的关系。我们在前期的研究中亦发现,E2F-1在胃癌的发生发展过程中是一个非常稳定、保守的基因,突变的发生率极低,而且E2F-1在胃癌组织中呈高表达,提示E2F-1在胃癌的发生发展过程中具有重要作用。通过构建E2F-1沉默的真核表达载体转染胃癌细胞MGC-803,我们发现转染后的胃癌细胞生长速度明显减慢,细胞凋亡明显增加,并促使MGC-803细胞停滞于M期,提示了E2F-1基因在体外具有癌基因的特性。为了进一步探讨E2F-1沉默后的体内抗肿瘤效应,本课题构建E2F-1基因RNA干扰(RNAi)慢病毒载体,并运用E2F-1的RNAi慢病毒载体进行了初步的动物实验,同时,应用半定量RT-PCR、Western blot和酵母双杂交等技术初步探讨其发生机制,以期为胃癌的基因治疗打下坚实的基础。
2方法
2.1根据前期的研究结果,选择干扰E2F-1基因效率最高的siRNA寡核苷酸序列,制备双链DNA oligo (E2F-1-shRNA),应用基因重组技术克隆到pLentiLox3.7(pLL3.7)'慢病毒表达载体中,XbaI和NotI进行双酶切和DNA测序鉴定重组克隆。在脂质体的介导下将慢病毒的混合包装载体质粒和包含E2F-1基因RNAi的重组慢病毒载体(pLL3.7-shRNA-E2F-1)共转染293T细胞,包装成病毒48小时后,收集病毒颗粒(Lv-shRNA-E2F-1),孔稀释法测定病毒滴度。另外,设计一条现有基因文库中基因均无同源性的非特异性siRNA序列作为阴性对照序列,同法构建阴性对照组慢病毒载体(pLL3.7-shRNA-NC),同法包装收集阴性对照组慢病毒颗粒(Lv-shRNA-NC),孔稀释法测定病毒滴度。
2.2取对数生长期的人胃癌细胞MGC-803常规消化,随机选取5只裸鼠,向其右腋皮下注射200μ1(1×106个细胞)单细胞悬液。待皮下移植瘤最长径长约1cm时,切取移植瘤,剪碎至1-2mm3,再移植回剩余裸鼠的右腋皮下,建立人胃癌裸鼠移植瘤模型。构建人胃癌裸鼠移植瘤模型成功后,将裸鼠编号,随机分为实验组(Lv-shRNA-E2F-1组)、阴性对照组(Lv-shRNA-NC组)和PBS组。隔天向各组分别注射相应的病毒颗粒100μl(5×108TU/ml),共6次,同时测量各组肿瘤的长径和短径以计算肿瘤的体积大小,并绘制其生长曲线；11天后颈椎脱臼处死裸鼠,称量肿瘤重量,取瘤体HE染色后观察其形态改变,应用免疫组化sp法、半定量逆转录-聚合酶链反应(RT-PCR)和蛋白印迹(Western Blot)分别检测肿瘤组织中E2F-1的mRNA和蛋白的表达,原位末端转移酶标记技术(TUNEL)检测皮下移植瘤细胞凋亡指数,探索基因治疗的效果。
2.3根据前期研究中基因芯片的结果,提取移植瘤组织的RNA及总蛋白,采用半定量RT-PCR检测移植瘤组织凋亡相关基因PTEN、Caspase-3、 Caspase-9和NF-κB mRNA的表达情况。采用Western blot检测胃癌细胞内PTEN、Caspase-3前体(pro-caspase-3)、Caspase-9前体(pro-caspase-9)、激活型Caspase-3(cleaved caspase-3)、激活型Caspase-9(cleaved caspase-9)和NF-κB的蛋白含量。
2.4根据前期预实验结果,设计引物,以pCMV-E2F-1-HA为模板,去掉E2F-1激活域,PCR扩增E2F-1截短体(309-1241bp),引入酶切位点EcoRI和BamHI,将E2F-1截短体连接至载体pGBKT7中,将构建成功的诱饵表达载体pGBKT7-E2F-1截短体运用化学转化法转化到酵母菌AH109中,采用选择性培养皿分析检测重组质粒的自激活活性和细胞毒性。再将pGBKT7-E2F-1和来源于人正常胃黏膜细胞的cDNA文库质粒顺序转化酵母AH109细胞中,通过营养缺陷型培养基进行阳性克隆的筛选。随后将所得的阳性克隆通过多次营养筛选、X-gal分析、酵母双杂交回转实验对获得的假定相互作用的可靠性加以验证。并对验证后的阳性克隆进行测序。最后借助数据库中的BLAST工具对候选阳性克隆的测序结果进行同源性分析,确定可能发生相互作用的蛋白。
3结果
3.1双酶切证实所设计的E2F-1及阴性对照组shRNA正确插入慢病毒载体,DNA测序证实插入的序列正确；293T细胞成功包装E2F-1-shRNA的重组慢病毒载体及其阴性对照组的重组慢病毒载体；收集的细胞培养上清液中,Lv-shRNA-E2F-1病毒的滴度为3×109TU/ml,Lv-shRNA-NC病毒的滴度为2×109TU/ml。
3.2成功构建人胃癌裸鼠移植瘤模型,三组裸鼠成瘤情况为Lv-shRNA-E2F-1组9只、Lv-shRNA-NC组9只、PBS组8只；各组皮下移植瘤生长曲线表明：与PBS组和Lv-shRNA-NC组比较,Lv-shRNA-E2F-1组肿瘤生长速度明显减慢(P<0.05),PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)；移植瘤重量称量结果显示：Lv-shRNA-E2F-1组肿瘤重量(0.637±0.061g)显著小于Lv-shRNA-NC组(2.225±0.079g)和PBS组(2.334±0.087g),(P<0.05),PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)。HE染色后显微镜下确认切取肿瘤为胃癌皮下移植瘤组织；免疫组化E2F-1特殊染色后可见Lv-shRNA-NC组和PBS组的移植瘤细胞细胞核内出现较多的棕褐色颗粒,而Lv-shRNA-E2F-1组的肿瘤细胞细胞核内均未见或偶见棕褐色颗粒(P<0.05)；半定量RT-PCR和Western blot结果均显示Lv-shRNA-E2F-1组E2F-1mRNA及蛋白表达较Lv-shRNA-NC组和PBS组明显下调(P<0.05),PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)；TUNEL结果显示：Lv-shRNA-E2F-1组的凋亡指数(18.7±3.6%)明显高于Lv-shRNA-NC组(6.5±0.7%)和PBS组(5.8±0.3%)(P<0.05),PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)。
3.3在移植瘤组织中,半定量RT-PCR结果显示：与PBS组和Lv-shRNA-NC组比较,Lv-shRNA-E2F-1组的PTEN、Caspase-3、Caspase-9mRNA表达明显上调,NF-κB mRNA表达明显下调(P＜0.05), PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)；Western-blot结果显示：Lv-shRNA-E2F-1组PTEN、cleaved caspase-9、cleaved caspase-3的蛋白表达量显著高于Lv-shRNA-NC组和PBS组(P<0.05),pro-caspase-9、 pro-caspase-3、NF-κB的蛋白表达量显著低于Lv-shRNA-NC组和PBS组(P＜0.05), PBS组和Lv-shRNA-NC组比较差异无统计学意义(P>0.05)。
3.4成功扩增了E2F-1截短体,并连接至载体pGBKT7中,成功构建E2F-1截短体的诱饵载体(pGBKT7-E2F-1),经测序比对结果正确。将pGBKT7-E2F-1转化到酵母菌AH109中,经检测无自激活活性和细胞毒性。AH109(pGBKT7-E2F-1)交配筛选人正常胃黏膜细胞的cDNA文库,在四缺培养基上进行筛选,经反复划线和X-gal检测后获得21个阳性克隆。将获得的阳性质粒一对一与诱饵质粒pGBKT7-E2F1重新共转化AH109酵母菌进行验证,获得阳性候选克隆20个,将阳性克隆测序后进行同源性分析鉴定,最终得到18个不同的相关蛋白,分别为：IRF7、CB、GUK1、ATPIF1、 RPSA、SERBP1、ASS1、MESDC2、WISP2、RCN1、CDC42EP1、EXOSC7、 EFEMP1、FSTL3、MT2A、LGALS1、SIVA1、未知基因。
4结论
4.1成功构建人E2F-1基因RNAi慢病毒载体,为下一步探讨E2F-1沉默对于胃癌生长的体内实验体内实验提供了理论基础和前期准备。为开展靶向E2F-1基因的胃癌基因治疗研究提供了必须的实验工具。
4.2瘤内注射靶向沉默E2F-1基因慢病毒颗粒Lv-shRNA-E2F-1可显著抑制人胃癌细胞MGC-803皮下移植瘤的生长速度,降低肿瘤重量,并明显下调移植瘤组织中E2F-1mRNA及蛋白表达水平。该慢病毒表达系统可能是胃癌治疗的一种有效工具。
4.3瘤内注射慢病毒颗粒Lv-shRNA-E2F-1抑制移植瘤组织中E2F-1的表达后,可引起PTEN的表达量上升,并激活Caspase-9和Caspase-3,降低NF-κB的表达。提示E2F-1沉默极有可能直接或间接通过PTEN/NF-κB信号传导通路,促进胃癌细胞凋亡,抑制胃癌的生长。
4.4成功构建了E2F-1截短体的诱饵载体,经检测无自激活活性和细胞毒性,应用酵母双杂交技术筛选出18个可与E2F-1相互作用的蛋白,结合前期的实验结果,为阐明E2F-1沉默抑制胃癌生长的作用机制提供了新的思路。
创新点
1目前,对于利用RNA干扰技术使E2F-1基因沉默后,探讨其对胃癌进展过程的影响的相关研究未见报道。本课题从体内实验及E2F-1的蛋白间相互作用出发,初步明确E2F-1基因沉默后对胃癌进展的影响,结合前期体外实验,有助于全面阐明E2F-1沉默在胃癌生长中的作用及其功能机制,为E2F-1作为胃癌治疗的一个新的治疗性和预后性靶基因提供理论和实验依据。
2酵母双杂交技术是研究新基因功能的有效手段,本研究拟采用此技术筛查与E2F-1相互作用的蛋白,这在国内外尚属首次。以寻找E2F-1的相互作用蛋白入手,有助于客观真实地阐明其作用的功能机制。
1. Background and Objective
Gastric cancer is a common human gastrointestinal cancer which is a serious threat to the health of the people of Guangxi. Currently, the primary treatment for gastric cancer is surgery, chemotherapy, radiotherapy and so on. Despite diagnosis and chemotherapy drugs have made significant progress, the therapeutic effects of gastric cancer remains poorly. Gene therapy has become a new tool in the field of treatment and research in the cancer. As one of the most important transcription factors during the cell cycle and apoptosis, E2F-1is a well-characterized member of the E2F gene family. A lot of studies have demonstrated that disorders of E2F-1signaling pathways will lead to the cancer. In our previous studies, we found that E2F-1was a very stable and conservative gene, and it was overexpressed in gastric cancer tissues. This research suggests that E2F-1may play an important role in the occurrence and development of gastric cancer. Moreover, we constructed a silencing eukaryotic vector targeting E2F-1, and transfected into human gastric cancer cell line MGC-803. The result showed that growth rate of gastric cancer cells was slower than control group, and apoptosis rate of cells increased significantly. We also found that more gastric cancer cells were stoped at M phase when E2F-1was suppressed. It can be concluded that E2F-1gene served as an oncogene in vitro. In order to further investigate anti-tumor effects of silencing E2F-1gene in vivo, lentiviral vector of RNA interference (RNAi) of the E2F-1gene will be constructed and used in preliminary animal experiments. At the same time, we will use semi-quantitative reverse transcription PCR (RT-PCR), Western blot and yeast two-hybrid system to reveal its mechanism.
