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1、-介导性shRNA能抑制肺癌细胞中livin沉默基因的表达从而促进SGC-7901细胞凋亡背景由于肿瘤细胞抑制凋亡增殖,特定凋亡的抑制因素会对于开展新的治疗策略提供一个合理途径。Livin是一种凋亡抑制蛋白家族成员，在多种恶性肿瘤的表达中具有意义。但是, 在有关胃癌方面没有可利用的数据。在本研究中,我们发现livin基因在人类胃癌中的表达并调查了介导的shRNA能抑制肺癌细胞中livin沉默基因的表达，从而促进SGC-7901细胞凋亡。方法mRNA及蛋白质livin基因的表达用逆转录聚合酶链反响技术及西方吸干化验进展了分析。小干扰RNA真核表达载体具体到livin基因采用基因重组、测序核酸。然
2、后用Lipofectamin2000转染进入SGC-7901细胞。逆转录聚合酶链反响技术和西方吸干化验用来验证的livin基因在SGC-7901细胞中使沉默基因生效。所得到的稳定的复制品用G418来筛选。细胞凋亡用应用流式细胞仪(FCM)来评估。细胞生长状态和5-FU的50%抑制浓度(IC50)和顺铂都由MTT比色法来决定。结果livin mRNA和蛋白质的表达检测40例中有19例(47.5%)有胃癌和SGC-7901细胞。没有livin基因表达的是在肿瘤邻近组织和良性胃溃疡病灶。相关发现在livin基因的表达和肿瘤的微小分化和淋巴结转移一样(P 0.05)。4个小干扰RNA真核表达矢量具体到
3、基因重组的livin基因建立。其中之一,能有效地减少livin基因的表达,抑制基因不少于70%(P 0.01)。重组的质粒被提取和转染到胃癌细胞。G418筛选所得到的稳定的复制品被放大讲究。当livin基因沉默,胃癌细胞的生殖活动明显低于对照组(P 0.05)。研究还说明,IC50上的5-Fu和顺铂在胃癌细胞的治疗上是通过shRNA减少以及刺激这些细胞(5-Fu proapoptotic和顺铂)(P 0.01)。结论livin基因在胃癌中的过分表达与肿瘤分化与淋巴结转移建立联系,建议了治疗胃癌病例分子预后因素之一。ShRNA可以抑制在SGC-7901细胞中的livin基因表达,诱导细胞凋亡。L
4、ivin可以作为治疗胃癌凋亡的新目标。1介绍胃癌是世界上最常见的恶性肿瘤之一。大多数患者被诊断为这个疾病的阶段,在最正确时间的时机错失了手术治愈。尽管有很大改善,但处于晚期胃癌的化疗患者的总体存活率仍然很低。癌症细胞化疗耐抗性可能导致手术失败。在耐药的原因中,抑制细胞凋亡的过程会起重要作用。癌细胞常有抗凋亡增长的特征1,介导其增加的阻力不同来刺激的细胞凋亡,如DNA损伤、缺氧、营养损失2、3。此外, 在临床实践中细胞凋亡抵抗被认为是肿瘤手术失败的主要原因,因此许多化疗药物和/或放射线疗法都是通过诱导凋亡肿瘤死亡实现的4。酶抑制剂 (IAPs),是一种新型的凋亡蛋白抑制基因家族5,6,包括病毒感
5、染,化疗药物, ,生长因子和肿瘤坏死因子-(TNF-凋亡信号通路)/ Fas信号通路7 - 9。IAPs是由一组有凋亡特性的构造相关的蛋白质构成10，在预防肿瘤细胞凋亡方面可能扮演一个重要的角色,并已成为近年来研究的热点。这个家庭的新成员是ML-IAP/livin/KIAP/BIRC7 (以下称为livin)有两个种类、livin和livin11-14。有证据说明, livin的过分表达能阻止 由多种刺激诱导的细胞凋亡12。有趣的是, livin基因被发现在肿瘤细胞中限制性表达,但是不存在或是很少数量存在于正常成人体组织中11-15,并且通过允许恶性细胞,以防止凋亡细胞死亡的方式导致肿瘤形成,
6、。所以抑制livin基因表达可能会呈现出一个有趣的治疗策略。在目前的研究中,我们调查了livin的表达在胃cancinomas及其邻近组织。livin的表达和临床病理参数之间的关系进展了分析。此外,我们探索了在抑制livin基因表达的shRNA可行性和胃癌易感性的凋亡细胞由shRNA介导的 livin沉默基因。2患者和方法2.1患者和肿瘤样本胃癌中四十个病例及承受胃切除手术的患者收集到的胃癌组织中13个病例(患者年龄从2977岁)。其中良性胃溃疡的13个病例(慢性浅表胃炎)患者在承受了胃视镜检查(患者年龄从3377岁)。这些病例均来自医科大学第一附属医院。胃癌患者被诊断为TNM级的14阶段(U
7、ICC ，2002)。手术之后肿瘤标本就立即被冻结在液态氮中,储存在-80直到使用为止。这是在所有的病人知情同意的情况下获得的。2.2逆转录聚合酶链反响技术程序总共RNA(2毫克)提取冷冻组织反转录进展，最后的体积2微升是用100 pmol of oligo(dT)15和200U M-MLV与逆转录酶(promega、美国) ,根据制造商的说明。Aliquots对应的250微升cDNA被放大在PCR缓冲容器中，在最终的50微升中含25pmol / ml处理剂和1U聚合酶。每一个放大了35周期,一个周期的变性曲线在30 s到达94。热处理在30s59livin and b-actin扩大到30s
8、72。没有RNA的病例作为阴性对照物包含在RTPCR中。一系列常用的的 livin和-actin处理剂如下： livin / upstream，5-TCCACAGTGTGCAGGAGACT-3;livin/ downstream;5-TCCACAGTGTGCAGGAGACT-3;-actin upstream,5-ACGGCACAAAGACGATGGAC-3-actin downstream,5-AGCGCAAGTACTCCGTGTG-3。产品的尺寸分别为livin/是312/258 bp ，-actin 是501bp。2.3 西方吸干技术分析病变同质性与冲力缓冲50mM Tris-HCl (
