应用反义寡核苷酸技术研究Skp2基因在胃癌细胞SGC-7901生长增殖中的作用
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摘要
目的：
反义寡核苷酸技术(antisense oligonucleotides technique)在1978年首次应用于抑制鸡肉瘤病毒的复制，此后逐渐成为公认的具有高度特异性和低毒性作用的基因干预手段。反义寡核苷酸能通过碱基配对原则与细胞内核酸特异结合形成杂交分子，从而在转录或翻译水平抑制特定基因表达。随着分子生物学和遗传工程的发展，现在已经能够快速、简易地合成较短的核苷酸片断，即寡核苷酸。此项技术不仅能应用于生命科学的研究，它在医学上的应用也越来越广泛，如治疗恶性肿瘤、病毒性疾病、炎症性疾病及减少疤痕的形成。尽管反义寡核苷酸技术在国外已进入临床实验治疗阶段，国内也已开始研究，但反义寡核苷酸要作为一类新型药物普遍应用于治疗有待于更深入的研究，尤其对其靶标基因作用机制的研究。
选择对肿瘤有治疗作用的特定目的基因靶标是反义寡核苷酸治疗的关键所在。癌变细胞的共同特征是与细胞增殖、分裂或分化有关的基因出现异常，因此应用反义寡核苷酸技术抑制异常表达的基因，能逆转或去除肿瘤细胞恶性增殖和扩散的行为。S期激酶相关蛋白2(S-phase kinase-associated protain 2，Skp2)是在恶变细胞中发现的细胞周期相关蛋白，它经泛素—蛋白酶体途径(ubiquitin-proteasome pathway，UPP)降解一些细胞周期调节因子，从而在细胞增殖调控中起重要作用。同时，Skp2有癌基因的潜能，在多种人类恶性肿瘤中高表达，并与肿瘤恶性程度及预后不良有密切联系。
胃癌是世界上最常见的消化道恶性肿瘤之一，其死亡率一直居恶性肿瘤之首，而我国胃癌患病及死亡率超过世界平均水平的两倍。胃癌和其它恶性肿瘤一样都是多因素、多阶段的获得性基因疾病，包括原癌基因的激活和抑癌基因的失活，并伴随着细胞增殖的异常。Skp2作为细胞增殖调控基因和潜在癌基因，它同样在胃癌中高表达并与胃癌的恶性程度相关。因此，Skp2基因在胃癌的发生、发展中有着重要的地位，有望成为胃癌治疗的靶点。近年来，针对胃癌风险基因的反义寡核苷酸治疗手段逐渐成为一种具有潜在价值的胃癌治疗手段。然而，针对Skp2基因的恶性肿瘤治疗研究刚刚起步，而采用Skp2反义寡核苷酸治疗胃癌的研究还未见报道。鉴于上述原因，本研究通过合成互补于Skp2 mRNA核苷酸序列的反义寡核苷酸(antisense oligodeoxy-nucleotides，ASODN)，研究Skp2基因在胃癌细胞SGC-7901生长和增殖中的作用及其可能机制，从而为针对Skp2基因的肿瘤治疗提供初步的理论依据。
方法：
根据实验设计，以细胞的不同处理因素分组：正常对照组，Skp2无义寡核苷酸(nonsense oligodeoxy-nucleotides，NSODN)处理组，以及Skp2反义寡核苷酸(antisense oligodeoxy-nucleotides，ASODN)处理组，转染浓度分为50 nmol／L，200 nmol／L和400 nmol／L。实验方法如下：采用阳离子脂质体Lipofectamine 2000 Reagent包裹Skp2寡核苷酸转染SGC-7901细胞；通过显微镜和MTT实验观察细胞的生长和增殖情况；应用流式细胞术检测细胞周期；提取细胞总RNA，应用逆转录聚合酶链反应检测细胞Skp2、p27 mRNA表达变化；提取细胞总蛋白，应用Western-blot法检测细胞Skp2蛋白、p27蛋白及p53蛋白表达变化。
结果：
1．转染200 nmol／L Skp2 ASODN后的SGC-7901细胞生长速度明显减慢，细胞边界不清，连接松散，失去克隆性生长特性。
2．200 nmol／L的ASODN在24 h、48 h、72 h等各个时间段上对胃癌细胞的增殖均有明显的抑制作用，p＜0.01。但在高浓度(400 nmol／L)、长时间(48 h以上)作用下的NSODN和ASODN对胃癌细胞均有较大的非反义性抑制效应，两者无显著性差异，p＞0.05。
3．200 nmol／L NSODN组细胞周期与正常对照组相比无明显变化，而200 nmol／LASODN组细胞周期发生改变：G_0／G_1期细胞所占比例略有降低趋势，而S期细胞所占比例却明显高于正常对照组细胞，p＜0.05。
4．200 nmol／L NSODN组Skp2 mRNA表达量与正常对照组相比无显著性差异，p＞0.05；200 nmol／L ASODN组Skp2 mRNA表达量明显低于正常对照组，p＜0.05；200 nmol／L的NSODN组和ASODN组p27表达量与正常对照组相比均无显著性差异，p＞0.05。
5．200 nmol／LASODN和NSODN转染细胞后：ASODN组Skp2蛋白表达量明显低于正常对照组，p＜0.05；ASODN组p27及p53蛋白表达量明显高于正常对照组，p＜0.05；NSODN组Skp2、p27及p53蛋白表达量与正常对照组相比均无显著性差异，p＞0.05。
结论：
