let-7a miRNA治疗胃癌的实验性研究及机制探讨
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摘要
肿瘤是一种多步骤基因组改变的疾病。至目前,对肿瘤基因组的研究主要关注的是蛋白编码基因,而对有功能的非编码序列之改变的认识很缺乏。miRNA是近年发现的一类内源性的、非编码RNA分子,长约22个核苷酸,调控转录后的翻译,并已被证实参与许多诸如细胞分化、增值等过程。据估计,在人类基因组中约有1000个miRNA,尽管要去确切地认定某个miRNA的目标基因还有困难,用计算机预测人类1／3的蛋白编码基因受miRNA调控,这提示miRNA与人类的很多疾病相关,包括肿瘤。的确,随着近五年的深入研究,miRNA对肿瘤基因的调节作用已成为肿瘤生物学一新领域。也有报道,miRNA可能是比蛋白编码基因更为有力的指标用以区分正常与肿瘤组织。尤其值得关注的是lethal-7(let-7),于九十年代在人和鼠中被发现。也有报道,1et-7可作为诸如肺癌、大肠癌等疾病中的一个新的肿瘤抑制基因候选者。
胃癌(Gastric carcinoma)是危害人类健康最常见的恶性肿瘤,据WHO报告,全世界每年约有765000人死于胃癌,每年新发病例925000,患者数达3715000。我国胃癌死亡率与其他国家相比处于较高水平达25.16／10万,占恶性肿瘤死亡之首为23%。非典型性增生是公认的癌前病变,慢性胃炎-胃黏膜萎缩-肠化生-不典型增生-胃癌这一演进模式已为国内外多数学者认同。在本研究中用原位杂交检测let-7a在胃粘膜、慢性萎缩性胃炎、胃癌中的表达。实验还显示了慢病毒携带的短发夹RNA能特异地、稳定地、有效地在体内、外表达let-7a,并能快速、有效地用于基因功能分析。最后以蛋白组学技术分析了let-7a过表达后胃癌细胞蛋白表达的改变,以发现let-7a影响胃癌细胞SGC-7901的相关功能蛋白,探讨其在胃癌细胞中产生作用的分子机制。本实验为.let-7a治疗胃癌提供新的实验和理论依据,指导进一步的临床应用。
实验内容包括以下五部分：
第一部分let-7a在胃粘膜、慢性萎缩胃炎、胃癌组织中的表达及其意义
目的：研究let-7a在胃粘膜、慢性萎缩性胃炎、胃癌组织中的表达,并分析其表达强度与胃粘膜癌变演进的关系。
方法：原位杂交法检测组织芯片上11例正常胃粘膜、17例慢性萎缩性胃炎、52胃癌组织中let-7a的表达,分析三者阳性率及表达强度差异性。
结果：let-7a在正常胃粘膜中(10例阳性)、慢性萎缩性胃炎(15例阳性)、胃癌组织(43例阳性)中的阳性表达率差异无显著性(P>0.05),而表达强度随着胃粘膜癌变的演进逐渐减弱,采用有序分组资料的线性趋势检验(Chi-Square and Correlations Test)分析(P<0.05)。
结论：let-7a表达的减弱与胃粘膜癌变演进过程相关,let-7a在胃癌的发生过程中可能起重要作用。第二部分let-7a慢病毒载体的构建与鉴定
目的：构建人let-7a基因慢病毒载体,用于后续试验。
方法：扩增出let-7a基因的全长,构建Pwpxl-MOD2-let-7a真核表达载体。将四质粒将Pwpxl-MOD2-let-7a、pRsv-REV、pMDlg-pRRE、pMD2G共转染包装细胞293 T,包装产生慢病毒。经超速离心收集病毒颗粒,转导HEK293 T细胞,以流式细胞仪鉴定的EGFP的表达,计算出慢病毒的功能滴度。
结果：成功构建Pwpxl-MOD2-let-7a真核表达载体,四质粒PwpxMOD2-let-7a、pRsv-REV、pMDLg-pRRE和pMD2G共转染HEK 293 T细胞后包装产生病毒。
结论：构建成let-7a表达载体Pwpxl-MOD2-let-7a;成功包装出人let-7a基因慢病毒载体,并包装出高滴度病毒：为通过let-7a治疗恶性肿瘤的研究奠定了基础。
第三部分let-7a对胃癌细胞SGC-7901生物学行为影响的体外实验
目的：筛选稳定过表达let-7a的胃癌细胞,观察let-7a在体外实验中在对胃癌细胞的影响。
方法：将let-7a用重组慢病毒转导入人胃癌SGC-7901细胞,Real-Time RT-PCR的方法测定目的基因分子拷贝数。通过细胞增殖、黏附、侵袭试验及细胞周期的检测等来观察let-7a对胃癌细胞的影响。
结果：(1)let-7a能被重组慢病毒高效地转导入靶细胞,转导效率几乎达100%,同时达到稳定的表达,荧光定量RT-PCR能检测到let-7a在靶细胞中的高表达；(2)在SGC-7901细胞株,转导入let-7a后的细胞的增殖明显的比亲代细胞慢(P<0.01)；(3)细胞黏附试验提示let-7a能明显抑制胃癌细胞的黏附能力；(4)细胞侵袭力试验提示let-7a能明显抑制胃癌细胞的侵袭能力；(5)用流式细胞仪对细胞周期分析显示let-7a对SGC-901的细胞周期有明显的滞留作用并(P<0.01)
结论：慢病毒载体作为转基因的工具,能高效转导和稳定表达let-7a；let-7a能够抑制SGC-7901细胞的增殖、黏附、侵袭能力和阻滞细胞周期进展,提示let-7a能有效的抑制胃癌细胞。
第四部分let-7a对裸鼠人胃癌皮下移植瘤生长的影响
目的：构建裸鼠的人胃癌皮下移植瘤模型,观察let-7a裸鼠胃癌皮下瘤对生长情况影响。
方法：将稳定过表达let-7a的胃癌细胞株注入裸鼠皮下,建立裸鼠人胃癌移植瘤模型,并用亲代、导入空载体的SGC-7901细胞组作对照,6周后比较三组形成的皮下移植瘤体积及重量差异。
结果：成功构建裸鼠人胃癌皮下移植瘤模型：6周后取下三组裸鼠皮下移植瘤率,实验组、空载体组、亲代细胞组的平均体积分别为：1100±50mm3、1300±68mm3、1340±55mm3。平均重量分别为：1250±20mg、1330±32mg、1350±31mg。实验组与亲代细胞组的平均体积、重量存在显著差异(P<0.05),但空载体组与亲代细胞组的平均体积、重量的差异无显著性(P>0.05)。
结论：过表达let-7a在体内可抑制胃癌生长,证实其在胃癌具有肿瘤基因治疗的潜力。
第五部分应用蛋白组学技术分离鉴定let-7a影响胃癌SGC-7901细胞功能的相关蛋白
目的：比较胃癌细胞SGC-7901、SGC-7901／EV、SGC-7901／let-7a之间差异表达的蛋白质,筛选let-7a影响SGC-7901细胞生物学行为的相关的功能蛋白,丰富let-7a对SGC-7901细胞生物学行为影响机制的理论。
方法：利用固相pH梯度双向凝胶电泳技术,分离胃癌细胞SGC-7901、SGC-7901／EV、SGC-7901／let-7a的总蛋白,建立SGC-7901、SGC-7901／EV、SGC-7901／let-7a的蛋白表达谱：用Image Master 2D Elite4.01图像分析软件,比较这三种组织中蛋白的表达差异,对差异表达蛋白进行质谱分析和Swiss-prot数据库搜索。Western blot分析部分蛋白质如细胞周期素依赖性激酶抑制蛋白1、Skp2蛋白和纤维连接蛋白在三株细胞系中的差异表达水平。
结果：建立了胃癌细胞SGC-7901、SGC-7901／EV、SGC-7901／let-7a的蛋白表达图谱,用扫描仪扫描后经Image Master 2 D Elite 4.01软件分析发现,蛋白质点数分别为1043、1046、1026,获得的大部分蛋白质的表达模式是相同的。其中SGC-7901、SGC-7901／EV两张胶中蛋白基本相同。在SGC-7901／EV、SGC-7901/let-7a胶中,可以在预期等电点和分子量大小处找到GFP蛋白,并经质谱验证。SGC-7901／let-7a细胞和其他两组细胞相比,有较多不同,差异点79个：其中11个蛋白点仅在SGC-7901／let-7a细胞中表达,31个蛋白点在SGC-7901／let-7a细胞中表达上调,21个点在SGC-7901／let-7a细胞中表达缺失,16个点在SGC-7901／let-7a细胞中为表达下调(表达量低于其他的5倍以上)。通过筛选,选择差异比较显著的6个点仅在在SGC-7901／let-7a细胞中表达和4个SGC-7901／let-7a细胞中缺失的点打质谱分析。经质谱分析鉴定出10种差异蛋白。其中：抗氧化蛋白2、胰岛素样生长因子结合蛋白2、二硫化蛋白异构酶A2、四氢叶酸合成酶、细胞周期素依赖性激酶抑制蛋白1、Rho-GTPsae激活蛋白4等6种蛋白质在SGC-7901/let-7a细胞中高表达；Skp2蛋白、血小板黏附蛋白CD41、纤维连接蛋白、Cks1蛋白等4种蛋白质在SGC-7901/let-7a细胞中低表达。通过搜索Swiss-port数据库,明确了这些蛋白的基本生物学信息,它们可能与细胞周期调控、肿瘤的侵袭转移及细胞代谢有关。通过Western blot分析,验证了SGC-7901、SGC-7901／EV、SGC-7901/let-7a三组细胞中的细胞周期素依赖性激酶抑制蛋白1、Skp2蛋白和纤维连接蛋白表达水平差异。