9、pH 7.5), 250 mM NaCl,0.1% NP40和5mM EGTA包含50mM氟化钠，60mM -丙三醇-磷酸盐,0.5mM钒酸钠,0.1mM苯甲基磺醯化氟10g/ml亮抑蛋白酶肽。用考马斯亮蓝微盘比色法测定蛋白质含量。蛋白质样品电泳10% 变性SDS凝胶并转移到PVDF膜(Roche、美国)。膜是培养特定的主要的抗体,与过氧化物酶继发性抗体反响 (细胞信号技术、美国),最后通过增强化学荧光到达可视化 (细胞信号技术、美国)。Ale*esis(美国) 和散塔克鲁兹生物技术(美国)购置的单克隆抗体livin (1:250) and actin (1:400)24细胞系和细胞培养我们选
10、了一个人类胃腺癌细胞系在这一研究中。SGC-7901(细胞研究所,中国,)是一种附着中度分化胃腺的人类细胞株。线是胃癌细胞上皮细胞,并成长为附着细胞RPMI 1640 (Hyclone Inc, USA)含10%FCS (Life Technologies, Inc.),每毫升100个单位的青霉素和毫升100微克的链霉素(BioWhittaker)。在含有5%CO2空气的条件下的37湿润培养器中保存SGC-7901细胞。溶解顺铂和氟尿嘧啶(齐鲁制药厂、中国)在DMSO并且4储存。2.5。ShRNA的合成和PGPU / GFP /Neo/livin质粒的制造通过siRNASequence-Sel
11、ector软件设计并合成了Livin的ShRNA序列(生物技术*公司。集团公司、中国)。序列如下(表1),然后被插入pGPU/GFP/Neo(GenePharma股份。中国) BbsI and BamH地址产生pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control质子。2.6SGC-7901的建立稳定表达pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control转染实验,SGC-7901细胞被镀成6孔板(3105孔密度), , 96孔板(1 104孔密度)和12孔板(1.5 105孔密度)转染之前培养24小时。按照制造商的说明这些细胞
12、被调控子用Li-pofectAMINE 2000转染4毫克/孔空的pGPU/GFP/Neo/vector， pGPU/GFP/Neo/livin或pGPU/GFP/Neo/Control 质粒 (生命技术公司、大岛屿,NY)。转染48小时后,这些细胞被转移在 1:15 (v/v)并用Geneticin (G418) 1000克/毫升培养4周。稳定转染的克隆体取出并保存在媒介容器400 g/ml G418用作另外的研究。27依赖贴壁细胞细胞生长的测定亲本细胞和细胞稳定表达pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control
13、被种到6孔盘子中。每隔一天收集三孔的细胞。使用计数器确定细胞数目(Coulter Electronics, Miami, FL).。种植一些天数后用平均SD记录每孔细胞的数量。2.8MTT测定通过MTT对细胞毒性进展了测量。呈几何数增长的细胞被镀在密度为10000细胞/孔的96孔板上作用24小时。接下来这些细胞被以不同浓度的药物治疗48小时。每孔参加100微升MTT溶液 (1毫克/毫升),并且这些细胞放在37下培养四小时。上层清液用异丙醇代替溶解有色甲品。用micro-ELISA测量到吸光率的波长为595nm(ClinBio-128 SLT, ,奥地利)。治疗细胞的吸光率相当于计算控制细胞的吸
14、光率并用细胞死亡百分率显示出来。2.9流式细胞术细胞被收集并参加冰冷的70%乙醇于PBS缓冲液中储存在-4摄暖直到使用。悬浮后后，100 ml核糖核酸酶I (1 mg/ml) and 100 ml 的聚酰亚胺(400 g/ml) 37下培养细胞并用流式细胞术(BD,美国)进展分析。2.10统计分析数据的展现要用至少三个不同实验SD的方法。实验结果用学生的t检验来分析和当P 0.05时被认为是具有统计学显著性。3结果3.1。livin在胃肠癌中的表达在目前的研究中,我们第一次验证了逆转录聚合酶链反响技术和西方吸干技术的存在在40胃癌中,13 癌组织和13良性病变胃粘膜损伤。在癌组织和良性病变胃粘
15、膜损伤中, 每个mRNA亚型不可见水平被发现后,在肿瘤组织中,19/40(47.5%)显示出mRNA及livina和livin蛋白质表达(Figs. 1 and 2). 。livin表达与预后变量相关,如组织学恶性度和淋巴结转移,但包括年龄、性别、阶段和肿瘤细胞浸润程度(表2)。3.2。稳定转染表达pGPU/GFP/Neo/livin与pGPU/GFP/Neo/Control的特征我们建立了SGC-7901 与任一pGPU/GFP/Neo/livin稳定转染，pGPU/GFP/Neo /Control 质粒，或空pGPU/GFP/Neo/vector (图3)。用西方吸干技术和逆转录聚合酶链反