1．Skp2反义寡核苷酸能使胃癌细胞Skp2 mRNA水平和蛋白表达水平均下降，表明在体外实验情况下，应用反义寡核苷酸技术能有效抑制目的基因的表达。
2．Skp2反义寡核苷酸能改变胃癌细胞生长状态和细胞周期，抑制细胞增殖，从而将在一定程度上逆转胃癌细胞扩散转移的恶性行为，可能对胃癌起到一定的治疗作用。
3．Skp2反义寡核苷酸能显著下调胃癌细胞内Skp2蛋白水平，使得泛素—蛋白酶体系统不能有效识别和降解细胞周期负性调控因子p27蛋白，高水平的p27抑制胃癌细胞生长和增殖；同时，Skp2蛋白水平的降低使细胞DNA合成受干扰，抑癌基因p53蛋白水平升高，可能使细胞发生凋亡。
Objective:
In 1978, antisense oligonucleotides technique was first used to inhibit the formation of the Rous sarcoma virus in chicken, and then this technique has been generally accepted as a gene intervention method which has superiority of high specificity and low toxicity. Antisense oligonucleotides are specifically designed to hybridise to the complementary RNA following the Watson-Crick binding rules, and inhibit expression of specific genes at the level of transcription or translation. Molecular biology and genetic engineering are developing so fast that shot nucleotides could be synthesized quickly and simplely. Antisense oligonucleotides technique can be used not only in the research of life science, but also in the treatment of malignant tumour, viral infection, inflammatory diseases and in reducing scar formation. Antisense oligonucleotides technology has entered into clinical trials in some countries whereas just started in China. Whether antisense oligonucleotides can be a new class of drugs for therapy still needs further researches, especially the mechanisms of how it target genes.
The key problem of antisense therapy for cancer is how to choose a target gene.
The common features of malignant cells are the abnormal functions of some specific genes which control cell proliferation, division or differentiation. So inhibition the translation of abnormal gene can reverse or eliminate the malignant behaviors of proliferation and metastasis in cancer cells. S-phase kinase-associated protain 2(Skp2) was first found in malignant cells, and played an important role in controlling cell proliferation. Skp2 could degradate some cell cycle regulators through ubiquitin-proteasome pathway to control the cell cycle progression. Meanwhile, Skp2 may be an oncogene, it has a high expression level in many malignant carcinomas and is close interrelated with maligant phenotypes and poor prognosis.