结论：胃癌细胞SGC-7901、SGC-7901／EV、SGC-7901／let-7a之间存在多种差异表达的蛋白,这些蛋白可能通过影响细胞周期调控、肿瘤的侵袭与转移及细胞代谢等,与SGC-7901细胞的生物学行为密切相关。对这些蛋白的研究探讨有助于丰富let-7a对SGC-7901细胞生物学行为影响机制的理论。
Cancer is a multi-step disease involving dynamic changes in the genome. However, studies on cancer genome so far have focused most heavily on protein-coding genes, and our knowledge on alterations of the functional noncoding sequences in cancer is largely absent. miRNA are a recently discovered class of short noncoding RNA genes that act posttranscriptionally as negative regulators of gene expression. A large body of research shows that animal miRNAs play fundamental roles in many biological processes, such as cell differentiation and cell proliferation. About 1,000 miRNA genes are thought to be encoded in the human genome. Although it is still difficult to identify accurately individual miRNA/target interactions, computational predictions of miRNA target genes indicate that as many as one third of all human protein-encoding genes may be regulated by miRNAs. This suggests that miRNAs could be involved in a wide variety of human diseases, including cancer. Indeed, as a result of intense research within the last 5 years, miRNA-mediated regulation of tumorigenesis is emerging as a new paradigm in the field of cancer biology. It has also been reported that these miRNA signatures may be a more robust tool than expression patterns of protein-encoding genes for distinguishing normal from tumor tissues. One particular miRNA, let-7, was discovered in the early 1990's humans and mice. It has been reported that let-7 as a novel candidate of tumor suppressor gene involving several cancers such as lung carcinoma and colon carcinoma.
Gastric carcinoma, which is the second most prevalent cancer, is a complex, inadequately understood, and often fatal disease when not detected at early stages. Atypical hyperplasia is generally accepted as a precancerous lesion. Cancerization progression from normal gastric mucosa, chronic atrophic gastritis to gastric carcinoma has been well acknowledged. In this study, the expression of let-7a in normal gastric tissue, chronic atrophic gastritis (atypical hyperplasia)and gastric carcinoma was detected by in situ hybridization. It has been demonstrated that the lentivirus-delivered short hairpin RNAs (shRNAs) are capable of specific, highly stable and functional overexpression of let-7a gene expression both in vitro and in vivo, enabling rapid and efficient analysis of gene function. Then,analysis of different Proteins expressed in SGC-7901、SGC-7901/EV and SGC-7901/let-7a Cells by Proteomics was performed to explore the mechanism of let-7a and provide a new experimental and theoretical basis gene therapy for gastric cancer to guide further clinical application.
Including the following five parts:
Part 1 Study of in situ Expression of let-7a in The Gastric Mucosa Cancerization Tissue Array and Its Clinical Significance
Objective To investigate the expression profile of novel candidate of tumor suppressor gene let-7a in gastric carcinoma, chronic atrophic gastritis (atypical hyperplasia) and normal gastric tissue, analyze the correlation between reduced let-7a and progression of gastric mucosa cancerization.
Methods Human gastric carcinoma, chronic atrophic gastritis (atypical hyperplasia) and normal gastric tissure microarrays were constructed previously and tissue microarays combined with in situ hybridization were used to detect the expression of let-7a.