16、响技术选择每个转染的克隆并分析决定livin mRNA,和蛋白质表达。其他的都被选择作为扩展和另外的研究。(如图4及5)显示， livinmRNA和蛋白质的水平在SGC-7901pGPU/GFP/Neo/livin2中转染降低了90%以上。Livin表达抑制在pGPU/GFP/Neo/livin1转染和消极控制中没有被发现。所以SGC-7901 pGPU/GFP/Neo/livin2被用来做后续实验。3.3稳定转染中抑制细胞生长SGC-7901的增长率pGPU / GFP /Neo/ livin2均有显著的抑制转染。如图显示，SGC-7901 Pgpu/GFP/尼欧/ livin2 / GFP
17、细胞数目有显著下降转染在72小时到96小时后镀(P 0.01),而阴性对照和家长的细胞。3.4。稳定的转染容易受细胞凋亡因子刺激我们认为SGC-7901 pGPU/GFP/Neo/livin2 转染和阴性对照细胞同细胞毒顺铂的增长速率明显抑制，图表6中显示SGC-7901 pGPU/GFP/Neo/livin2转染细胞数量培养后72小时和96小时与消极控制和亲本细胞相比明显降低阴性对照细胞和细胞毒药物(5 -氟尿嘧啶和顺铂)。pGPU MTT测定说明,SGC-7901 /Neo/ livin2 / GFP更敏感转染顺铂和氟尿嘧啶比消极的控制和亲本细胞(Figs. 7A, 6B)。由顺铂和5氟尿
18、嘧啶诱导凋亡细胞数增加至约2.5 - 3倍的pGPU/GFP/Neo/livin2转相比，其控制细胞P0.001;图7C条。此外，经历了稳定转染无自发性凋亡更容易比对照细胞P0.05。图7C条凋亡刺激。讨论在本研究中,我们说明,推荐的新成员:一个新的IAP家族成员，被认为是不符合非癌胃组织的表达，只有在胃癌患者47.5的比例计算，也说明，抑制Livin的表达或功能的原因自发性细胞凋亡和对SGC - 7901细胞生长的抑制，使细胞更容易凋亡刺激。据认为，活着有两个亚型，A和B虽然这两个亚型在阻止肿瘤坏死因子诱导细胞凋亡参与- A和抗CD95的在体外，他们表现出一些不同的抗凋亡的特性。活着b似乎是
19、在阻断DNA损伤剂诱导细胞凋亡13超过活着有效。一些组织中Livin分布研究说明，最近都活着升高亚型A和B已发现在心脏，胎盘，肺，脾，卵巢，而活着balone特别是在已检测到胎儿组织和dult肾脏和Livin一单是在脑，骨骼肌和外周血淋巴细胞的检测11-14。此外，虽然Livin的表达是在一个癌细胞的细胞株和肿瘤组织中的一些品种检测14-18和反活着抗体在胃癌和肺癌患者血清识别19,20，没有数据有关亚型中Livin的表达在胃癌肿瘤组织。我们的第一次研究说明，活着亚型A和B几乎都在胃癌组织47.5和Livin表达与一些的预后因素，如分级，淋巴结转移，相关的比例计算。从文献资料说明，这两个活着亚
20、型参与了阻止细胞凋亡，并可能给活着的过度表达与细胞的强烈抵抗化疗诱导细胞凋亡。胃癌一般具有高度抗癌症放化疗和中抗凋亡21。这些结果说明，Livin的高表达可能对*些癌症患者和胃癌患者预后化疗的责任。与促进肿瘤细胞的凋亡抵抗可能提供一个合理干预策略在癌症治疗的根底上开展新的特定因素干扰22,23。由于Livin的表达可能有助于肿瘤细胞和肿瘤的特异性表达及其在细胞凋亡的抗性表型可以让活着的一个有趣的肿瘤治疗靶点的具体干预措施的战略，我们选择了作为一个分子靶点的Livin基因。的shRNA技术representiong一个极其有力的工具，抑制源性基因表达24,25作出抑制Livin基因，并试图纠正胃
21、癌细胞凋亡的缺乏。 沉默的shRNA成效的tageted基因的表达是不同的，与半的生活和丰富的基因产物与靶mRNA作为24-27，以及无障碍的关系。在这项研究中，我们观察到硅livin1是经常更强烈的沉默比硅livin2 Livin基因。我们的研究结果还说明，沉默Livin基因的表达可能存在强烈的增加或几个凋亡的代理人在场下的SGC - 7901细胞凋亡反响，抑制细胞的生长，这说明，与美好生活的干扰导致了对凋亡刺激的敏感性。对HeLa细胞类似的结果报告了Crnkovic -梅坦斯18。总之，我们的结果说明，Livin的表达和功能抑制自发性细胞凋亡和抑制细胞生长的体外敏感性增强化疗药物的结果。由
22、于在胃癌中的表达，但活着的优惠在正常组织中，这些数据说明，针对活着途径单独或与细胞毒性药物可能在胃癌的治疗作用。尽管他们的治疗潜力，主要技术障碍仍有待克制，才能申请成为毒品的shRNA。在治疗方面，将不得不满足基因治疗的方法，如高效输送到目标细胞的免疫反响或躲避，一般的挑战。值得注意的是，在最近的研究说明，体的shRNA可以直接应用到出生后小鼠脏器highpressure尾静脉注射，导致靶基因特异性抑制28-30。这些数据说明，一个活泼的shRNA通过血液的直接应用是主要可行的。英文翻译：对照版E*pression of livin in gastric cancer and inductio
23、n of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene Background-Because of increased resistance to apoptosis in tumor cells, inhibition of specific antiapoptotic factors may provide a rational approach for the development of novel therapeutic strategies.Livin, a novel inhibitor