2Methods
2.1According to our previous research, the double-stranded DNA oligo (E2F-1-shRNA) which can cause most RNA interference (RNAi) effect to E2F-1was made. The shRNA sequences were annealed and linked with linearized into the pLentiLox3.7lentiviral vector (pLL3.7). The recombinants were named as pLL3.7-shRNA-E2F-1, and were identified by double restriction digestion with XbaI/NotI and DNA sequencing. Moreover, the recombinant lentivirus (Lv-shRNA-E2F-1) was harvested from293T cells when the pLL3.7-shRNA-E2F-1co-transfected with lentiviral packing materials into them after48h. The virus particles were collected. Virus titer was determined by hole dilution method. In addition, the negative control sequences which had no significant homology to any human gene sequences were constructed. This complementary hairpin DNA oligo were synthesized, annealed, and ligated into linearized pLL3.7vector, and virus particles were collected by criteria method mentioned above. The negative control recombinants were named as pLL3.7-shRNA-NC. The negative control virus particles were named as Lv-shRNA-NC.
2.2Human gastric cancer cells MGC-803in logarithmic growth phase were obtained by centrifugation and stored. Five BALB/C nude mice were inplanted subcutaneously in flanks with200μl (1×106cells per nude) MGC-803cells to produce the tumor. When the maximum diameter of the tumor was longer than1cm, we pulled out the tumor, and cut it into several small pieces (1-2mm3) which were tansplanted into the rest of nude mouse subcutaneously to establish the nude mouse gastric cell carcinoma model. They were randomly divided into experimental group (Lv-shRNA-E2F-1group), negative control group (Lv-shRNA-NC group) and PBS group. The corresponding agents were injected100μl per time (5×108TU/ml) for a consecutive of6times on every two days, and measured the tumor volumes at the same time. The animals were sacrificed at11days, and the subcutaneous tumors were analyzed. Firstly, the tumor growth-curve was drawn, and the weight of tumor was measured. Secondly, the morphological changes of tumor cells were observed by the optical microscope. Thirdly, the expression of E2F-1in tumor was detected by immunohistochemistry, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting respectively. The apoptosis of tumor was measured by in situ end labeling technique (TUNEL).
2.3In our previous studies, we found that PTEN expression was increased when the E2F-1was downregulated in MGC-803cells in virto by using the gene chip assay. Therefore, to investigate the mechanisms of E2F-1-shRNA induction of transplant tumor cell apoptosis, we observed the expression of some apoptosis-associated genes by semi-quantitative RT-PCR and western blot, such as PTEN, caspase-3, caspase-9and NF-κB in this research.
2.4According to the results of our pilot experiments, we decide to remove the E2F-1activatied domain, and construct a bait vector of truncated E2F-1gene (309-1241bp) for yeast two-hybrid system. Above all, PCR primers were designed, and the gene sequence of truncated E2F-1was amplified from the PCMV-E2F-1-HA which was conserved in our lab by using PCR. Secondly, the truncated fragment was cloned into pGBKT7, and resulting recombinant plasmid was named as pGBKT7-E2F-1. This recombinant plasmid was transfered into AH109, and the autonomic activation and cytotoxicity of recombinants was determined by selective petri dish method. After that, The cDNA library of human gastric mucosa which was also conserved in our lab was screened with pGBKT7-E2F1as a bait plasmid by yeast two-hybrid system, and His+/Leu+/Trp+/Ade+/LacZ+positive yeast clones were obtained. In addtion, the inserted fragments of identified positive clones were confirmed and sequenced by using yeast turning test and BLAST tools.
3Results
3.1The small hairpin RNA sequences (shRNA) were successfully inserted into pLentiLox3.7vector by double restriction digestion, and the sequences were identified by DNA sequencing. The shRNA of E2F-1gene of the recombinant lentiviral vector was successfully packed into293T cells. The recombinant lentivirus was harvested from293T cells, and the titer of the virus of Lv-shRNA-E2F-1and Lv-shRNA-NC was3×109TU/ml and2×109TU/ml respectively.
3.2The model of human gastric cancer in nude mice was successfully established by subcutaneous transplantation. The formation rate of transplanted tumour in Lv-shRNA-E2F-1, Lv-shRNA-NC and PBS group was90%,90%and80%respectively. The tumor growth-curve showed that the tumor growth rate in the Lv-shRNA-E2F-1treatment group was much slower than that in the Lv-shRNA-NC and PBS group (P<0.05).The weight of transplant tumor in Lv-shRNA-E2F-1group (0.637±0.061g) was lighter than Lv-shRNA-NC (2.225±0.079g) and PBS group (2.334±0.087g). The tissues got from the nude mice haven been diagnosed that was malignant tumor under microscope after HE stain. Immunohistochemistry results showed that there were a few of brown granules in Lv-shRNA-NC and PBS group, while they were almost not found in the Lv-shRNA-E2F-1group (P＜0.05). Compared with Lv-shRNA-NC and PBS group, the expression level of E2F-1mRNA and protein were decreased in the Lv-shRNA-E2F-1group (P＜0.05). The apoptotic rate in the Lv-shRNA-E2F-1treatment group was18.7±3.6%, significantly higher than that in the other two groups (6.5±0.7%for Lv-shRNA-NC and5.8±0.3%for PBS, P＜0.05). There was no difference to find between the Lv-shRNA-NC and PBS group in all the experiment mention above (P＞0.05).
3.3To investigate the mechanisms of E2F-1-shRNA induction of transplant tumor cell apoptosis, we used semi-quantitative RT-PCR and western blot to examine the mRNA and protein expression of PTEN, Caspase-3, Caspase-9and NF-κB. Densitometry showed that PTEN, Caspase-3and Caspase-9mRNA expression in the Lv-shRNA-E2F-1group was higher while NF-κB was lower than that of the Lv-shRNA-NC and PBS groups (P＜0.05), and no difference was found between Lv-shRNA-NC and PBS groups (P＞0.05). In addition, E2F-1-shRNA induced cleavage of pro-caspase-3(35kDa) and pro-caspase-9(47kDa) into other multiple, cleaved, maturation products, but only the17kDa form of cleaved caspase-3and37kDa form of cleaved caspase-9were observered in tumor tissue. Densitometry showed that PTEN, p17cleaved caspase-3and p37cleaved caspase-9protein expression in the Lv-shRNA-E2F-1group was higher while NF-κB, pro-caspase-3and pro-caspase-9expression was lower than that in the Lv-shRNA-NC and PBS groups (P<0.05), and no difference was found between the Lv-shRNA-NC and PBS groups (P>0.05).