Results Results showed that there was no significant difference of positive rates of let-7a among normal gastric tissue, chronic atrophic gastritis and gastric carcinoma (P>0.05).However, the degree of let-7a expression in the groups are closely related to the progression of gastric mucosa cancerization (P<0.05).
Conclusions The results suggested that the gastric carcinoma had relatively lowered expression of let-7a. Reduced let-7a may be a fundamental factor in the formation of gastric carcinoma.
Part 2 Construction and identification of the lentiviral Vector of human let-7a gene
Objective To construct a lentiviral Vector of human let-7a gene,and use it in the following experiences.
Methods Pwpxl-MOD2-let-7a eukaryotic expression plasmid was constructed by DNA recombinant method. Pwpxl-MOD2-let-7a was constructed along with pRsv-REV、pMD1g-pRRE、pMD2G into HEK 293 T to package lentivirus particles. According to the EGFP expression, the functional titer was determined by FCM after transduction into HEK 293 T cells.
Results The eukaryotic expression vector Pwpxl-MOD2-let-7a has been successfully constructed. After cotransfection, lentiviral vector can be Packaged in HEK 293 T cell and high functional titer was detected.
Conclusions Lentivirus vector of human let-7a gene has been constructed, which is the essential building block required for the let-7a related gene therapy research.
Part 3 in vitro Study on the Effect of let-7a in Human Gastric Carcinoma Cell Line
Objective To study the effects of the let-7a in human gastric carcinoma cell line by constructing a gastric cancer cell line SGC-7901 stably overexpressing let-7a gene.
Methods The let-7a was transferred to the human gastric carcinoma cell lines SGC-7901 by recombinant lentiviruses which carrying let-7a. The infected cells were quantified by Real-Time RT-PCR for the expression level of let-7a, then characterized by cell proliferation assays, cell adhesion assays and cell invasion assays. Cell cycle was analysised by flow cytometry (FCM).
Results (1) The efficiency of infection of the cell lines using the lentiviral vectors was almost 100 % allowing the stable expression of let-7a in nearly all cells. Over expression of let-7a was confirmed by quantitative Real-time PCR analysis. (2) SGC-7901 cells overexpressing let-7a grew slowly compared to SGC-7901 parental cells or cell transfected with empty vector.(P<0.01). (3)The adhesion ability of SGC-7901 cells overexpressing let-7a reduced when compared to their parental or cell transfected with empty vector. (4) Cell invasion assays indicated that let-7a can decrease the capability of SGC-7901. (5)Flow cytometry based cell cycle assays showed that overexpression of let-7a can arrest SGC-7901 cells (P< 0.01).
Conclusion The use of lentiviral vectors allowed the efficient transfection of let-7a in nearly 100 % of target cells. Overxpression of the let-7a resulted in a decrease of cell proliferation, adhesion, invasion and arrest cell cycle progression in SGC-7901 cells. It suggests that as a novel tumor suppressor gene let-7a can effectively inhibit gastric carcinoma.
Part 4 Lentiviral vector-mediated up-regulation of let-7a therapy for human gastric carcinoma xenogeneic graft in nude mouse subcutaneous
Objective By forming subcutaneous xenograft tumors in nude mouse to observe affection of let-7a on human gastric carcinoma.
Method Gastric carcinoma cells SGC-7901 stably overexpressing let-7a(SGC-7901/let-7a) were injected in nude mouse subcutaneous. SGC-7901/EV and parental SGC-7901 cells were used as the controls. After 6 weeks of observation, the weight and value of xenografts among the three groups were surveyed.
Result The xenografts were successfully formed in the three groups. On the 42th day after inoculation all the mice were sacrificed. The tumor masses were weighted and their volumes were measured. The average tumor weight of SGC-7901/let-7a, SGC-7901/NS and parental SGC-7901 were 1250±20mg、1330±32mg、1350±31mg respectively. Their average tumor volume were 1100±50mm3、1300±68mm3、1340± 55mm3.The tumor weight and volume of SGC-7901/let-7a were significantly lower than SGC-7901/NS and parental SGC-7901(P< 0.05),while there is no significant difference between the laters (P> 0.05).
Conclusion Overexpression of let-7a can inhibit tumor growth of transplanted human gastric cancer in nude mice and has a curative effect on gastric carcinoma as genetic therapy.
Part 5 Analysis of different proteins expression in SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells by proteomics
Objective In order to better understand the basic mechanisms of let-7a influncing gastric carcinoma, the different protein expression were investigated in SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells.
Mathods The total proteins of SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells were separated by immobilized pH gradient (IPG)-based two-dimensional gel electrophorsis (2-DE),the 2-DE maps were established for SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells respectively. The different expression protein spots were analyzed with Image Master 2D Elite4.01 analysis software. The peptide mass fingerprinting of different expressed proteins were verified by MALDI-TOF-MS. The function of the different expressed proteins were identified through swiss-port database searching. Western blot analysis was used to confirm the differential expression levels of the Partial Proteins.
Rsult We established the 2-DE maps of SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells respectively.79 different protein spots were found by image software. Among the total,6 overexpressed proteins and 4 underexpressed proteins of SGC-7901/let-7a cells were recognized with MALDI-TOF MS. The biological information of these proteins was identified through Swiss-port database search. The overexpressed proteins including Antioxidant protein 2, Insulin-like growth factor binding protein 2, Protein disulfide isomerase A2, C-1-tetrahydrofolate synthase, Cyclin-dependent kinase inhibitorl and Rho-GTPase activating protein 4 so on. The underexpressed proteins were consisted of S-phase kinase-associated protein 2, Platelet membrane glycoprotein, Fibronectin and Cksl protein. Some of those proteins were related to cell cycle control, cellular metabolism invasion and metastasis of tumor. The differential expression levels of the Partial Proteins (Cyclin-dependent kinase inhibitorl, S-phase kinase-associated protein 2, Fibronectin) were verified by western blot analysis.
Conclusion Many different expressed proteins among SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells were identified. These different expressed proteins perhaps have closed relation to the function of let-7a in genesis and development of gastric carcinoma through influence of the cell cycle control, invasion, metastasis and cellular metabolism. Investigating these proteins is helpful to explore the basic mechanisms of let-7a in gastric carcinoma.We could better understand the function of let-7a in gastric carcinoma and enrich its theory.