24、 of apoptosis protein family, has been found to be e*pressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detected the e*pression of livin in human gastric car
25、cinoma and investigated the apoptotic susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene. Methods-The mRNA and protein e*pression of livin were analyzed by RT-PCR and western blot assay.The relationship between livin e*pression and clinical pathologic parameters was investi
26、gated. The small interfering RNA eukaryotic e*pression vector specific to livin was constructed by gene rebination, and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay were used to validate gene-silencing efficiency of l
27、ivin in SGC-7901 cells. Stable clones were obtained by G418 screening. The cell apoptosis was assessed by flow cytometry (FCM). Cell growth state and 50 % inhibition concentration (IC50) of 5-FU and cisplatin was determined by MTT method. Results-The e*pression of livin mRNA and protein were detecte
28、d in 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No e*pression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin e*pression and poor differentiation of tumors as well as lymph node metastases (P 0.05). Four sm
29、all interfering RNA eukaryotic e*pressionvector specific to livin were constructed by gene rebination. And one of them can efficiently decrease the e*pression of livin, the inhibition of the gene was not less than 70% (P 0.01). The rebinated plasmids were e*tracted and transfected gastric cancer cel
30、ls. The stable clones were obtained by G418 screening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantly lower than the control groups(P 0.05). The study also showed that IC50 of 5-Fu and cisplatin on gastric cancer
31、 cells treated by shRNA was decreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P 0.01). Conclusions-C Livin is overe*pressed in gastric carcinoma with a relationship to tumor differentiation and lymph node metastases, which is suggested to be one of the molec
32、ular prognostic factors for some cases of gastric cancer. ShRNA can inhibit livin e*pression in SGC-7901 cells and induce cell apoptosis.Livin may serve as a new target for apoptosis-inducing therapy of gastric cancer.1. Introduction Gastric cancer is one of the most mon malignancies in the world. M