3.4The bait vector pGBKT7-E2F-1was constructed successfully. The result of selective petri dish method showed pGBKT7-E2F-1could not autonomously activate the reporter gene, and it did not have cytotoxicity on AH109yeasts. After the cDNA library of human gastric mucosa was screened with pGBKT7-E2F1, we could obtain20His+/Leu+/Trp+/Ade+/LacZ+positive yeast clones.18different candidate gene sequences were confirmed by yeast turning test and BLAST analysis in NCBI, including IRF7, CB, GUK1, ATPIF1, RPSA, SERBP1, ASS1, MESDC2, WISP2, RCN1, CDC42EP1, EXOSC7, EFEMP1, FSTL3, MT2A, LGALS1, SIVA1and one unknown gene.
4Conclusions
4.1Lentiviral shRNA expression vector targeting E2F-1gene has been successfully constructed, which provides a stable vector for further study of the relationship between gastric cancer and E2F-1gene.
4.2Intra-tumor injection of retrovirus Lv-shRNA-E2F-1has significantly inhibitory effect on the growth of human gastric subcutaneous tumor of Nude Mice. This lentiviral expression vetor would be an effective tool for gastric cancer treatment.
4.3E2F-1-shRNA could increase PTEN, Caspase-3and Caspase-9expression while NF-κB expression was decreased. This result indicate that blocking E2F-1expression probably upregulates PTEN expression directly or indirectly, decreases NF-κB expression, activates Caspase-3and Caspase-9via PTEN/NF-Kb signaling pathway, and inhibits the growth of MGC-803cells in vivo.
4.4The bait vector pGBKT7-E2F-1was constructed successfully. There was not self-activation activity and cytotoxicity.18different proteins interacting with E2F-1have been successfully screened from the cDNA library of human gastric mucosa by yeast two-hybrid system. At the same time, the prophase work related, we could illuminate mechanism How E2F-1silencing inhibited the growth of gastric cancer.
Innovation
1. So far, the effect of RNA interference targeting E2F-1on gastric cancer is still not clear. In present study, we investigated how E2F-1-shRNA affected the human gastric cancer cells growth in vivo by using lentivirus-mediated RNA interference system. Moreover, this study also for the first time searched the protein interacting with E2F-1by yeast two-hybrid system analysis. Combining with our previous research, the mechanism how E2F-1silencing inhibited the growth of gastric cancer could be illuminated. This study lays the foundation for treatment of gastric cancer through downregulation of E2F-1expression.
2. Yeast two-hybrid sysstem is an effective tool to explore the gene function. This study for the first time searched the protein interacting with E2F-1at home and abroad by using yeast two-hybrid system, and helped clarify the role of E2F-1in gastric cancer.
引文
[1]宋海峰,钱海利,张雪艳,等.复制缺陷型腺病毒Ad-p14ARF治疗肝细胞癌的实验研究及作用机制的探讨.中华肿瘤杂志,2006,28(9)：641-645.
[2]Tanaka H, Yashiro M, Sunami T, etc. ICAM-2 gene therapy for peritoneal dissemination of scirrhous gastric carcinoma. Clin Cancer Res,2004, 10(14):4885-4892.
[3]Carneiro F, Oliveira C, Leite M. Molecular targers and biological modifiers in gastric cancer. Semin Diagn Pathol,2008,25(4):274-287.
[4]Vachani A, Moon E, Wakeam E, etc. Gene therapy for mesothelioma and lung cancer. Respir. Am J Respir Cell Mol Biol,2010,42(4):385-393.
[5]胡品津.基因多态性与胃癌遗传易感性的关系.中华消化杂志,2006,26(4)：278-279.
[6]李诚,周健,裘炯良.胃癌流行病学与分子生物学病因的研究进展.肿瘤防治研究,2004,31(2)：115-118.
[7]Martin GS. Cell signaling and cancer. Cancer Cell,2003,4 (3):167-174.
[8]Logan N, Graham A, Zhao X, etc. E2F-8:an E2F family memher with a similar organization of DNA binding domains to E2F-7. Oncogene,2005, 24(31):5000-5004.
[9]Helin K, Lees JA,Vidal M, etc. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell,1992,70(2):337-350.
[10]Martinsson HS, Starborg M, Erlandsson F, etc. Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb. Exp Cell Res,2005,305(2):383-391.
[11]Muller H, Bracken AP, Vernell R, etc. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev,2001,15(3):267-85.
[12]苑汐子,尹永硕,李雷,等.RNA干扰E2F-1表达对胃癌MGC803细胞生物学行为的影响及其机制.中华实验外科杂志,2009,26(11)：1467-1469.
[13]王晓通,李雷,肖强,等.E2F-1过表达对胃癌细胞生物学行为影响.广东医学,2009,30(11)：1600-1603.
[14]Yang XH, Sladek TL. Overexpression of the E2F-1 transcription factor gene mediates cell transformation. Gene Expr,1995,4(4-5):195-204.
[15]Johnson DG, Schwarz JK, Cress WD, el al. Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature,1993, 365(6444):349-352.
[16]Suh DS, Yoon MS, Choi KU, etc. Significance of E2F-1 overexpression in epithelial ovarian cancer. Int J Gynecol Cancer,2008,18(3):492-498.
[17]Alla V, Engelmann D, Niemetz A, etc. E2F1 in melanoma progression and metastasis. J Natl Cancer Inst,2010,102(2):127-133.
[18]张学文,孟子辉,赵吉生,等.E2F-1启动子调控下肿瘤靶向性作用的研究.中华外科杂志,2006,44(23)：1636-1639.
[19]Komori H, Enomoto M, Nakamura M, etc. Distinct E2F-mediated transcrip tional program regulates p14ARF gene expression. EMBO J,2005,24(21): 3724-3736.
[20]Li Z, Kreutzer M, Mikkat S, etc. Proteomic analysis of the E2F1 response in p53-negative cancer cells:new aspects in the regulation of cell survival and death. Proteomics,2006,6(21):5735-5745.
[21]Vorburger SA, Hetrakul N, Xia W, etc. Gene therapy with E2F-1 up-regulates the protein kinase PKR and inhibits growth of leiomyosarcoma in vivo. Mol Cancer Ther,2005,4(11):1710-1716.
[22]张峰,刘哗,刘三光,等.RNA干预研究及其在肿瘤基因治疗中的应用.中华实验外科杂志,2006,23(3)：381-382.
[23]Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA interference. Nature,2004,431(7006):371-378.
[24]Mello CC, Conte D Jr. Revealing the world of RNA interference. Nature, 2004,431(7006):338-342.
[25]Pushparaj PN, Arthi JJ, Manikandan J, etc. siRNA, miRNA, and shRNA:in vivo applications. J Dent Res,2008,87(11):992-1003.
[26]靳西凤,冉志华.RNA干扰技术与结肠癌.世界华人消化消化杂志,2006,14(20)：2003-2008.
[27]Nakase M, Inui M, Okumura K, etc. P53 gene therapy of human osteosarcoma using a transferring-modified cationic liposome. Mol Cancer Ther,2005,4(4):625-631.
[28]Neschadim A, Mcart JA, Keating A, etc. A roadmap to safe, efficient and stable lentivirus-mediated gene therapy with hematopoietic cell transplantation. Biol Blood Marrow Tansplant,2007,13(12):1407-1416.
[29]肖强,徐君毅,谢玉波.E2F-1在胃癌组织中的突变、表达及其意义.中华实验外科杂志,2006,23(9)：1138.
[30]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[1]Engelmann D, Piitzer BM. The dark side of E2F1:in transit beyond apoptosis. Cancer Res,2012,72(3):571-575.
[2]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[3]尹永硕,李雷,谢玉波,等.小分子干扰RNA对胃癌MGC803细胞E2F-1表达及其生物学行为的影响.中华实验外科杂志,2009,26(1)：17-19.
[4]Picanco-Castro V, de Sousa Russo-Carbolante EM, Tadeu Covas D. Advances in lentiviral vectors:a patent review. Recent Pat DNA Gene Seq, 2012,6(2):82-90.
[5]Pellinen R, Hakkarainen T, Walllfbrs T, et al. Cancer cells as targets for lentiviros-mediated gene transfer and gene therapy. Int J Oncol,2004,25(6): 1753-1762.
[6]尹永硕.RNA干扰对胃癌MGC803细胞E2F-1基因表达及其生物学行 为的研究[学位论文].广西,广西医科大学,2008.
[7]Zielske SP, Stevenson M. Importin 7 may be dispensable for human immunodeficiency virus type 1 and simian immunodeficiency virus infection of primary macrophages. J Virol,2005,79(17):11541-11546.
[8]Pullmann R Jr, Juhaszova M, Lopez de Silanes I, etc. Enhanced proliferation of cultured human vascular smooth muscle cells linked to increased function of RNA-binding protein HuR. J Biol Chem,2005, 280(24):22819-22826.
[9]Kovesdi I, Reichel R, Nevins JR. Identification of a cellular transcription factor involved in E1A trans-activation. Cell,1986,45(2):219-228.
[10]柯爱武,刘康达.c-Met和整合素信号转导的交互作用与肿瘤转移.中华肿瘤防治杂志,2007,14(7)：560-563.
[11]Baldini E, Camerini A, Sgambato A, etc. Cyclin A and E2F1 overexpression correlate with reduced disease-free survival in nodenegative breast cancer patients. Anticancer Res,2006,26(6B):4415-4421.
[12]Zacharatos P, Kotsinas A, Evangelou K, etc. Distinct expression patterns of the transcription factor E2F-1 in relation to tumour growth paramters in common human carcinomas. J Pothol,2004,203(3):744-753.