胃癌(Gastric carcinoma)是危害人类健康最常见的恶性肿瘤,据WHO报告,全世界每年约有765000人死于胃癌,每年新发病例925000,患者数达3715000。我国胃癌死亡率与其他国家相比处于较高水平达25.16／10万,占恶性肿瘤死亡之首为23%。非典型性增生是公认的癌前病变,慢性胃炎-胃黏膜萎缩-肠化生-不典型增生-胃癌这一演进模式已为国内外多数学者认同。在本研究中用原位杂交检测let-7a在胃粘膜、慢性萎缩性胃炎、胃癌中的表达。实验还显示了慢病毒携带的短发夹RNA能特异地、稳定地、有效地在体内、外表达let-7a,并能快速、有效地用于基因功能分析。最后以蛋白组学技术分析了let-7a过表达后胃癌细胞蛋白表达的改变,以发现let-7a影响胃癌细胞SGC-7901的相关功能蛋白,探讨其在胃癌细胞中产生作用的分子机制。本实验为.let-7a治疗胃癌提供新的实验和理论依据,指导进一步的临床应用。
实验内容包括以下五部分：
第一部分let-7a在胃粘膜、慢性萎缩胃炎、胃癌组织中的表达及其意义
目的：研究let-7a在胃粘膜、慢性萎缩性胃炎、胃癌组织中的表达,并分析其表达强度与胃粘膜癌变演进的关系。
方法：原位杂交法检测组织芯片上11例正常胃粘膜、17例慢性萎缩性胃炎、52胃癌组织中let-7a的表达,分析三者阳性率及表达强度差异性。
结果：let-7a在正常胃粘膜中(10例阳性)、慢性萎缩性胃炎(15例阳性)、胃癌组织(43例阳性)中的阳性表达率差异无显著性(P>0.05),而表达强度随着胃粘膜癌变的演进逐渐减弱,采用有序分组资料的线性趋势检验(Chi-Square and Correlations Test)分析(P<0.05)。
结论：let-7a表达的减弱与胃粘膜癌变演进过程相关,let-7a在胃癌的发生过程中可能起重要作用。第二部分let-7a慢病毒载体的构建与鉴定
目的：构建人let-7a基因慢病毒载体,用于后续试验。
方法：扩增出let-7a基因的全长,构建Pwpxl-MOD2-let-7a真核表达载体。将四质粒将Pwpxl-MOD2-let-7a、pRsv-REV、pMDlg-pRRE、pMD2G共转染包装细胞293 T,包装产生慢病毒。经超速离心收集病毒颗粒,转导HEK293 T细胞,以流式细胞仪鉴定的EGFP的表达,计算出慢病毒的功能滴度。
结果：成功构建Pwpxl-MOD2-let-7a真核表达载体,四质粒PwpxMOD2-let-7a、pRsv-REV、pMDLg-pRRE和pMD2G共转染HEK 293 T细胞后包装产生病毒。
结论：构建成let-7a表达载体Pwpxl-MOD2-let-7a;成功包装出人let-7a基因慢病毒载体,并包装出高滴度病毒：为通过let-7a治疗恶性肿瘤的研究奠定了基础。
第三部分let-7a对胃癌细胞SGC-7901生物学行为影响的体外实验
目的：筛选稳定过表达let-7a的胃癌细胞,观察let-7a在体外实验中在对胃癌细胞的影响。
方法：将let-7a用重组慢病毒转导入人胃癌SGC-7901细胞,Real-Time RT-PCR的方法测定目的基因分子拷贝数。通过细胞增殖、黏附、侵袭试验及细胞周期的检测等来观察let-7a对胃癌细胞的影响。
结果：(1)let-7a能被重组慢病毒高效地转导入靶细胞,转导效率几乎达100%,同时达到稳定的表达,荧光定量RT-PCR能检测到let-7a在靶细胞中的高表达；(2)在SGC-7901细胞株,转导入let-7a后的细胞的增殖明显的比亲代细胞慢(P<0.01)；(3)细胞黏附试验提示let-7a能明显抑制胃癌细胞的黏附能力；(4)细胞侵袭力试验提示let-7a能明显抑制胃癌细胞的侵袭能力；(5)用流式细胞仪对细胞周期分析显示let-7a对SGC-901的细胞周期有明显的滞留作用并(P<0.01)
结论：慢病毒载体作为转基因的工具,能高效转导和稳定表达let-7a；let-7a能够抑制SGC-7901细胞的增殖、黏附、侵袭能力和阻滞细胞周期进展,提示let-7a能有效的抑制胃癌细胞。
第四部分let-7a对裸鼠人胃癌皮下移植瘤生长的影响
目的：构建裸鼠的人胃癌皮下移植瘤模型,观察let-7a裸鼠胃癌皮下瘤对生长情况影响。
方法：将稳定过表达let-7a的胃癌细胞株注入裸鼠皮下,建立裸鼠人胃癌移植瘤模型,并用亲代、导入空载体的SGC-7901细胞组作对照,6周后比较三组形成的皮下移植瘤体积及重量差异。
结果：成功构建裸鼠人胃癌皮下移植瘤模型：6周后取下三组裸鼠皮下移植瘤率,实验组、空载体组、亲代细胞组的平均体积分别为：1100±50mm3、1300±68mm3、1340±55mm3。平均重量分别为：1250±20mg、1330±32mg、1350±31mg。实验组与亲代细胞组的平均体积、重量存在显著差异(P<0.05),但空载体组与亲代细胞组的平均体积、重量的差异无显著性(P>0.05)。
结论：过表达let-7a在体内可抑制胃癌生长,证实其在胃癌具有肿瘤基因治疗的潜力。
第五部分应用蛋白组学技术分离鉴定let-7a影响胃癌SGC-7901细胞功能的相关蛋白
目的：比较胃癌细胞SGC-7901、SGC-7901／EV、SGC-7901／let-7a之间差异表达的蛋白质,筛选let-7a影响SGC-7901细胞生物学行为的相关的功能蛋白,丰富let-7a对SGC-7901细胞生物学行为影响机制的理论。
方法：利用固相pH梯度双向凝胶电泳技术,分离胃癌细胞SGC-7901、SGC-7901／EV、SGC-7901／let-7a的总蛋白,建立SGC-7901、SGC-7901／EV、SGC-7901／let-7a的蛋白表达谱：用Image Master 2D Elite4.01图像分析软件,比较这三种组织中蛋白的表达差异,对差异表达蛋白进行质谱分析和Swiss-prot数据库搜索。Western blot分析部分蛋白质如细胞周期素依赖性激酶抑制蛋白1、Skp2蛋白和纤维连接蛋白在三株细胞系中的差异表达水平。
结果：建立了胃癌细胞SGC-7901、SGC-7901／EV、SGC-7901／let-7a的蛋白表达图谱,用扫描仪扫描后经Image Master 2 D Elite 4.01软件分析发现,蛋白质点数分别为1043、1046、1026,获得的大部分蛋白质的表达模式是相同的。其中SGC-7901、SGC-7901／EV两张胶中蛋白基本相同。在SGC-7901／EV、SGC-7901/let-7a胶中,可以在预期等电点和分子量大小处找到GFP蛋白,并经质谱验证。SGC-7901／let-7a细胞和其他两组细胞相比,有较多不同,差异点79个：其中11个蛋白点仅在SGC-7901／let-7a细胞中表达,31个蛋白点在SGC-7901／let-7a细胞中表达上调,21个点在SGC-7901／let-7a细胞中表达缺失,16个点在SGC-7901／let-7a细胞中为表达下调(表达量低于其他的5倍以上)。通过筛选,选择差异比较显著的6个点仅在在SGC-7901／let-7a细胞中表达和4个SGC-7901／let-7a细胞中缺失的点打质谱分析。经质谱分析鉴定出10种差异蛋白。其中：抗氧化蛋白2、胰岛素样生长因子结合蛋白2、二硫化蛋白异构酶A2、四氢叶酸合成酶、细胞周期素依赖性激酶抑制蛋白1、Rho-GTPsae激活蛋白4等6种蛋白质在SGC-7901/let-7a细胞中高表达；Skp2蛋白、血小板黏附蛋白CD41、纤维连接蛋白、Cks1蛋白等4种蛋白质在SGC-7901/let-7a细胞中低表达。通过搜索Swiss-port数据库,明确了这些蛋白的基本生物学信息,它们可能与细胞周期调控、肿瘤的侵袭转移及细胞代谢有关。通过Western blot分析,验证了SGC-7901、SGC-7901／EV、SGC-7901/let-7a三组细胞中的细胞周期素依赖性激酶抑制蛋白1、Skp2蛋白和纤维连接蛋白表达水平差异。
结论：胃癌细胞SGC-7901、SGC-7901／EV、SGC-7901／let-7a之间存在多种差异表达的蛋白,这些蛋白可能通过影响细胞周期调控、肿瘤的侵袭与转移及细胞代谢等,与SGC-7901细胞的生物学行为密切相关。对这些蛋白的研究探讨有助于丰富let-7a对SGC-7901细胞生物学行为影响机制的理论。
Cancer is a multi-step disease involving dynamic changes in the genome. However, studies on cancer genome so far have focused most heavily on protein-coding genes, and our knowledge on alterations of the functional noncoding sequences in cancer is largely absent. miRNA are a recently discovered class of short noncoding RNA genes that act posttranscriptionally as negative regulators of gene expression. A large body of research shows that animal miRNAs play fundamental roles in many biological processes, such as cell differentiation and cell proliferation. About 1,000 miRNA genes are thought to be encoded in the human genome. Although it is still difficult to identify accurately individual miRNA/target interactions, computational