Gastric cancer is one of the most common gastrointestinal cancers and has the highest mortality rate among all malignant carcinomas. It is noticeable that the prevalence and mortality rates of gastric cancer in China are twice as high as the average of the world. Like other malignant carcinomas, gastric cancer is an acquired genopathy of multi-factor and multi-stage, including activation of proto-oncogene, inactivation of tumor suppressor gene, and abnormal proliferation at the cellular level. As a regulator of cell proliferation and a potential oncogene, Skp2 also has a high expression level and correlate with malignangt phenotypes in gastric carcinomas. So, Skp2 gene plays an important role in carcinogenesis and development of gastric cancer, and may be a target for therapy. In recent years, antisense oligonucleotides technique targeting cancer risk genes has become a prospective therapeutical strategy for gastric cancer. However, cancer treatment research by targeting Skp2 gene has just begun, and research of using Skp2 antisense oligonucleotides in the treatment of gastric cancer has not been reported. Therefore, antisense oligodeoxy-nucleotide (ASODN), which hybridises to the Skp2 mRNA nucleotides sequence, was used in the present study. The functions and mechanisms of Skp2 gene in controlling growth and proliferation of gastric cancer SGC-7901 cells were explored for providing preliminary information about mechanism of cancer treatment targeting Skp2 gene.
Methods:
According to the experiment design, different treatment methods were performed in three groups: normal control group, nonsense oligodeoxy-nucleotides(NSODN) treated group and antisense oligodeoxy-nucleotides(ASODN) treated group. The concentrations of transfection were 50 nmol/L, 200 nmol/L and 400 nmol/L. The Skp2 oligodeoxy-nucleotides were delivered into SGC-7901 cells using lipofectamine 2000 Reagent. The growth and proliferation of cells were observed with light microscopy, and MTT assay. The cell cycle was detected by flow cytometry. The total RNA of cells was extracted and the expression levels of Skp2 and p27 mRNA were detected by reverse transcription-polymerase chain reaction. The total protein of cells was extracted and the expression levels of Skp2 protein, p27 protein and p53 protein were detected by western-blot.
Results:
1. After transfected with 200 nmol/L Skp2 ASODN, the growth of SGC-7901 cells was obviously slowed down, unclear cell borders and loose cell junctions were observed, the characteristics of clonal growth in cancer cells were lost.
2. 200 nmol/L Skp2 ASODN significantly inhibited the proliferation of gastric cancer cells at 24, 48 and 72 h, p < 0.01. But after transfected with high concentration(400 nmol/L) of oligodeoxy-nucleotides for more than 48 h, both NSODN and ASOND had serious non-antisense inhibition effect, and there was no significant difference between both, p > 0.05.
3. The cell cycle of 200 nmol/L NSODN group cells was similar with that of normal control group, but the cell cycle of 200 nmol/L ASODN group cells was changed: cells in the G_0/G_1 phase were decreased slightly, and cells in the S phase were obviously increased compared with that of normal control group, p < 0.05.
4. At the concentration of 200 nmol/L, Skp2 mRNA level of NSODN group was not
changed compared with that of normal control group, p > 0.05; but Skp2 mRNA level of ASODN group was obviously lower than that of normal control group, p < 0.05; p27 mRNA levels of NSODN group and ASODN group were not changed compared with that of normal control group, p > 0.05.
5. After transfected with 200 nmol/L Skp2 ASODN and Skp2 NSODN, Skp2 protein level of ASODN group was obviously lower than that of normal control group, p < 0.05; both p27 and p53 protein levels of ASODN group were obviously higher than that of normal control group, p < 0.05. As expected, Skp2、p27 and p53 protein levels of NSODN group were not changed compared with that of normal control group, p > 0.05.
Conclusion:
1. After treatment with Skp2 ASODN, the decrease of both mRNA and protein levels of Skp2 indicated that application of antisense oligonucleotides technique could inhibit expression of target genes effectively in vitro.
2. Skp2 antisense oligodeoxy-nucleotides could change phenotype and cell cycle of gastric cancer cells, and the growth and proliferation of cells were inhibited. So it will partly reverse the malignant behaviors of proliferation and metastasis of gastric cancer cells, and promisingly have a certain therapeutic effect for gastric cancer.
3. Skp2 antisense oligodeoxy-nucleotides could significantly reduce Skp2 protein level of gastric cancer cells, it makes ubiquitin-proteasome system have an ineffective identification and degradation of negative cell-cycle regulatory protein p27, so high level of p27 protein inhibits the growth and proliferation rates of cells; meanwhile, low level of Skp2 protein interacts DNA replication, leads to high expression level of tumor suppressor gene p53, and may induce apoptosis.