33、ost patients with this disease are diagnosed in advanced stages, and lose the chance of surgical eradication. Despite much progress in chemotherapy, the overall survival of the patients with gastric cancer in advanced stage is still poor. Resistance of cancer cells to chemoagents may contribute to f
34、ailure of the treatment. Among the reasons of drug resistance, inhibited process of cell apoptosis may play an important role.Cancer cells are often characterized by increased resistance to apoptosis 1, which mediates their increased resistance to various stimuli of cell apoptosis, such as DNA damag
35、e, hypo*ia, nutrient-deprivation 2,3. Moreover, apoptosis resistance is considered to be a major cause of therapeutic failure for tumors in clinical practice, since many chemo- and/or radiotherapeutic agents function through the induction of apoptotic tumor death 4.Inhibitor of apoptosis protein (IA
36、Ps) is a novel family of intracellular proteins which suppress apoptosis induced by a variety of stimuli 5,6, including viral infection, chemotherapeutic drugs, staurosporin, growth factor withdrawal, and by ponents of the tumor necrosis factor-a (TNF-a)/Fas apoptotic signaling pathways 7C9. The IAP
37、s consists of a group of structurally related proteins with antiapoptotic properties 10, and may play a substantial role in preventing tumor cell from apoptosis, and has bee the focus of research in recent years. A novel member of this family is ML-IAP/livin/KIAP/BIRC7 (in the following termed livin
38、) which has two isoforms, livin a and livin b 11C14. It has been shown that over-e*pression of the livin can block apoptosis induced by a variety of proapoptotic stimuli 12. Interestingly, livin gene has been found to be restrictively e*pressed in tumor cells, but not, or to lesser amounts in most n
39、ormal adult tissues 11C15, and may contribute to tumorigenesis by allowing malignant cell to avoid apoptotic cell death. So inhibition of livin e*pression may represent an interesting therapeutic strategy. In the present study, we investigated the e*pression of livin in gastric cancinomas and their
40、adjacent tissues. The relationship between livin e*pression and clinical pathologic parameters was analyzed. Furthermore, we e*plored the feasibility of shRNA in inhibiting livin gene e*pression and the apoptotic susceptibility of gastric cancer cell by shRNA-mediated silencing of the livin gene.2.