predictions of miRNA target genes indicate that as many as one third of all human protein-encoding genes may be regulated by miRNAs. This suggests that miRNAs could be involved in a wide variety of human diseases, including cancer. Indeed, as a result of intense research within the last 5 years, miRNA-mediated regulation of tumorigenesis is emerging as a new paradigm in the field of cancer biology. It has also been reported that these miRNA signatures may be a more robust tool than expression patterns of protein-encoding genes for distinguishing normal from tumor tissues. One particular miRNA, let-7, was discovered in the early 1990's humans and mice. It has been reported that let-7 as a novel candidate of tumor suppressor gene involving several cancers such as lung carcinoma and colon carcinoma.
Gastric carcinoma, which is the second most prevalent cancer, is a complex, inadequately understood, and often fatal disease when not detected at early stages. Atypical hyperplasia is generally accepted as a precancerous lesion. Cancerization progression from normal gastric mucosa, chronic atrophic gastritis to gastric carcinoma has been well acknowledged. In this study, the expression of let-7a in normal gastric tissue, chronic atrophic gastritis (atypical hyperplasia)and gastric carcinoma was detected by in situ hybridization. It has been demonstrated that the lentivirus-delivered short hairpin RNAs (shRNAs) are capable of specific, highly stable and functional overexpression of let-7a gene expression both in vitro and in vivo, enabling rapid and efficient analysis of gene function. Then,analysis of different Proteins expressed in SGC-7901、SGC-7901/EV and SGC-7901/let-7a Cells by Proteomics was performed to explore the mechanism of let-7a and provide a new experimental and theoretical basis gene therapy for gastric cancer to guide further clinical application.
Including the following five parts:
Part 1 Study of in situ Expression of let-7a in The Gastric Mucosa Cancerization Tissue Array and Its Clinical Significance
Objective To investigate the expression profile of novel candidate of tumor suppressor gene let-7a in gastric carcinoma, chronic atrophic gastritis (atypical hyperplasia) and normal gastric tissue, analyze the correlation between reduced let-7a and progression of gastric mucosa cancerization.
Methods Human gastric carcinoma, chronic atrophic gastritis (atypical hyperplasia) and normal gastric tissure microarrays were constructed previously and tissue microarays combined with in situ hybridization were used to detect the expression of let-7a.
Results Results showed that there was no significant difference of positive rates of let-7a among normal gastric tissue, chronic atrophic gastritis and gastric carcinoma (P>0.05).However, the degree of let-7a expression in the groups are closely related to the progression of gastric mucosa cancerization (P<0.05).
Conclusions The results suggested that the gastric carcinoma had relatively lowered expression of let-7a. Reduced let-7a may be a fundamental factor in the formation of gastric carcinoma.
Part 2 Construction and identification of the lentiviral Vector of human let-7a gene
Objective To construct a lentiviral Vector of human let-7a gene,and use it in the following experiences.
Methods Pwpxl-MOD2-let-7a eukaryotic expression plasmid was constructed by DNA recombinant method. Pwpxl-MOD2-let-7a was constructed along with pRsv-REV、pMD1g-pRRE、pMD2G into HEK 293 T to package lentivirus particles. According to the EGFP expression, the functional titer was determined by FCM after transduction into HEK 293 T cells.
Results The eukaryotic expression vector Pwpxl-MOD2-let-7a has been successfully constructed. After cotransfection, lentiviral vector can be Packaged in HEK 293 T cell and high functional titer was detected.
Conclusions Lentivirus vector of human let-7a gene has been constructed, which is the essential building block required for the let-7a related gene therapy research.
Part 3 in vitro Study on the Effect of let-7a in Human Gastric Carcinoma Cell Line
Objective To study the effects of the let-7a in human gastric carcinoma cell line by constructing a gastric cancer cell line SGC-7901 stably overexpressing let-7a gene.