反义寡核苷酸技术(antisense oligonucleotides technique)在1978年首次应用于抑制鸡肉瘤病毒的复制，此后逐渐成为公认的具有高度特异性和低毒性作用的基因干预手段。反义寡核苷酸能通过碱基配对原则与细胞内核酸特异结合形成杂交分子，从而在转录或翻译水平抑制特定基因表达。随着分子生物学和遗传工程的发展，现在已经能够快速、简易地合成较短的核苷酸片断，即寡核苷酸。此项技术不仅能应用于生命科学的研究，它在医学上的应用也越来越广泛，如治疗恶性肿瘤、病毒性疾病、炎症性疾病及减少疤痕的形成。尽管反义寡核苷酸技术在国外已进入临床实验治疗阶段，国内也已开始研究，但反义寡核苷酸要作为一类新型药物普遍应用于治疗有待于更深入的研究，尤其对其靶标基因作用机制的研究。
选择对肿瘤有治疗作用的特定目的基因靶标是反义寡核苷酸治疗的关键所在。癌变细胞的共同特征是与细胞增殖、分裂或分化有关的基因出现异常，因此应用反义寡核苷酸技术抑制异常表达的基因，能逆转或去除肿瘤细胞恶性增殖和扩散的行为。S期激酶相关蛋白2(S-phase kinase-associated protain 2，Skp2)是在恶变细胞中发现的细胞周期相关蛋白，它经泛素—蛋白酶体途径(ubiquitin-proteasome pathway，UPP)降解一些细胞周期调节因子，从而在细胞增殖调控中起重要作用。同时，Skp2有癌基因的潜能，在多种人类恶性肿瘤中高表达，并与肿瘤恶性程度及预后不良有密切联系。
胃癌是世界上最常见的消化道恶性肿瘤之一，其死亡率一直居恶性肿瘤之首，而我国胃癌患病及死亡率超过世界平均水平的两倍。胃癌和其它恶性肿瘤一样都是多因素、多阶段的获得性基因疾病，包括原癌基因的激活和抑癌基因的失活，并伴随着细胞增殖的异常。Skp2作为细胞增殖调控基因和潜在癌基因，它同样在胃癌中高表达并与胃癌的恶性程度相关。因此，Skp2基因在胃癌的发生、发展中有着重要的地位，有望成为胃癌治疗的靶点。近年来，针对胃癌风险基因的反义寡核苷酸治疗手段逐渐成为一种具有潜在价值的胃癌治疗手段。然而，针对Skp2基因的恶性肿瘤治疗研究刚刚起步，而采用Skp2反义寡核苷酸治疗胃癌的研究还未见报道。鉴于上述原因，本研究通过合成互补于Skp2 mRNA核苷酸序列的反义寡核苷酸(antisense oligodeoxy-nucleotides，ASODN)，研究Skp2基因在胃癌细胞SGC-7901生长和增殖中的作用及其可能机制，从而为针对Skp2基因的肿瘤治疗提供初步的理论依据。
方法：
根据实验设计，以细胞的不同处理因素分组：正常对照组，Skp2无义寡核苷酸(nonsense oligodeoxy-nucleotides，NSODN)处理组，以及Skp2反义寡核苷酸(antisense oligodeoxy-nucleotides，ASODN)处理组，转染浓度分为50 nmol／L，200 nmol／L和400 nmol／L。实验方法如下：采用阳离子脂质体Lipofectamine 2000 Reagent包裹Skp2寡核苷酸转染SGC-7901细胞；通过显微镜和MTT实验观察细胞的生长和增殖情况；应用流式细胞术检测细胞周期；提取细胞总RNA，应用逆转录聚合酶链反应检测细胞Skp2、p27 mRNA表达变化；提取细胞总蛋白，应用Western-blot法检测细胞Skp2蛋白、p27蛋白及p53蛋白表达变化。
结果：
1．转染200 nmol／L Skp2 ASODN后的SGC-7901细胞生长速度明显减慢，细胞边界不清，连接松散，失去克隆性生长特性。
2．200 nmol／L的ASODN在24 h、48 h、72 h等各个时间段上对胃癌细胞的增殖均有明显的抑制作用，p＜0.01。但在高浓度(400 nmol／L)、长时间(48 h以上)作用下的NSODN和ASODN对胃癌细胞均有较大的非反义性抑制效应，两者无显著性差异，p＞0.05。
3．200 nmol／L NSODN组细胞周期与正常对照组相比无明显变化，而200 nmol／LASODN组细胞周期发生改变：G_0／G_1期细胞所占比例略有降低趋势，而S期细胞所占比例却明显高于正常对照组细胞，p＜0.05。
4．200 nmol／L NSODN组Skp2 mRNA表达量与正常对照组相比无显著性差异，p＞0.05；200 nmol／L ASODN组Skp2 mRNA表达量明显低于正常对照组，p＜0.05；200 nmol／L的NSODN组和ASODN组p27表达量与正常对照组相比均无显著性差异，p＞0.05。
5．200 nmol／LASODN和NSODN转染细胞后：ASODN组Skp2蛋白表达量明显低于正常对照组，p＜0.05；ASODN组p27及p53蛋白表达量明显高于正常对照组，p＜0.05；NSODN组Skp2、p27及p53蛋白表达量与正常对照组相比均无显著性差异，p＞0.05。
结论：
1．Skp2反义寡核苷酸能使胃癌细胞Skp2 mRNA水平和蛋白表达水平均下降，表明在体外实验情况下，应用反义寡核苷酸技术能有效抑制目的基因的表达。