[13]Huang CL, Liu D, Nakano J, etc. E2F1 overexpression correlates with thymidylate synthase and survivin gene expressions and tumor proliferation in non small-cell lung cancer. Clin Cancer Res,2007,13(23):6938-6946.
[14]Sowers R, Toguchida J, Qin J, etc. mRNA expression levels of E2F transcription factors correlate with dihydrofolate reductase, reduced folate carrier, and thymidylate synthase mRNA expression in osteosarcoma. Mol Cancer Ther,2003,2(6):535-541.
[15]Molina-Privado I, Rodriguez-Martinez M, Rebollo P, etc. E2F1 expression is deregulated and plays an oncogenic role in sporadic Burkitt's lymphoma. Cancer Res,2009,69(9):4052-4058.
[16]Zhang SY, Liu SC, Johnson DG, etc. E2F-1 gene transfer enhances invasiveness of human head and neck carcinoma cell line. Cancer Res, 2000,60(21):5972-5976.
[17]Xiao Q, Li L, Xie Y, etc. Transcription factor E2F-1 is upregulated in human gastric cancer tissues and its overexpression suppresses gastric tumor cell proliferation. Cell Oncol,2007,29(4):335-349.
[18]Singhania R, Khairuddin N, Clarke D, etc. RNA interference for the treatment of papillomavirus disease. Open Virol J,2012,6:204-215.
[19]Chang SS, Zhang Z, Liu Y. RNA interference pathways in fungi: mechanisms and functions. Annu Rev Microbiol.2012,66:305-323.
[20]Hannon GJ. RNA interference. Nature,2002,418(6894):244-251.
[21]Tijsterman M, Okihara KL, Thijssen K, etc. PPW-1, a PAZ/PIWI protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans. Curr Biol,2002,12(17):1535-1540.
[22]Palauqui JC, Elmayan T, Pollien JM, etc. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J,1997,16(15): 4738-4745.
[23]Sijen T, Fleenor J, Simmer F, etc. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell,2001,107(4):465-476.
[24]Scott RC, Juhsz G, Neufeld TP. Direct induction of autophagy by Atgl inhibits cell growth and induces apoptotic cell death. Curr Biol,2007,17(1): 1-11.
[25]Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol,2004,36(12):2503-2518.
[26]Podolska K, Stachurska A, Hajdukiewicz K, etc. Gene therapy prospects--intranasal delivery of therapeutic genes. Adv Clin Exp Med, 2012,21(4):525-534.
[27]Rossetti M, Cavarelli M, Gregori S, etc. HIV-derived vectors for gene therapy targeting dendritic cells. Adv Exp Med Biol,2013,762:239-261.
[28]Schuldt NJ, Amalfitano A. Malar vaccines:focus on adenovirus based vectors. Vaccine,2012,30(35):5191-5198.
[29]Xiao PJ, Lentz TB, Samulski RJ. Recombinant adeno-associated virus: clinical application and development as a gene-therapy vector. Ther Deliv, 2012,3(7):835-856.
[30]Reiser J, Lai Z, Zhang XY, etc. Development of multigene and regulated lentivirus vectors. J Virol,2000,74(22):10589-10599.
[1]Herszenyi L, Tulassay Z. Epidemiology of gastrointestinal and liver tumors. Eur Rev Med Pharmacol Sci,2010,14(4):249-258.
[2]Herszenyi L, Tulassay Z. Epidemiology of gastrointestinal and liver tumors. Eur Rev Med Pharmacol Sci,2010,14(4):249-258.
[3]Moscova M, Marsh DJ, Baxter RC, etc. Protein chip discovery of secreted proteins regulated by the phosphatidylinositol 3-kinase pathway in ovarian cancer cell lines. Cancer Res,2006,66(3):1376-1383.
[4]Verdaguer E, Susana Gde A, Clemens A, etc. Implication of the transcription factor E2F-1 in the modulation of neuronal apoptosis. Biomed Pharmacother,2007,61(7):390-399.
[5]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[6]Capozza F, Williams TM, Schubert W, etc. Absence of caveolin-1 sensitizes mouse skin to carcinogen-induced epidermal hyperplasia and tumor formation. Am J Pathol,2003,162(6):2029-2039.
[7]Kato M, Asaka M. Recent development of gastric cancer prevention. Jpn J Clin Oncol,2012,42(11):987-994.
[8]Cervantes A, Roda D, Tarazona N, etc. Current questions for the treatment of advanced gastric cancer. Cancer Treat Rev,2013,39(1):60-67.
[9]Olowoyeye A, Okwundu CI. Gene therapy for sickle cell disease. Cochrane Database Syst Rev,2012,11:CD007652.
[10]Neschadim A, McCart JA, Keating A, etc. A roadmap to safe, efficient, and stable lentivirus-mediated gene therapy with hematopoietic cell transplantation. Biol Blood Marrow Transplant,2007,13(12):1407-1416.
[11]Zhang Y, Chen L, Yang S, etc. E2F1:a potential negative regulator of hTERT transcription in normal cells upon activation of oncogenic c-Myc. Med Sci Monit,2012,18(1):RA12-15.
[12]Johnson DG, Cress WD, Jakoi L, etc. Oncogenic capacity of the E2F1 gene. Proc Natl Acad Sci U S A,1994,91(26):12823-12827.
[13]Zhang SY,Liu SC, Al-Saleem LF, etc. E2F-1:a proliferative marker of breast neoplasia. Cancer Epidemiol Biomarkers Prev,2000,9(4):395-401.
[14]Banerjee D, Gorlick R, Liefshitz A, etc. Levels of E2F-1 expression are higher in lung metastasis of colon cancer as compared with hepatic metastasis and correlate with levels of thymidylate synthase. Cancer Res,2000,60(9):2365-2367.
[15]Xiao Q, Li L, Xie Y, etc. Transcription factor E2F-1 is upregulated in human gastric cancer tissues and its overexpression suppresses gastric tumor cell proliferation. Cell Oncol,2007,29(4):335-349.
[16]Yamasaki L, Bronson R, Williams BO, etc. Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rbl(+/-)mice. Nat Genet,1998, 18(4):360-364.
[17]尹永硕,李雷,谢玉波,等.小分子干扰RNA对胃癌MGC803细胞E2F-1表达及其生物学行为的影响.中华实验外科杂志,2009,26(1)：17-19.
[18]Zhang SY, Liu SC, Johnson DQ etc. E2F-1 gene transfer enhances invasiveness of human head and neck carcinoma cell line. Cancer Res, 2000,60(21):5972-5976.
[19]刘春斌,唐振勇,肖强,等.转录因子E2F-1在胃癌中的表达及临床意义.广东医学,2009,30(7)：1065-1067.
[20]苑汐子,尹永硕,李雷,等.RNA干扰E2F-1表达对胃癌MGC803细胞生物学行为的影响及其机制.中华实验外科杂志,2009,26(11)：1467-1469.
[1]苑汐子,尹永硕,李雷,等.RNA干扰E2F-1表达对胃癌MGC803细胞生物学行为的影响及其机制.中华实验外科杂志,2009,26(11)：1467-1469.
[2]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[3]欧海玲,覃宇周,张锡流,等.P120连环蛋白在鼻咽癌中的表达及其与细胞凋亡的关系.实用医学杂志,2009,25(22)：3749-3752.
[4]Hlobilkova A, Knillova J, Bartek J, etc. The mechanism of action of the tumour suppressor gene PTEN. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub,2003,147(1):19-25.
[5]Haier J, Nicolson GL. Time-dependent dephosphorylation through serine/threonine phosphatases is required for stable adhesion of highly and poorly metastatic HT-29 colon carcinoma cell lines to collagen. Anticancer Res,2000,20(4):2265-2271.
[6]Li J, Yen C, Liaw D, etc. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science,1997, 275(5308):1943-1947.
[7]Sato K, Tamura G, Tsuchiya T, etc. Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer. Virchows Arch,2002, 440(2):160-165.
[8]Forgacs E, Biesterveld EJ, Sekido Y, etc. Mutation analysis of the PTEN/MMAC1 gene in lung cancer. Oncogene,1998,17(12):1557-1565.
[9]Favaloro B, Allocati N, Graziano V, etc. Role of apoptosis in disease. Aging (Albany NY),2012,4(5):330-349.
[10]Wurstle ML, Laussmann MA, Rehm M. The central role of initiator caspase-9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome. Exp Cell Res,2012,318(11): 1213-1220.
[11]Chappell WH, Steelman LS, Long JM, etc. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Inhibitors:Rationale and Importance to Inhibiting These Pathways in Human Health. Oncotarget,2011,2(3):135-164.
[12]Yamakawa N, Takahashi A, Mori E, etc. High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation. Cancer Sci,2008,99(7):1455-1460.
[13]Reinhard K, Huber M, Lohoff M, etc. The role of NF-κB activation during protection against Leishmania infection. Int J Med Microbiol,2012, 302(4-5):230-235.
[14]邓元,张学斌,王鸿雁,等.乳腺癌组织中核转录因子NF-κB和抑癌基因PTEN的表达及其意义.现代肿瘤医学,2008,16(7)：1132-1134.