Methods The let-7a was transferred to the human gastric carcinoma cell lines SGC-7901 by recombinant lentiviruses which carrying let-7a. The infected cells were quantified by Real-Time RT-PCR for the expression level of let-7a, then characterized by cell proliferation assays, cell adhesion assays and cell invasion assays. Cell cycle was analysised by flow cytometry (FCM).
Results (1) The efficiency of infection of the cell lines using the lentiviral vectors was almost 100 % allowing the stable expression of let-7a in nearly all cells. Over expression of let-7a was confirmed by quantitative Real-time PCR analysis. (2) SGC-7901 cells overexpressing let-7a grew slowly compared to SGC-7901 parental cells or cell transfected with empty vector.(P<0.01). (3)The adhesion ability of SGC-7901 cells overexpressing let-7a reduced when compared to their parental or cell transfected with empty vector. (4) Cell invasion assays indicated that let-7a can decrease the capability of SGC-7901. (5)Flow cytometry based cell cycle assays showed that overexpression of let-7a can arrest SGC-7901 cells (P< 0.01).
Conclusion The use of lentiviral vectors allowed the efficient transfection of let-7a in nearly 100 % of target cells. Overxpression of the let-7a resulted in a decrease of cell proliferation, adhesion, invasion and arrest cell cycle progression in SGC-7901 cells. It suggests that as a novel tumor suppressor gene let-7a can effectively inhibit gastric carcinoma.
Part 4 Lentiviral vector-mediated up-regulation of let-7a therapy for human gastric carcinoma xenogeneic graft in nude mouse subcutaneous
Objective By forming subcutaneous xenograft tumors in nude mouse to observe affection of let-7a on human gastric carcinoma.
Method Gastric carcinoma cells SGC-7901 stably overexpressing let-7a(SGC-7901/let-7a) were injected in nude mouse subcutaneous. SGC-7901/EV and parental SGC-7901 cells were used as the controls. After 6 weeks of observation, the weight and value of xenografts among the three groups were surveyed.
Result The xenografts were successfully formed in the three groups. On the 42th day after inoculation all the mice were sacrificed. The tumor masses were weighted and their volumes were measured. The average tumor weight of SGC-7901/let-7a, SGC-7901/NS and parental SGC-7901 were 1250±20mg、1330±32mg、1350±31mg respectively. Their average tumor volume were 1100±50mm3、1300±68mm3、1340± 55mm3.The tumor weight and volume of SGC-7901/let-7a were significantly lower than SGC-7901/NS and parental SGC-7901(P< 0.05),while there is no significant difference between the laters (P> 0.05).
Conclusion Overexpression of let-7a can inhibit tumor growth of transplanted human gastric cancer in nude mice and has a curative effect on gastric carcinoma as genetic therapy.
Part 5 Analysis of different proteins expression in SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells by proteomics
Objective In order to better understand the basic mechanisms of let-7a influncing gastric carcinoma, the different protein expression were investigated in SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells.
Mathods The total proteins of SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells were separated by immobilized pH gradient (IPG)-based two-dimensional gel electrophorsis (2-DE),the 2-DE maps were established for SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells respectively. The different expression protein spots were analyzed with Image Master 2D Elite4.01 analysis software. The peptide mass fingerprinting of different expressed proteins were verified by MALDI-TOF-MS. The function of the different expressed proteins were identified through swiss-port database searching. Western blot analysis was used to confirm the differential expression levels of the Partial Proteins.
Rsult We established the 2-DE maps of SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells respectively.79 different protein spots were found by image software. Among the total,6 overexpressed proteins and 4 underexpressed proteins of SGC-7901/let-7a cells were recognized with MALDI-TOF MS. The biological information of these proteins was identified through Swiss-port database search. The overexpressed proteins including Antioxidant protein 2, Insulin-like growth factor binding protein 2, Protein disulfide isomerase A2, C-1-tetrahydrofolate synthase, Cyclin-dependent kinase inhibitorl and Rho-GTPase activating protein 4 so on. The underexpressed proteins were consisted of S-phase kinase-associated protein 2, Platelet membrane glycoprotein, Fibronectin and Cksl protein. Some of those proteins were related to cell cycle control, cellular metabolism invasion and metastasis of tumor. The differential expression levels of the Partial Proteins (Cyclin-dependent kinase inhibitorl, S-phase kinase-associated protein 2, Fibronectin) were verified by western blot analysis.
Conclusion Many different expressed proteins among SGC-7901、SGC-7901/EV and SGC-7901/let-7a cells were identified. These different expressed proteins perhaps have closed relation to the function of let-7a in genesis and development of gastric carcinoma through influence of the cell cycle control, invasion, metastasis and cellular metabolism. Investigating these proteins is helpful to explore the basic mechanisms of let-7a in gastric carcinoma.We could better understand the function of let-7a in gastric carcinoma and enrich its theory.
引文
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[1]Bing Z, Pat M, Howard J, et al. The significance of controlled conditions in Lentiviral vector titration and in the use of multiplicity of infection (MOI) for Prediction gene transfer events. Genetic Vaccine and Therapy.2004,2:6.
[2]Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediated RNA interference in mammalian cell culture [J].Nature,2001, 411(6836):494-498.
[3]Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene,2002,21:5716-5724.
[4]Jacque JM, Triques K. Steveenson M. Modulation of HIV-1 replication by RNA interference. Nature,2002,418:435-438.
[5]Shlomai A, Shaul Y.Inhibition of hepatitis B virus expression replication by RNA interference. Hepatology,2003,37(4):764-770.
[6]Lagos-Quintana M, Rauhut R, Lendeckel W, et al:Identification of novel genes coding for small expressed RNAs. Science,2001,294:853-858.
[7]Brennecke J, Hipfner D R, Stark A, et al. Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell,2003,113:25-36.
[8]李继霞,周克元.miRNA,研究进展.生物化学与生物物理进展,2003；30(5)：702-704.
[9]Michaela Seherr, Michael A, Morgan and Matthial Eder. Gene silencing Mediated by small interfering RNAs in mammalian cells.Current Medicinal Chemistry,2003, 10,245-256.
[10]Shen C, Buck AK, Liu X, et al. Gene silencing by adenovirus-delivered siRNA.FEBS Lett,2003,539(1-3):111-4.
[11]Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell,2002,2(3):243-7.
[12]Tomar RS,Matta H,Chaudhary PM. Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene,2003,22(36):5712-5.
[13]Sheila A, Stewart, Derek M, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA,2003,9:493-501.