2．Skp2反义寡核苷酸能改变胃癌细胞生长状态和细胞周期，抑制细胞增殖，从而将在一定程度上逆转胃癌细胞扩散转移的恶性行为，可能对胃癌起到一定的治疗作用。
3．Skp2反义寡核苷酸能显著下调胃癌细胞内Skp2蛋白水平，使得泛素—蛋白酶体系统不能有效识别和降解细胞周期负性调控因子p27蛋白，高水平的p27抑制胃癌细胞生长和增殖；同时，Skp2蛋白水平的降低使细胞DNA合成受干扰，抑癌基因p53蛋白水平升高，可能使细胞发生凋亡。
Objective:
In 1978, antisense oligonucleotides technique was first used to inhibit the formation of the Rous sarcoma virus in chicken, and then this technique has been generally accepted as a gene intervention method which has superiority of high specificity and low toxicity. Antisense oligonucleotides are specifically designed to hybridise to the complementary RNA following the Watson-Crick binding rules, and inhibit expression of specific genes at the level of transcription or translation. Molecular biology and genetic engineering are developing so fast that shot nucleotides could be synthesized quickly and simplely. Antisense oligonucleotides technique can be used not only in the research of life science, but also in the treatment of malignant tumour, viral infection, inflammatory diseases and in reducing scar formation. Antisense oligonucleotides technology has entered into clinical trials in some countries whereas just started in China. Whether antisense oligonucleotides can be a new class of drugs for therapy still needs further researches, especially the mechanisms of how it target genes.
The key problem of antisense therapy for cancer is how to choose a target gene.
The common features of malignant cells are the abnormal functions of some specific genes which control cell proliferation, division or differentiation. So inhibition the translation of abnormal gene can reverse or eliminate the malignant behaviors of proliferation and metastasis in cancer cells. S-phase kinase-associated protain 2(Skp2) was first found in malignant cells, and played an important role in controlling cell proliferation. Skp2 could degradate some cell cycle regulators through ubiquitin-proteasome pathway to control the cell cycle progression. Meanwhile, Skp2 may be an oncogene, it has a high expression level in many malignant carcinomas and is close interrelated with maligant phenotypes and poor prognosis.