[15]Mayo MW, Madrid LV, Westerheide SD, etc. PTEN blocks tumor necrosis factor-induced NF-kappa B-dependent transcription by inhibiting the transactivation potential of the p65 subunit. J Biol Chem,2002,277(13): 11116-11125.
[16]苏剑东,吴灵飞.NF-κB与细胞凋亡.世界华人消化杂志,2007,15(12)：1411-1416
[1]孙秀娣,牧人,周有尚,等.中国胃癌死亡率20年变化情况分析及其发展趋势预测.中华肿瘤杂志,2004,26(1)：4-10.
[2]Knoll S, Emmrich S, Putzer BM. The E2F1-miRNA Cancer Progression Network. Adv Exp Med Biol,2013,774:135-1347.
[3]肖强,徐君毅,谢玉波.E2F-1在胃癌组织中的突变、表达及其意义.中华实验外科杂志,2006,23(9)：1138.
[4]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[5]Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature,1989,340(6230):245-246.
[6]Rajagopala SV, Sikorski P, Caufield JH, etc. Studying protein complexes by the yeast two-hybrid system. Methods,2012,58(4):392-399.
[7]何震.人正常胃粘膜细胞全长cDNA文库的构建及鉴定[学位论文].广西,广西医科大学,2010.
[8]王长青.E2F-1基因对胃癌细胞增殖、凋亡、迁移、侵袭和化疗药物敏感性影响的研究[学位论文].广西,广西医科大学,2008.
[9]Agaton C, Uhlen M, Hober S. Genome-based proteomics. Electrophoresis, 2004,25(9):1280-1288.
[10]Miller J, Stagljar I. Using the yeast two-hybrid system to identify interacting proteins. Methods Mol Biol,2004,261:247-262.
[11]Traven A, Jelicic B, Sopta M. Yeast Gal4:a transcriptional paradigm revisited. EMBO Rep,2006,7(5):496-499.
[12]Vong QP, Liu Z, Yoo JG, etc. A role for borg5 during trophectoderm differentiation. Stem Cells,2010,28(6):1030-1038.
[13]Kouklis P, Konstantoulaki M, Vogel S, etc. Cdc42 regulates the restoration of endothelial barrier function. Circ Res,2004,94(2):159-166.
[14]Li Z, Dong X, Wang Z, etc. Regulation of PTEN by Rho small GTPases. Nat Cell Biol,2005,7(4):399-404.
[15]Pod gorski I, Sloane BF. Cathepin B and its role(s) in cancer progression. Biochem Soc Symp,2003,70(22):263-276.
[16]Yide Mei, Mian Wu. Multifaceted functions of Siva-1:more than an Indian God of Destruction. Protein Cell,2012,3(2):117-122.
[17]Fortin, MacLaurin JG, Arbour N, etc. The proapoptotic gene SIVA is a direct transcriptional target for the tumor suppressors p53 and E2F1. J Biol Chem,2004,279(27):28706-28714.
[18]Ray RM, Bhattacharya S, Johnson, etc. LR. Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73- and E2F1-mediated expression of PUMA and Siva-1. Apoptosis,2011,16(1): 35-44.
[19]王晓通,李雷,肖强,等.E2F-1过表达对胃癌细胞生物学行为影响.广东医学,2009,30(11)：1600-1603.
[20]林存刚,徐慧,陈玉成.酵母双杂交系统研究及其应用进展.微生物学杂志,2005,25(6)：85-89.
[1]Engelmann D, Putzer BM. The dark side of E2F1:in transit beyond apoptosis. Cancer Res,2012,72(3):571-575.
[2]Helin K, Lees JA,Vidal M, etc. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell,1992,70(2):337-350.
[3]Muller H, Helin K. The E2F transcripation factors:key regulators of cell Proliferation. Biochim Biophys Acta,2000,1470(1):M1-12.
[4]Johnson DG, Walker CL. Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol,1999,39:295-312.
[5]Gewirtz DA, Di YM, Randolph JK, etc. Rb dephosphorylation and suppression of E2F activity in human breast tumor cells exposed to a pharmacyological concentration of estradiol. Arch Biochem Biophys,2001, 388(2):243-252.
[6]Trimarchi JM, Lees JA. Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol,2002,3(1):11-20.
[7]Banerjee D, Gorlic R, Liefshitz A, etc. Levels of E2F-1 expression are higher in lung metastasis of colon cancer as compared with hepatic metastasis and correlate with levels of thymidylate synthase. Cancer Res, 2000,60(9):2365-2367.
[8]Tsukuda K, Wiewrodt R, Molnar-Kimber K, etc. An E2F-responsive replication-selective adenovirus targeted to the defective cell cycle in cancer cells:potent antitumoral efficacyu but no toxicity to normal cell. Cancer Research,2002,62(12):3438-3447.
[9]Shu HK, Julin CM, Furman F, etc. Overexpression of E2F1 in glioma-derived cell lines induces a p53-independent apoptosis that is further enhanced by ionizing radiation. Neuro-oncol,2000,2(1):16-21.
[10]Sun B, Wingate H, Swisher SG, etc. Absence of pRb facilitates E2F1-induced apoptosis in breast cancer cells. Cell Cycle,2010,9(6): 1122-1130.
[11]Gomez-Gutierrez JG, Garcia-Garcia A, Hao H, etc. Adenovirus-mediated expression of truncated E2F-1 suppresses tumor growth in vitro and in vivo. Cancer,2010,116(18):4420-4432.
[12]Ramalho RM, Ribeiro PS, Sola S, etc. Inhibition of the E2F-1/p53/Bax pathway by tauroursodeoxycholic acid in amyloid beta-peptide-induced apoptosis of PC12 cells. J Neurochem,2004,90(3):567-575.
[13]Kherrouche Z, Blais A, Ferreira E, etc. ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene. Biochem J,2006,396(3):547-556.
[14]Suh DS, Yoon MS, Choi KU, etc. Significance of E2F-1 overexpression in epithelial ovarian cancer. Int J Gynecol Cancer,2008,18(3):492-498.
[15]Alla V, Engelmann D, Niemetz A, etc. E2F1 in melanoma progression and metastasis. J Natl Cancer Inst,2010,102(2):127-133.
[16]王晓通,钱倩,李雷,等.RNA干扰介导的转录因子E2F-1沉默对人胃癌裸鼠移植瘤生长的影响.中华实验外科杂志,2011,28(3)：340-342.
[17]Xiao Q, Li L, Xie Y, etc. Transcription factor E2F-1 is upregulated in human gastric cancer tissues and its overexpression suppresses gastric tumor cell proliferation. Cellular Oncol,2007,29(4):335-349.
[18]Wang XT, Xie YB, Xiao Q. Lentivirus-mediated RNA interference targeting E2F-1 inhibits human gastric cancer MGC-803 cell growth in vivo. Exp Mol Med,2011,43(11):638-645.
[19]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[20]Xie Y, Wang C, Li L, etc. Overexpression of E2F-1 inhibits progression of gastric cancer in vitro. Cell Biol Int,2009,33(6):640-649.
[21]Suzuki T, Yasui W, Yokozaki H, etc. Expression of the E2F family in human gastrointestinal carcinomas. Int J Cancer,1999,81(4):535-538.
[22]Vuaroqueaux V, Urban P, Labuhn M, etc. Low E2F1 transcript levels are a strong determinant of favorable breast cancer outcome. Breast Cancer Res, 2007,9(3):R33.
[23]Molina-Privado I, Rodriguez-Martinez M, Rebollo P, etc. E2F1 expression is deregulated and plays an oncogenic role in sporadic Burkitt's lymphoma. Cancer Res,2009,69(9):4052-4058.
[24]Zacharatos P, Kotsinas A, Evangelou K, etc. Distinct expression patterns of the transcription factor E2F-1 in relation to tumour growth parameters in common human carcinomas. J Pathol,2004,203(3):744-753.
[25]Rabbani F, Richon VM, Orlow I, etc. Prognostic significance of transcription factor E2F-1 in bladder cancer, genotypic and phenotypic characterization. J Natl Cancer Inst,1999,91(10):874-871.
[26]Han S, Park K, Bae BN, etc. E2F-1 expression is related with the poor survival of lymph node-positive breast patients treated with fluorouracil, doxorubicin and cyclophosphamide. Breast Cancer Res Treat,2003,82(1): 11-16.
[27]Kelly-Spratt KS, Gurley KE, Yasui Y, etc. p19Arf suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways. PLoS Biol,2004,2(8):E242.
[28]Olowoyeye A, Okwundu CI. Gene therapy for sickle cell disease. Cochrane Database Syst Rev,2012,11:CD007652.
[29]Li WJ, Gu YY, Zhang HJ, etc. Induction of p14ARF by E2F1 contributes to 8-chloro-adenosine-induced apoptosis in human lung cancer H1299 cells. Chemotherapy,2009,55(5):335-343.
[30]Ray RM, Bhattacharya S, Johnson LR. Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73-and E2F1-mediated expression of PUMA and Siva-1. Apoptosis,2011,16(1): 35-44.
[31]Vorburger SA, Hetrakul N, Xia W, etc. Gene therapy with E2F-1 up-regulates the protein kinase PKR and inhibits growth of leiomyosarcoma in vivo. Mol Cancer Ther,2005,4(11):1710-1716.