[14]Hommel JD, Sears RM, Georgescu D, et al. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med 2003,9:1539-1544.
[15]Shinagawa T, Ishii S. Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA Polymerase Ⅱ Promoter. Genes Dev.2003, 17:1340-1345
[1]Hanawa H, Kelly PF, Nat hwani AC, et al. Comparison of various envelope proteins for t heir ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood. Mol Ther,2002,5 (3):242-251.
[2]PhiliP S. Bernard, Carl T. Wittwer. Real-Tmie PCR technology for cancer diagnostics. Clinical Chemistry.2002,48:1178-1185.
[3]Julie Clayton, The silent treatment, NATURE,2004,431:599-605.
[4]Zhang XN, Xiong W, Wang JD, et al, siRNA-mediated inhibition of HBV replication and expression, World J Gastroenterol.2004,10(20):2967-2971.
[5]Barton GM, Medzhitov R. Retroviral delivery of small interfering RNA into Primary cells. Proc Natl Acad Sei USA,2002,99(23):14943-14945.
[6]Abbas-Terki T, Blaneo-Bose W, Deglon N, et al. Lentiviral-mediated RNA interference..Hum Gene Ther,2002,13(18):2197-2201.
[7]Patriek J, Paddison, Amy A, et al.. Stable suppression of gene expression by RNAi in mammalian cells. PNAS,2002,993:1443-1448.
[8]朱益民,刘志明.癌基因微小RNA在肿瘤中的作用.国际肿瘤学杂志,2008,12：883-885.
[9]Akao Y, Nakagawa Y, Naoe T. let-7 microRNA functions as a potential growth suppressor in human colon cancer cell. Biol Pharm Bull,2006,29(5):903.
[10]Fengyan Yu, Herui Yao, Pengcheng Zhu, et al. let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells. Cell,2007,131,1109-1123.
[1]Nemoto T, Kubota S, lshida H, et al. Ornithine decarboxylase, Mitogen-activated Protein kinaseand matrix metalloproteinase-2 expressions in human colontumors.World J Gastroenterol2005,11(20):3065-3069.
[2]崔龙,曹广文,王元和等。Hsv-TK基因体外特异性前体药转换抗大肠癌作用研究。中华外科杂志,1997,35：356-358。
[3]Cao GW, Qi ZT, Pan X, et al. Gene therapy for human colorectal carcinoma using human CEA promoter controlled bacterial ADP-ribosylating toxin genes human CEA:PEA&DAT gene transfer. World J Gastroenterol,1998,4:388-391.
[4]Tumor targeted gene therapy with Plasmid expressing human tumor necrosis factor alpha in vitro and in vivo. Neoplasma.2005,52(4).344-51.
[5]Wu DH, Liu L, Chen LH. Antitumor effects and radiosensitization of cytosine deaminase and thymidine kinase fusion suicide gene on colorectal carcinoma cellS. World J Gastroenterol..2005, 11(20):3051-5.
[6]Jiang YR, Liu CS, Chen XL. et al. Establishment of lentivirus-mediated system of double suicide genes and its killing effects on K562 cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi.2004.12(1):29-34.
[7]Freytag SO, Strieker H, Pegg J,et al. Phase I study of replication-competent adenovirus-mediated double-suicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate-to high-risk prostate cancer. Cancer Res,2003, 63(21):7497-506.
[8]Aurora Esquela-Kerscher, Phong Trang, Jason F. Wiggins, The let-7 microRNA reduces tumor growth in mouse models of lung cancer. Cell Cycle 20087:6, 759-764.
[9]Sui G, Soohoo C, Affar I B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sei USA 2002, 99:5515—20.
[10]Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hair Pin RNAs in mammalian cells. Proc Natl Acad Sei USA 2002:99:6047-52.
[11]Shen Pang, Mo K, Kang, Sam Kung, et al. Anticancer Effector of Lentiviral Veetor Capable Expressing HIV-1 Vpr. Clinical Cancer Research.2001, 7:3567-3573.
[1]Blackstock WP, Weir MP, Proteomics:quantitative and Physical mapping of cellular Proteins. Trends Biotechnol 1999;17(3):121-7.
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[1]Berezikov E, Guryev V, Vande Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell,2005,120 (1): 21-24.
[2]Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science,2001,294:853-858.
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[4]Cho WC. Contribution of oncoproteomics to cancer biomarker discovery. Mol Cancer 2007,6:25.
[5]Cho WC, Cheng CH. Oncoproteomics:current trends and future perspectives. Expert Rev Proteomics,2007,4:401-410.
[6]Frantz G D, Pham T Q, Peale F V Jr, et al. Detection of novel gene expression in paraffin——embedded tissues by isotopic in situ hybridization in tissue microarrays. J Pathol,2001,195(1):87-96.
.[7] Jerome Torrisani, Laurie Parmentier, Louis Buscail, et al. Enjoy the Silence:The Story of let-7 MicroRNA and Cancer. Current Genomics,2007,8,229.
[8]Zamore PD., Tuschl T., Sharp PA et al.RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell,2000, 101:25-33.
[9]Hammond SM., Bernstein E., Beach D, et al. An RNA-directed nuclease mediates gene silencing in Drosophila cells. Nature,2000,404,293-296.
[10]Johnson SM, Grosshans H, Shingara J, et al. RAS is regulated by the let-7 microRNA family. Cell,2005,120:635-647.
[11]Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res,2004,64:3753-3756.
[12]Lorenzo F, Sempere, Mette Christensen, et al. Altered MicroRNA Expression Confined to Specific Epithelial Cell Subpopulations in Breast Cancer. Cancer Res,2007,67(24):11615-11619.
[13]Kloosterman W P, Wienholds E, Bruijn E, et al. In situ detection of miRNAs in animal embryos using LNA 2 modified oligonucleotide probes. Nat Methods, 2006,3(1):27-29.
[14]Akao Y, Nakagawa Y, Naoe T. let-7 microRNA functions as a potential growth suppressor in human colon cancer cell. Biol Pharm Bull,2006,29(5):903.
[15]Fengyan Yu, Herui Yao, Pengcheng Zhu, et al. let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells. Cell,2007,131,1109-1123.
[1]Bing Z, Pat M, Howard J, et al. The significance of controlled conditions in Lentiviral vector titration and in the use of multiplicity of infection (MOI) for Prediction gene transfer events. Genetic Vaccine and Therapy.2004,2:6.
[2]Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediated RNA interference in mammalian cell culture [J].Nature,2001, 411(6836):494-498.
[3]Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene,2002,21:5716-5724.