Gastric cancer is one of the most common gastrointestinal cancers and has the highest mortality rate among all malignant carcinomas. It is noticeable that the prevalence and mortality rates of gastric cancer in China are twice as high as the average of the world. Like other malignant carcinomas, gastric cancer is an acquired genopathy of multi-factor and multi-stage, including activation of proto-oncogene, inactivation of tumor suppressor gene, and abnormal proliferation at the cellular level. As a regulator of cell proliferation and a potential oncogene, Skp2 also has a high expression level and correlate with malignangt phenotypes in gastric carcinomas. So, Skp2 gene plays an important role in carcinogenesis and development of gastric cancer, and may be a target for therapy. In recent years, antisense oligonucleotides technique targeting cancer risk genes has become a prospective therapeutical strategy for gastric cancer. However, cancer treatment research by targeting Skp2 gene has just begun, and research of using Skp2 antisense oligonucleotides in the treatment of gastric cancer has not been reported. Therefore, antisense oligodeoxy-nucleotide (ASODN), which hybridises to the Skp2 mRNA nucleotides sequence, was used in the present study. The functions and mechanisms of Skp2 gene in controlling growth and proliferation of gastric cancer SGC-7901 cells were explored for providing preliminary information about mechanism of cancer treatment targeting Skp2 gene.
Methods:
According to the experiment design, different treatment methods were performed in three groups: normal control group, nonsense oligodeoxy-nucleotides(NSODN) treated group and antisense oligodeoxy-nucleotides(ASODN) treated group. The concentrations of transfection were 50 nmol/L, 200 nmol/L and 400 nmol/L. The Skp2 oligodeoxy-nucleotides were delivered into SGC-7901 cells using lipofectamine 2000 Reagent. The growth and proliferation of cells were observed with light microscopy, and MTT assay. The cell cycle was detected by flow cytometry. The total RNA of cells was extracted and the expression levels of Skp2 and p27 mRNA were detected by reverse transcription-polymerase chain reaction. The total protein of cells was extracted and the expression levels of Skp2 protein, p27 protein and p53 protein were detected by western-blot.
Results:
1. After transfected with 200 nmol/L Skp2 ASODN, the growth of SGC-7901 cells was obviously slowed down, unclear cell borders and loose cell junctions were observed, the characteristics of clonal growth in cancer cells were lost.
2. 200 nmol/L Skp2 ASODN significantly inhibited the proliferation of gastric cancer cells at 24, 48 and 72 h, p < 0.01. But after transfected with high concentration(400 nmol/L) of oligodeoxy-nucleotides for more than 48 h, both NSODN and ASOND had serious non-antisense inhibition effect, and there was no significant difference between both, p > 0.05.
3. The cell cycle of 200 nmol/L NSODN group cells was similar with that of normal control group, but the cell cycle of 200 nmol/L ASODN group cells was changed: cells in the G_0/G_1 phase were decreased slightly, and cells in the S phase were obviously increased compared with that of normal control group, p < 0.05.
4. At the concentration of 200 nmol/L, Skp2 mRNA level of NSODN group was not
changed compared with that of normal control group, p > 0.05; but Skp2 mRNA level of ASODN group was obviously lower than that of normal control group, p < 0.05; p27 mRNA levels of NSODN group and ASODN group were not changed compared with that of normal control group, p > 0.05.
5. After transfected with 200 nmol/L Skp2 ASODN and Skp2 NSODN, Skp2 protein level of ASODN group was obviously lower than that of normal control group, p < 0.05; both p27 and p53 protein levels of ASODN group were obviously higher than that of normal control group, p < 0.05. As expected, Skp2、p27 and p53 protein levels of NSODN group were not changed compared with that of normal control group, p > 0.05.
Conclusion:
1. After treatment with Skp2 ASODN, the decrease of both mRNA and protein levels of Skp2 indicated that application of antisense oligonucleotides technique could inhibit expression of target genes effectively in vitro.
2. Skp2 antisense oligodeoxy-nucleotides could change phenotype and cell cycle of gastric cancer cells, and the growth and proliferation of cells were inhibited. So it will partly reverse the malignant behaviors of proliferation and metastasis of gastric cancer cells, and promisingly have a certain therapeutic effect for gastric cancer.