[32]Kuhn H, Liebers U, Gessner C, etc. Adenovirus-mediated E2F-1 gene transfer in nonsmall-cell lung cancer induces cell growth arrest and apoptosis. Eur Respir J,2002,20(3):703-709.
[33]Qin G, Kishore R, Dolan CM, etc. Cell cycle regulator E2F1 modulates angiogenesis via p53-dependent transcriptional control of VEGF. Proc Natl Acad Sci U S A,2006,103(29):11015-11020.
[34]汪听,唐滔,陈波,等.E2F1的siRNA真核表达载体转染对胆管癌细胞的作用.中国普通外科杂志,2008,17(2)：130-133
[35]Johnson DG The paradox of E2F1:oncogene and tumor suppressor gene. Mol Carcinog,2000,27(3):151-157.
[36]Yang XH, Sladek TL, Overexpression of the E2F-1 transcription factor gene mediates cell transformation. Gene Expr,1995,4(4-5):195-204.
[37]Olson MV, Johnson DG, Jiang H, etc. Transgenic E2F1 expression in the mouse brain induces a human-like bimodal pattern of tumors. Cancer Res, 2007,67(9):4005-4009.
[38]Rounbehler RJ, Rogers PM, Conti CJ, etc. Inactivation of E2f1 enhances tumorigenesis in a Myc transgenic model. Cancer Res,2002,62(11): 3276-3281.
[2]Tanaka H, Yashiro M, Sunami T, etc. ICAM-2 gene therapy for peritoneal dissemination of scirrhous gastric carcinoma. Clin Cancer Res,2004, 10(14):4885-4892.
[3]Carneiro F, Oliveira C, Leite M. Molecular targers and biological modifiers in gastric cancer. Semin Diagn Pathol,2008,25(4):274-287.
[4]Vachani A, Moon E, Wakeam E, etc. Gene therapy for mesothelioma and lung cancer. Respir. Am J Respir Cell Mol Biol,2010,42(4):385-393.
[5]胡品津.基因多态性与胃癌遗传易感性的关系.中华消化杂志,2006,26(4)：278-279.
[6]李诚,周健,裘炯良.胃癌流行病学与分子生物学病因的研究进展.肿瘤防治研究,2004,31(2)：115-118.
[7]Martin GS. Cell signaling and cancer. Cancer Cell,2003,4 (3):167-174.
[8]Logan N, Graham A, Zhao X, etc. E2F-8:an E2F family memher with a similar organization of DNA binding domains to E2F-7. Oncogene,2005, 24(31):5000-5004.
[9]Helin K, Lees JA,Vidal M, etc. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell,1992,70(2):337-350.
[10]Martinsson HS, Starborg M, Erlandsson F, etc. Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb. Exp Cell Res,2005,305(2):383-391.
[11]Muller H, Bracken AP, Vernell R, etc. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev,2001,15(3):267-85.
[12]苑汐子,尹永硕,李雷,等.RNA干扰E2F-1表达对胃癌MGC803细胞生物学行为的影响及其机制.中华实验外科杂志,2009,26(11)：1467-1469.
[13]王晓通,李雷,肖强,等.E2F-1过表达对胃癌细胞生物学行为影响.广东医学,2009,30(11)：1600-1603.
[14]Yang XH, Sladek TL. Overexpression of the E2F-1 transcription factor gene mediates cell transformation. Gene Expr,1995,4(4-5):195-204.
[15]Johnson DG, Schwarz JK, Cress WD, el al. Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature,1993, 365(6444):349-352.
[16]Suh DS, Yoon MS, Choi KU, etc. Significance of E2F-1 overexpression in epithelial ovarian cancer. Int J Gynecol Cancer,2008,18(3):492-498.
[17]Alla V, Engelmann D, Niemetz A, etc. E2F1 in melanoma progression and metastasis. J Natl Cancer Inst,2010,102(2):127-133.
[18]张学文,孟子辉,赵吉生,等.E2F-1启动子调控下肿瘤靶向性作用的研究.中华外科杂志,2006,44(23)：1636-1639.
[19]Komori H, Enomoto M, Nakamura M, etc. Distinct E2F-mediated transcrip tional program regulates p14ARF gene expression. EMBO J,2005,24(21): 3724-3736.
[20]Li Z, Kreutzer M, Mikkat S, etc. Proteomic analysis of the E2F1 response in p53-negative cancer cells:new aspects in the regulation of cell survival and death. Proteomics,2006,6(21):5735-5745.
[21]Vorburger SA, Hetrakul N, Xia W, etc. Gene therapy with E2F-1 up-regulates the protein kinase PKR and inhibits growth of leiomyosarcoma in vivo. Mol Cancer Ther,2005,4(11):1710-1716.
[22]张峰,刘哗,刘三光,等.RNA干预研究及其在肿瘤基因治疗中的应用.中华实验外科杂志,2006,23(3)：381-382.
[23]Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA interference. Nature,2004,431(7006):371-378.
[24]Mello CC, Conte D Jr. Revealing the world of RNA interference. Nature, 2004,431(7006):338-342.
[25]Pushparaj PN, Arthi JJ, Manikandan J, etc. siRNA, miRNA, and shRNA:in vivo applications. J Dent Res,2008,87(11):992-1003.
[26]靳西凤,冉志华.RNA干扰技术与结肠癌.世界华人消化消化杂志,2006,14(20)：2003-2008.
[27]Nakase M, Inui M, Okumura K, etc. P53 gene therapy of human osteosarcoma using a transferring-modified cationic liposome. Mol Cancer Ther,2005,4(4):625-631.
[28]Neschadim A, Mcart JA, Keating A, etc. A roadmap to safe, efficient and stable lentivirus-mediated gene therapy with hematopoietic cell transplantation. Biol Blood Marrow Tansplant,2007,13(12):1407-1416.
[29]肖强,徐君毅,谢玉波.E2F-1在胃癌组织中的突变、表达及其意义.中华实验外科杂志,2006,23(9)：1138.
[30]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[1]Engelmann D, Piitzer BM. The dark side of E2F1:in transit beyond apoptosis. Cancer Res,2012,72(3):571-575.
[2]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[3]尹永硕,李雷,谢玉波,等.小分子干扰RNA对胃癌MGC803细胞E2F-1表达及其生物学行为的影响.中华实验外科杂志,2009,26(1)：17-19.
[4]Picanco-Castro V, de Sousa Russo-Carbolante EM, Tadeu Covas D. Advances in lentiviral vectors:a patent review. Recent Pat DNA Gene Seq, 2012,6(2):82-90.
[5]Pellinen R, Hakkarainen T, Walllfbrs T, et al. Cancer cells as targets for lentiviros-mediated gene transfer and gene therapy. Int J Oncol,2004,25(6): 1753-1762.
[6]尹永硕.RNA干扰对胃癌MGC803细胞E2F-1基因表达及其生物学行 为的研究[学位论文].广西,广西医科大学,2008.
[7]Zielske SP, Stevenson M. Importin 7 may be dispensable for human immunodeficiency virus type 1 and simian immunodeficiency virus infection of primary macrophages. J Virol,2005,79(17):11541-11546.
[8]Pullmann R Jr, Juhaszova M, Lopez de Silanes I, etc. Enhanced proliferation of cultured human vascular smooth muscle cells linked to increased function of RNA-binding protein HuR. J Biol Chem,2005, 280(24):22819-22826.
[9]Kovesdi I, Reichel R, Nevins JR. Identification of a cellular transcription factor involved in E1A trans-activation. Cell,1986,45(2):219-228.
[10]柯爱武,刘康达.c-Met和整合素信号转导的交互作用与肿瘤转移.中华肿瘤防治杂志,2007,14(7)：560-563.
[11]Baldini E, Camerini A, Sgambato A, etc. Cyclin A and E2F1 overexpression correlate with reduced disease-free survival in nodenegative breast cancer patients. Anticancer Res,2006,26(6B):4415-4421.
[12]Zacharatos P, Kotsinas A, Evangelou K, etc. Distinct expression patterns of the transcription factor E2F-1 in relation to tumour growth paramters in common human carcinomas. J Pothol,2004,203(3):744-753.
[13]Huang CL, Liu D, Nakano J, etc. E2F1 overexpression correlates with thymidylate synthase and survivin gene expressions and tumor proliferation in non small-cell lung cancer. Clin Cancer Res,2007,13(23):6938-6946.
[14]Sowers R, Toguchida J, Qin J, etc. mRNA expression levels of E2F transcription factors correlate with dihydrofolate reductase, reduced folate carrier, and thymidylate synthase mRNA expression in osteosarcoma. Mol Cancer Ther,2003,2(6):535-541.
[15]Molina-Privado I, Rodriguez-Martinez M, Rebollo P, etc. E2F1 expression is deregulated and plays an oncogenic role in sporadic Burkitt's lymphoma. Cancer Res,2009,69(9):4052-4058.
[16]Zhang SY, Liu SC, Johnson DG, etc. E2F-1 gene transfer enhances invasiveness of human head and neck carcinoma cell line. Cancer Res, 2000,60(21):5972-5976.
[17]Xiao Q, Li L, Xie Y, etc. Transcription factor E2F-1 is upregulated in human gastric cancer tissues and its overexpression suppresses gastric tumor cell proliferation. Cell Oncol,2007,29(4):335-349.
[18]Singhania R, Khairuddin N, Clarke D, etc. RNA interference for the treatment of papillomavirus disease. Open Virol J,2012,6:204-215.