41、Patients and methods2.1. Patients and tumor samples Forty samples of gastric carcinoma and 13 samples of paracancerous tissues were collected from the patients who received gastrectomy (age of patients ranging from 29-77 years). Thirteen samples of benign gastric lesion (chronic superficial gastriti
42、s) were gained from the patients undergoing gastric endoscopic e*amination (age of patients ranging from 33-77 years). These samples were collected from patients admitted to the First Affiliated Hospital of Nanjing Medical University. The patients with gastric cancer were diagnosed as being in stage
43、 I to IV based on TNM classification (UICC, 2002). Tumor specimens were immediately frozen in liquid nitrogen after surgery and stored at -80 until use. Informed consent was obtained from all patients.2.2. RT-PCR procedureTotal RNA (2 mg) e*tracted from frozen tissues was reverse transcribed in a fi
44、nal volume of 25 ml with 100 pmol of oligo(dT)15 and 200U M-MLV reverse transcriptase (promega, USA), according to the manufacturers guidelines. Aliquots corresponding to 2.5 ml cDNA were then amplified in PCR buffer containing 25pmol/ml each primer and 1 U Taq polymerase in a final volume of 50 ml.
45、 Each amplification was performed for 35 cycles, one cycle profile consisted of denaturationat 94 8C for 30 s, annealing at 59 8C (livin and b-actin) for 30 s and e*tension at 72 8C for 30 s. A sample without RNA was included in each RTCPCR as a negative control. Sequences of livin and -actin primer
46、s used are as follows:livina/b up stream,50-TCCACAGTGTGCAGGAGACT-30;livina/downstream,50-ACGGCACAAAGACGATGGAC-30;b-actinupstream,50-AGCGCAAGTACTCCGTGTG-30;-actin downstream, 50-AAGCAATGCTATCACCTCCC-30.The size of the amplified products were312/258 bp for livina/b and 501 bp for b-actin respectively.
47、2.3. Western Blot Analysis Tissues were homogenized with lysis buffer 50 mM Tris-HCl(pH 7.5), 250 mM NaCl, 0.1% NP40, and 5 mM EGTA containing 50 mM sodium fluoride, 60 mM b-glycerol-phosphate, 0.5 mM sodium vanadate, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin.
48、The total protein concentrationwas determined using Coomassie Brilliant Blue. Protein samples were electrophoresed in a 10% denaturing SDS gel and transferred to PVDF membrane (Roche, USA). The membranes were incubated with specific primary antibodies, reacted with a pero*idase-conjugated secondary antibody (Cell signaling technology,USA), and finally visualized by enhanced chemiluminescence (Cell signaling technology, USA). Monoclonal antibodies recognizing livin (1:250) and actin (1:400) were purchased from Ale*esis Inc. (USA) and Santa Cruz Biotechnology (USA).2.4. Ce