3. Skp2 antisense oligodeoxy-nucleotides could significantly reduce Skp2 protein level of gastric cancer cells, it makes ubiquitin-proteasome system have an ineffective identification and degradation of negative cell-cycle regulatory protein p27, so high level of p27 protein inhibits the growth and proliferation rates of cells; meanwhile, low level of Skp2 protein interacts DNA replication, leads to high expression level of tumor suppressor gene p53, and may induce apoptosis.
引文
邓超，包菊平．反义寡核苷酸技术的进展及在医学中的应用[J]．海南医学院学报，2003，9(6)：375-377
罗晓萍．反义寡核苷酸技术的进展及应用[J]．上海第二医科大学学报，2000，20(2)：185-187．
王小中，冯文莉，刘兴等．SKP2反义寡核苷酸对K562细胞生长和增殖的影响[J]．癌症，2003，22(9)：948
张宏，苏秀兰．抑癌基因p53及细胞周期素cyclinD1与胃癌的关系[J]．现代肿瘤医学，2007，15(1)：134-136
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罗晓萍．反义寡核苷酸技术的进展及应用[J]．上海第二医科大学学报，2000，20(2)：185-187．
王小中，冯文莉，刘兴等．SKP2反义寡核苷酸对K562细胞生长和增殖的影响[J]．癌症，2003，22(9)：948
张宏，苏秀兰．抑癌基因p53及细胞周期素cyclinD1与胃癌的关系[J]．现代肿瘤医学，2007，15(1)：134-136
Bordier, B., Perala-Heape, M., Degols, G., Lebleu, B., Litvak, S., Sarih-Cottin, L., and Helene, C. (1995). Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells. Proc NatlAcad Sci USA 92, 9383-9387.
Carrano, A. C., Eytan, E., Hershko, A., and Pagano, M. (1999). SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1, 193-199.
Cowsert, L. M. (1997). In vitro and in vivo activity of antisense inhibitors of ras: potential for clinical development. Anticancer Drug Des 12, 359-371.
Demetrick, D. J., Zhang, H., and Beach, D. H. (1996). Chromosomal mapping of the genes for the human CDK2/cyclin A-associated proteins p19 (SKP1A and SKP1B) and p45 (SKP2). Cytogenet Cell Genet 73, 104-107.
Fero, M. L., Rivkin, M., Tasch, M., Porter, P., Carow, C. E., Firpo, E., Polyak, K., Tsai, L. H., Broudy, V., Perlmutter, R. M., Kaushansky, K., and Roberts, J. M. (1996). A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85, 733-744.
Galizia, G., Ferraraccio, F., Lieto, E., Orditura, M., Castellano, P., Imperatore, V., La Manna, G., Pinto, M., Ciardiello, F., La Mura, A., and De Vita, F. (2006). p27 downregulation and metallothionein overexpression in gastric cancer patients are associated with a poor survival rate. J Surg Oncol 93, 241-252.
Ganoth, D., Bornstein, G, Ko, T. K., Larsen, B., Tyers, M., Pagano, M., and Hershko, A. (2001). The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nat Cell Biol 3, 321-324.
Hara, T., Kamura, T., Nakayama, K., Oshikawa, K., and Hatakeyama, S. (2001). Degradation of p27(Kip1) at the G(0)-G(1) transition mediated by a Skp2-independent ubiquitination pathway. J Biol Chem 276, 48937-48943.
Hatta, T., Nakagawa, Y, Takai, K., Nakada, S., Yokota, T., and Takaku, H. (1995). Inhibition of influenza virus RNA polymerase and nucleoprotein of gene expression by antisense oligonucleotides. Nucleic Acids Symp Ser, 129-130.
Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem 258, 8206-8214.
Honjo, S., Kase, S., Osaki, M., Ardyanto, T. D., Kaibara, N., and Ito, H. (2005). COX-2 correlates with F-box protein, Skp2 expression and prognosis in human gastric carcinoma. Int J Oncol 26, 353-360.
Kamata, Y, Watanabe, J., Nishimura, Y., Arai, T., Kawaguchi, M., Hattori, M., Obokata, A., and Kuramoto, H. (2005). High expression of skp2 correlates with poor prognosis in endometrial endometrioid adenocarcinoma. J Cancer Res Clin Oncol 131, 591-596.