[19]Chang SS, Zhang Z, Liu Y. RNA interference pathways in fungi: mechanisms and functions. Annu Rev Microbiol.2012,66:305-323.
[20]Hannon GJ. RNA interference. Nature,2002,418(6894):244-251.
[21]Tijsterman M, Okihara KL, Thijssen K, etc. PPW-1, a PAZ/PIWI protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans. Curr Biol,2002,12(17):1535-1540.
[22]Palauqui JC, Elmayan T, Pollien JM, etc. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J,1997,16(15): 4738-4745.
[23]Sijen T, Fleenor J, Simmer F, etc. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell,2001,107(4):465-476.
[24]Scott RC, Juhsz G, Neufeld TP. Direct induction of autophagy by Atgl inhibits cell growth and induces apoptotic cell death. Curr Biol,2007,17(1): 1-11.
[25]Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol,2004,36(12):2503-2518.
[26]Podolska K, Stachurska A, Hajdukiewicz K, etc. Gene therapy prospects--intranasal delivery of therapeutic genes. Adv Clin Exp Med, 2012,21(4):525-534.
[27]Rossetti M, Cavarelli M, Gregori S, etc. HIV-derived vectors for gene therapy targeting dendritic cells. Adv Exp Med Biol,2013,762:239-261.
[28]Schuldt NJ, Amalfitano A. Malar vaccines:focus on adenovirus based vectors. Vaccine,2012,30(35):5191-5198.
[29]Xiao PJ, Lentz TB, Samulski RJ. Recombinant adeno-associated virus: clinical application and development as a gene-therapy vector. Ther Deliv, 2012,3(7):835-856.
[30]Reiser J, Lai Z, Zhang XY, etc. Development of multigene and regulated lentivirus vectors. J Virol,2000,74(22):10589-10599.
[1]Herszenyi L, Tulassay Z. Epidemiology of gastrointestinal and liver tumors. Eur Rev Med Pharmacol Sci,2010,14(4):249-258.
[2]Herszenyi L, Tulassay Z. Epidemiology of gastrointestinal and liver tumors. Eur Rev Med Pharmacol Sci,2010,14(4):249-258.
[3]Moscova M, Marsh DJ, Baxter RC, etc. Protein chip discovery of secreted proteins regulated by the phosphatidylinositol 3-kinase pathway in ovarian cancer cell lines. Cancer Res,2006,66(3):1376-1383.
[4]Verdaguer E, Susana Gde A, Clemens A, etc. Implication of the transcription factor E2F-1 in the modulation of neuronal apoptosis. Biomed Pharmacother,2007,61(7):390-399.
[5]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[6]Capozza F, Williams TM, Schubert W, etc. Absence of caveolin-1 sensitizes mouse skin to carcinogen-induced epidermal hyperplasia and tumor formation. Am J Pathol,2003,162(6):2029-2039.
[7]Kato M, Asaka M. Recent development of gastric cancer prevention. Jpn J Clin Oncol,2012,42(11):987-994.
[8]Cervantes A, Roda D, Tarazona N, etc. Current questions for the treatment of advanced gastric cancer. Cancer Treat Rev,2013,39(1):60-67.
[9]Olowoyeye A, Okwundu CI. Gene therapy for sickle cell disease. Cochrane Database Syst Rev,2012,11:CD007652.
[10]Neschadim A, McCart JA, Keating A, etc. A roadmap to safe, efficient, and stable lentivirus-mediated gene therapy with hematopoietic cell transplantation. Biol Blood Marrow Transplant,2007,13(12):1407-1416.
[11]Zhang Y, Chen L, Yang S, etc. E2F1:a potential negative regulator of hTERT transcription in normal cells upon activation of oncogenic c-Myc. Med Sci Monit,2012,18(1):RA12-15.
[12]Johnson DG, Cress WD, Jakoi L, etc. Oncogenic capacity of the E2F1 gene. Proc Natl Acad Sci U S A,1994,91(26):12823-12827.
[13]Zhang SY,Liu SC, Al-Saleem LF, etc. E2F-1:a proliferative marker of breast neoplasia. Cancer Epidemiol Biomarkers Prev,2000,9(4):395-401.
[14]Banerjee D, Gorlick R, Liefshitz A, etc. Levels of E2F-1 expression are higher in lung metastasis of colon cancer as compared with hepatic metastasis and correlate with levels of thymidylate synthase. Cancer Res,2000,60(9):2365-2367.
[15]Xiao Q, Li L, Xie Y, etc. Transcription factor E2F-1 is upregulated in human gastric cancer tissues and its overexpression suppresses gastric tumor cell proliferation. Cell Oncol,2007,29(4):335-349.
[16]Yamasaki L, Bronson R, Williams BO, etc. Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rbl(+/-)mice. Nat Genet,1998, 18(4):360-364.
[17]尹永硕,李雷,谢玉波,等.小分子干扰RNA对胃癌MGC803细胞E2F-1表达及其生物学行为的影响.中华实验外科杂志,2009,26(1)：17-19.
[18]Zhang SY, Liu SC, Johnson DQ etc. E2F-1 gene transfer enhances invasiveness of human head and neck carcinoma cell line. Cancer Res, 2000,60(21):5972-5976.
[19]刘春斌,唐振勇,肖强,等.转录因子E2F-1在胃癌中的表达及临床意义.广东医学,2009,30(7)：1065-1067.
[20]苑汐子,尹永硕,李雷,等.RNA干扰E2F-1表达对胃癌MGC803细胞生物学行为的影响及其机制.中华实验外科杂志,2009,26(11)：1467-1469.
[1]苑汐子,尹永硕,李雷,等.RNA干扰E2F-1表达对胃癌MGC803细胞生物学行为的影响及其机制.中华实验外科杂志,2009,26(11)：1467-1469.
[2]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[3]欧海玲,覃宇周,张锡流,等.P120连环蛋白在鼻咽癌中的表达及其与细胞凋亡的关系.实用医学杂志,2009,25(22)：3749-3752.
[4]Hlobilkova A, Knillova J, Bartek J, etc. The mechanism of action of the tumour suppressor gene PTEN. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub,2003,147(1):19-25.
[5]Haier J, Nicolson GL. Time-dependent dephosphorylation through serine/threonine phosphatases is required for stable adhesion of highly and poorly metastatic HT-29 colon carcinoma cell lines to collagen. Anticancer Res,2000,20(4):2265-2271.
[6]Li J, Yen C, Liaw D, etc. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science,1997, 275(5308):1943-1947.
[7]Sato K, Tamura G, Tsuchiya T, etc. Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer. Virchows Arch,2002, 440(2):160-165.
[8]Forgacs E, Biesterveld EJ, Sekido Y, etc. Mutation analysis of the PTEN/MMAC1 gene in lung cancer. Oncogene,1998,17(12):1557-1565.
[9]Favaloro B, Allocati N, Graziano V, etc. Role of apoptosis in disease. Aging (Albany NY),2012,4(5):330-349.
[10]Wurstle ML, Laussmann MA, Rehm M. The central role of initiator caspase-9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome. Exp Cell Res,2012,318(11): 1213-1220.
[11]Chappell WH, Steelman LS, Long JM, etc. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Inhibitors:Rationale and Importance to Inhibiting These Pathways in Human Health. Oncotarget,2011,2(3):135-164.
[12]Yamakawa N, Takahashi A, Mori E, etc. High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation. Cancer Sci,2008,99(7):1455-1460.
[13]Reinhard K, Huber M, Lohoff M, etc. The role of NF-κB activation during protection against Leishmania infection. Int J Med Microbiol,2012, 302(4-5):230-235.
[14]邓元,张学斌,王鸿雁,等.乳腺癌组织中核转录因子NF-κB和抑癌基因PTEN的表达及其意义.现代肿瘤医学,2008,16(7)：1132-1134.
[15]Mayo MW, Madrid LV, Westerheide SD, etc. PTEN blocks tumor necrosis factor-induced NF-kappa B-dependent transcription by inhibiting the transactivation potential of the p65 subunit. J Biol Chem,2002,277(13): 11116-11125.
[16]苏剑东,吴灵飞.NF-κB与细胞凋亡.世界华人消化杂志,2007,15(12)：1411-1416
[1]孙秀娣,牧人,周有尚,等.中国胃癌死亡率20年变化情况分析及其发展趋势预测.中华肿瘤杂志,2004,26(1)：4-10.
[2]Knoll S, Emmrich S, Putzer BM. The E2F1-miRNA Cancer Progression Network. Adv Exp Med Biol,2013,774:135-1347.
[3]肖强,徐君毅,谢玉波.E2F-1在胃癌组织中的突变、表达及其意义.中华实验外科杂志,2006,23(9)：1138.
[4]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[5]Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature,1989,340(6230):245-246.
[6]Rajagopala SV, Sikorski P, Caufield JH, etc. Studying protein complexes by the yeast two-hybrid system. Methods,2012,58(4):392-399.
[7]何震.人正常胃粘膜细胞全长cDNA文库的构建及鉴定[学位论文].广西,广西医科大学,2010.
[8]王长青.E2F-1基因对胃癌细胞增殖、凋亡、迁移、侵袭和化疗药物敏感性影响的研究[学位论文].广西,广西医科大学,2008.
[9]Agaton C, Uhlen M, Hober S. Genome-based proteomics. Electrophoresis, 2004,25(9):1280-1288.