[4]Jacque JM, Triques K. Steveenson M. Modulation of HIV-1 replication by RNA interference. Nature,2002,418:435-438.
[5]Shlomai A, Shaul Y.Inhibition of hepatitis B virus expression replication by RNA interference. Hepatology,2003,37(4):764-770.
[6]Lagos-Quintana M, Rauhut R, Lendeckel W, et al:Identification of novel genes coding for small expressed RNAs. Science,2001,294:853-858.
[7]Brennecke J, Hipfner D R, Stark A, et al. Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell,2003,113:25-36.
[8]李继霞,周克元.miRNA,研究进展.生物化学与生物物理进展,2003；30(5)：702-704.
[9]Michaela Seherr, Michael A, Morgan and Matthial Eder. Gene silencing Mediated by small interfering RNAs in mammalian cells.Current Medicinal Chemistry,2003, 10,245-256.
[10]Shen C, Buck AK, Liu X, et al. Gene silencing by adenovirus-delivered siRNA.FEBS Lett,2003,539(1-3):111-4.
[11]Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell,2002,2(3):243-7.
[12]Tomar RS,Matta H,Chaudhary PM. Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene,2003,22(36):5712-5.
[13]Sheila A, Stewart, Derek M, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA,2003,9:493-501.
[14]Hommel JD, Sears RM, Georgescu D, et al. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med 2003,9:1539-1544.
[15]Shinagawa T, Ishii S. Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA Polymerase Ⅱ Promoter. Genes Dev.2003, 17:1340-1345
[1]Hanawa H, Kelly PF, Nat hwani AC, et al. Comparison of various envelope proteins for t heir ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood. Mol Ther,2002,5 (3):242-251.
[2]PhiliP S. Bernard, Carl T. Wittwer. Real-Tmie PCR technology for cancer diagnostics. Clinical Chemistry.2002,48:1178-1185.
[3]Julie Clayton, The silent treatment, NATURE,2004,431:599-605.
[4]Zhang XN, Xiong W, Wang JD, et al, siRNA-mediated inhibition of HBV replication and expression, World J Gastroenterol.2004,10(20):2967-2971.
[5]Barton GM, Medzhitov R. Retroviral delivery of small interfering RNA into Primary cells. Proc Natl Acad Sei USA,2002,99(23):14943-14945.
[6]Abbas-Terki T, Blaneo-Bose W, Deglon N, et al. Lentiviral-mediated RNA interference..Hum Gene Ther,2002,13(18):2197-2201.
[7]Patriek J, Paddison, Amy A, et al.. Stable suppression of gene expression by RNAi in mammalian cells. PNAS,2002,993:1443-1448.
[8]朱益民,刘志明.癌基因微小RNA在肿瘤中的作用.国际肿瘤学杂志,2008,12：883-885.
[9]Akao Y, Nakagawa Y, Naoe T. let-7 microRNA functions as a potential growth suppressor in human colon cancer cell. Biol Pharm Bull,2006,29(5):903.
[10]Fengyan Yu, Herui Yao, Pengcheng Zhu, et al. let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells. Cell,2007,131,1109-1123.
[1]Nemoto T, Kubota S, lshida H, et al. Ornithine decarboxylase, Mitogen-activated Protein kinaseand matrix metalloproteinase-2 expressions in human colontumors.World J Gastroenterol2005,11(20):3065-3069.
[2]崔龙,曹广文,王元和等。Hsv-TK基因体外特异性前体药转换抗大肠癌作用研究。中华外科杂志,1997,35：356-358。
[3]Cao GW, Qi ZT, Pan X, et al. Gene therapy for human colorectal carcinoma using human CEA promoter controlled bacterial ADP-ribosylating toxin genes human CEA:PEA&DAT gene transfer. World J Gastroenterol,1998,4:388-391.
[4]Tumor targeted gene therapy with Plasmid expressing human tumor necrosis factor alpha in vitro and in vivo. Neoplasma.2005,52(4).344-51.
[5]Wu DH, Liu L, Chen LH. Antitumor effects and radiosensitization of cytosine deaminase and thymidine kinase fusion suicide gene on colorectal carcinoma cellS. World J Gastroenterol..2005, 11(20):3051-5.
[6]Jiang YR, Liu CS, Chen XL. et al. Establishment of lentivirus-mediated system of double suicide genes and its killing effects on K562 cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi.2004.12(1):29-34.
[7]Freytag SO, Strieker H, Pegg J,et al. Phase I study of replication-competent adenovirus-mediated double-suicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate-to high-risk prostate cancer. Cancer Res,2003, 63(21):7497-506.
[8]Aurora Esquela-Kerscher, Phong Trang, Jason F. Wiggins, The let-7 microRNA reduces tumor growth in mouse models of lung cancer. Cell Cycle 20087:6, 759-764.
[9]Sui G, Soohoo C, Affar I B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sei USA 2002, 99:5515—20.
[10]Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hair Pin RNAs in mammalian cells. Proc Natl Acad Sei USA 2002:99:6047-52.
[11]Shen Pang, Mo K, Kang, Sam Kung, et al. Anticancer Effector of Lentiviral Veetor Capable Expressing HIV-1 Vpr. Clinical Cancer Research.2001, 7:3567-3573.
[1]Blackstock WP, Weir MP, Proteomics:quantitative and Physical mapping of cellular Proteins. Trends Biotechnol 1999;17(3):121-7.
[2]7.Mullner S, Neumann T, Lottspeich F. Proteomics-a new way for drug target djscovery. Arzneimittelforschung 1998;48(1):93-5.
[3]Noda A, Ning Y, Venable SF, et al. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen[J]. Exp Cell Res,1994,211 (1):90-98.
[4]Scott MT, Morrice N, Ball KL. Reversible phosphorylation at the C-terminal regulatory domain of p21 (Waf1/Cip1) modulates proliferating cell nuclear antigen binding [J]. J Biol Chem,2000,275 (15):11529-11537.
[5]Bi JX, Shuttleworth J, Al-RubeaiM. Uncoup ling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells [J]. Biotechnol Bioeng,2004,85 (7):741-749.
[6]Zhang H,Kobayashi R,Galaktionov K,Beach D. p19Skp1 and p45Skp2 are essential elements of the cyclin ACDK2 S phase kinase. Cell,1995,82(6):915～ 925
[7]Kornitzer D,Ciechanover A. Modes of regulation of biquitinmediated protein degradation. J Cell Physiol,2000,182 (1):1～11.
[8]DeSalle L M, Pagano M. Regulation of G1 to S transition by the ubiquitin pathway. FEBS Lett,2001,490(3):179-189
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