Katayose, Y, Kim, M., Rakkar, A. N., Li, Z., Cowan, K. H., and Seth, P. (1997). Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor p27. Cancer Res 57, 5441-5445.
Katner, A. L., Gootam, P., Hoang, Q. B., Gnarra, J. R., and Rayford, W. (2002). A recombinant adenovirus expressing p7(Kip1) induces cell cycle arrest and apoptosis in human 786-0 renal carcinoma cells. J Urol 168, 766-773.
Kausch, I., Ewerdwalbesloh, N., Jiang, H., Doehn, C., Kruger, S., and Jocham, D. (2005). [Combination treatment with antisense oligonucleotides and chemotherapy in vitro]. Urologe A 44, 909-914.
Kravtsov, V. G, Shakhmurov, M. G, Sukmanov, O. V., Zairat'iants, O. V., and Shirin, N. I. (2006). [Expression of cycline-dependent kinase p27 in the low differentiated gastric adenocarcinoma]. Arkh Patol 68,14-16.
Latres, E., Chiarle, R., Schulman, B. A., Pavletich, N. P., Pellicer, A., Inghirami, G, and Pagano, M. (2001). Role of the F-box protein Skp2 in lymphomagenesis. Proc Natl Acad Sci U S A 98, 2515-2520.
Li, J. Q., Wu, F., Masaki, T., Kubo, A., Fujita, J., Dixon, D. A., Beauchamp, R. D., Ishida, T., Kuriyama, S., and Imaida, K. (2004). Correlation of Skp2 with carcinogenesis, invasion, metastasis, and prognosis in colorectal tumors. Int J Oncol 25, 87-95.
Ma, X. M., Liu, J. H., Guo, J. W., Liu, Y., and Zuo, L. F. (2006). [Correlation of Skp2 expression in gastric carcinoma to expression of P27 and PTEN]. Ai Zheng 25, 56-61.
Ma, X. M., Liu, Y., Guo, J. W., Liu, J. H., and Zuo, L. F. (2005). Relation of overexpression of S phase kinase-associated protein 2 with reduced expression of p27 and PTEN in human gastric carcinoma. World J Gastroenterol 11, 6716-6721.
Masuda, T. A., Inoue, H., Sonoda, H., Mine, S., Yoshikawa, Y, Nakayama, K., and Mori, M. (2002). Clinical and biological significance of S-phase kinase-associated protein 2 (Skp2) gene expression in gastric carcinoma: modulation of malignant phenotype by Skp2 overexpression, possibly via p27 proteolysis. Cancer Res 62, 3819-3825.
Monia, B. P., Johnston, J. F., Geiger, T., Muller, M., and Fabbro, D. (1996). Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase. Nat Med 2, 668-675.
Nakayama, K. I., and Nakayama, K. (2005). Regulation of the cell cycle by SCF-type ubiquitin ligases. Semin Cell Dev Biol 16, 323-333.
Nakayama, K. I., and Nakayama, K. (2006). Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer 6, 369-381.
Signoretti, S., Di Marcotullio, L., Richardson, A., Ramaswamy, S., Isaac, B., Rue, M., Monti, F., Loda, M., and Pagano, M. (2002). Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer. J Clin Invest 110, 633-641.
Stahel, R. A., and Zangemeister-Wittke, U. (2003). Antisense oligonucleotides for cancer therapy-an overview. Lung Cancer 41 Suppl 1, S81-88.
Tamm, I. (2006). Antisense therapy in malignant diseases: status quo and quo vadis? Clin Sci (Lond) 110, 427-442.
Tamm, I., Dorken, B., and Hartmann, G. (2001). Antisense therapy in oncology: new hope for an old idea? Lancet 358, 489-497.
Tsukamoto, S., Sugio, K., Sakada, T., Ushijima, C, Yamazaki, K., and Sugimachi, K. (2001). Reduced expression of cell-cycle regulator p27(Kip1) correlates with a shortened survival in non-small cell lung cancer. Lung Cancer 34, 83-90.
Webb, A., Cunningham, D., Cotter, F., Clarke, P. A., di Stefano, F., Ross, P., Corbo, M., and Dziewanowska, Z. (1997). BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 349, 1137-1141.
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