[10]Miller J, Stagljar I. Using the yeast two-hybrid system to identify interacting proteins. Methods Mol Biol,2004,261:247-262.
[11]Traven A, Jelicic B, Sopta M. Yeast Gal4:a transcriptional paradigm revisited. EMBO Rep,2006,7(5):496-499.
[12]Vong QP, Liu Z, Yoo JG, etc. A role for borg5 during trophectoderm differentiation. Stem Cells,2010,28(6):1030-1038.
[13]Kouklis P, Konstantoulaki M, Vogel S, etc. Cdc42 regulates the restoration of endothelial barrier function. Circ Res,2004,94(2):159-166.
[14]Li Z, Dong X, Wang Z, etc. Regulation of PTEN by Rho small GTPases. Nat Cell Biol,2005,7(4):399-404.
[15]Pod gorski I, Sloane BF. Cathepin B and its role(s) in cancer progression. Biochem Soc Symp,2003,70(22):263-276.
[16]Yide Mei, Mian Wu. Multifaceted functions of Siva-1:more than an Indian God of Destruction. Protein Cell,2012,3(2):117-122.
[17]Fortin, MacLaurin JG, Arbour N, etc. The proapoptotic gene SIVA is a direct transcriptional target for the tumor suppressors p53 and E2F1. J Biol Chem,2004,279(27):28706-28714.
[18]Ray RM, Bhattacharya S, Johnson, etc. LR. Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73- and E2F1-mediated expression of PUMA and Siva-1. Apoptosis,2011,16(1): 35-44.
[19]王晓通,李雷,肖强,等.E2F-1过表达对胃癌细胞生物学行为影响.广东医学,2009,30(11)：1600-1603.
[20]林存刚,徐慧,陈玉成.酵母双杂交系统研究及其应用进展.微生物学杂志,2005,25(6)：85-89.
[1]Engelmann D, Putzer BM. The dark side of E2F1:in transit beyond apoptosis. Cancer Res,2012,72(3):571-575.
[2]Helin K, Lees JA,Vidal M, etc. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell,1992,70(2):337-350.
[3]Muller H, Helin K. The E2F transcripation factors:key regulators of cell Proliferation. Biochim Biophys Acta,2000,1470(1):M1-12.
[4]Johnson DG, Walker CL. Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol,1999,39:295-312.
[5]Gewirtz DA, Di YM, Randolph JK, etc. Rb dephosphorylation and suppression of E2F activity in human breast tumor cells exposed to a pharmacyological concentration of estradiol. Arch Biochem Biophys,2001, 388(2):243-252.
[6]Trimarchi JM, Lees JA. Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol,2002,3(1):11-20.
[7]Banerjee D, Gorlic R, Liefshitz A, etc. Levels of E2F-1 expression are higher in lung metastasis of colon cancer as compared with hepatic metastasis and correlate with levels of thymidylate synthase. Cancer Res, 2000,60(9):2365-2367.
[8]Tsukuda K, Wiewrodt R, Molnar-Kimber K, etc. An E2F-responsive replication-selective adenovirus targeted to the defective cell cycle in cancer cells:potent antitumoral efficacyu but no toxicity to normal cell. Cancer Research,2002,62(12):3438-3447.
[9]Shu HK, Julin CM, Furman F, etc. Overexpression of E2F1 in glioma-derived cell lines induces a p53-independent apoptosis that is further enhanced by ionizing radiation. Neuro-oncol,2000,2(1):16-21.
[10]Sun B, Wingate H, Swisher SG, etc. Absence of pRb facilitates E2F1-induced apoptosis in breast cancer cells. Cell Cycle,2010,9(6): 1122-1130.
[11]Gomez-Gutierrez JG, Garcia-Garcia A, Hao H, etc. Adenovirus-mediated expression of truncated E2F-1 suppresses tumor growth in vitro and in vivo. Cancer,2010,116(18):4420-4432.
[12]Ramalho RM, Ribeiro PS, Sola S, etc. Inhibition of the E2F-1/p53/Bax pathway by tauroursodeoxycholic acid in amyloid beta-peptide-induced apoptosis of PC12 cells. J Neurochem,2004,90(3):567-575.
[13]Kherrouche Z, Blais A, Ferreira E, etc. ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene. Biochem J,2006,396(3):547-556.
[14]Suh DS, Yoon MS, Choi KU, etc. Significance of E2F-1 overexpression in epithelial ovarian cancer. Int J Gynecol Cancer,2008,18(3):492-498.
[15]Alla V, Engelmann D, Niemetz A, etc. E2F1 in melanoma progression and metastasis. J Natl Cancer Inst,2010,102(2):127-133.
[16]王晓通,钱倩,李雷,等.RNA干扰介导的转录因子E2F-1沉默对人胃癌裸鼠移植瘤生长的影响.中华实验外科杂志,2011,28(3)：340-342.
[17]Xiao Q, Li L, Xie Y, etc. Transcription factor E2F-1 is upregulated in human gastric cancer tissues and its overexpression suppresses gastric tumor cell proliferation. Cellular Oncol,2007,29(4):335-349.
[18]Wang XT, Xie YB, Xiao Q. Lentivirus-mediated RNA interference targeting E2F-1 inhibits human gastric cancer MGC-803 cell growth in vivo. Exp Mol Med,2011,43(11):638-645.
[19]Xie Y, Yin Y, Li L, etc. Short interfering RNA directed against the E2F-1 gene suppressing gastric cancer progression in vitro. Oncol Rep,2009, 21(5):1345-1353.
[20]Xie Y, Wang C, Li L, etc. Overexpression of E2F-1 inhibits progression of gastric cancer in vitro. Cell Biol Int,2009,33(6):640-649.
[21]Suzuki T, Yasui W, Yokozaki H, etc. Expression of the E2F family in human gastrointestinal carcinomas. Int J Cancer,1999,81(4):535-538.
[22]Vuaroqueaux V, Urban P, Labuhn M, etc. Low E2F1 transcript levels are a strong determinant of favorable breast cancer outcome. Breast Cancer Res, 2007,9(3):R33.
[23]Molina-Privado I, Rodriguez-Martinez M, Rebollo P, etc. E2F1 expression is deregulated and plays an oncogenic role in sporadic Burkitt's lymphoma. Cancer Res,2009,69(9):4052-4058.
[24]Zacharatos P, Kotsinas A, Evangelou K, etc. Distinct expression patterns of the transcription factor E2F-1 in relation to tumour growth parameters in common human carcinomas. J Pathol,2004,203(3):744-753.
[25]Rabbani F, Richon VM, Orlow I, etc. Prognostic significance of transcription factor E2F-1 in bladder cancer, genotypic and phenotypic characterization. J Natl Cancer Inst,1999,91(10):874-871.
[26]Han S, Park K, Bae BN, etc. E2F-1 expression is related with the poor survival of lymph node-positive breast patients treated with fluorouracil, doxorubicin and cyclophosphamide. Breast Cancer Res Treat,2003,82(1): 11-16.
[27]Kelly-Spratt KS, Gurley KE, Yasui Y, etc. p19Arf suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways. PLoS Biol,2004,2(8):E242.
[28]Olowoyeye A, Okwundu CI. Gene therapy for sickle cell disease. Cochrane Database Syst Rev,2012,11:CD007652.
[29]Li WJ, Gu YY, Zhang HJ, etc. Induction of p14ARF by E2F1 contributes to 8-chloro-adenosine-induced apoptosis in human lung cancer H1299 cells. Chemotherapy,2009,55(5):335-343.
[30]Ray RM, Bhattacharya S, Johnson LR. Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73-and E2F1-mediated expression of PUMA and Siva-1. Apoptosis,2011,16(1): 35-44.
[31]Vorburger SA, Hetrakul N, Xia W, etc. Gene therapy with E2F-1 up-regulates the protein kinase PKR and inhibits growth of leiomyosarcoma in vivo. Mol Cancer Ther,2005,4(11):1710-1716.
[32]Kuhn H, Liebers U, Gessner C, etc. Adenovirus-mediated E2F-1 gene transfer in nonsmall-cell lung cancer induces cell growth arrest and apoptosis. Eur Respir J,2002,20(3):703-709.
[33]Qin G, Kishore R, Dolan CM, etc. Cell cycle regulator E2F1 modulates angiogenesis via p53-dependent transcriptional control of VEGF. Proc Natl Acad Sci U S A,2006,103(29):11015-11020.
[34]汪听,唐滔,陈波,等.E2F1的siRNA真核表达载体转染对胆管癌细胞的作用.中国普通外科杂志,2008,17(2)：130-133
[35]Johnson DG The paradox of E2F1:oncogene and tumor suppressor gene. Mol Carcinog,2000,27(3):151-157.
[36]Yang XH, Sladek TL, Overexpression of the E2F-1 transcription factor gene mediates cell transformation. Gene Expr,1995,4(4-5):195-204.
[37]Olson MV, Johnson DG, Jiang H, etc. Transgenic E2F1 expression in the mouse brain induces a human-like bimodal pattern of tumors. Cancer Res, 2007,67(9):4005-4009.
[38]Rounbehler RJ, Rogers PM, Conti CJ, etc. Inactivation of E2f1 enhances tumorigenesis in a Myc transgenic model. Cancer Res,2002,62(11): 3276-3281.