医药学类文献双语版：汉译英

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发展新的治 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应技术及西方 吸干化验 进行了分析。小干扰

2、RNA 真核表达载体具体到 livin 基因采用基因重组、 测序核酸。然后 用 Lipofectamin2000 转染进入 SGC-7901 细胞。逆转录聚合酶链 反应技术和西方吸干化 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。结果一 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

3、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)。研究还表 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

4、顺 铂)(P 0.01) o结论一 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。Livin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，在最佳时间的机会错失了手术治愈。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

5、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。酶抑制剂（lAPs）,是一种新型的凋亡蛋白抑制基因家族匚56,包括病毒感染，化疗药物，生长因子和肿瘤坏死因子-a（TNFa凋亡信号通路”Fas信号通路7 - 9。lAPs是由一组有凋亡特性的结构相关的蛋白质构成：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livin -14- 有证据表明,

6、 livin的过分表达能阻止由多种刺激诱导的细胞凋亡刃。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 织中匚11-15，并且通过允许恶性细胞，以避免凋亡细胞死亡的方式导致肿瘤形成，。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can ci no mas及其邻近组织。liv in的表 达和临床病理参数之间的关系进行了分析。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

7、病例及接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）患者在接受 了胃内视镜检 查（患者年龄从3377岁）。这些病例均来自南京医科大学第一附属医院。胃癌患者被诊断为TNM级的14阶段（UICC , 2002 ）。手术之后肿瘤标本 就立即被冻结在液态氮 中，储存在-80 C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逆转录聚合酶链反应技术程序总共RNA （2毫克）提取冷冻组织反转录进行，最后的体积2微升是用100 pmol ofoligo （dT） 15和200U M-MLV与逆转录酶（promega美国），根据

8、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内 到达94C。热处理在30s内59C （ livin and b-actin ）扩大到30s内72C。没有RNA的病 例作为阴性对照物包含在 RT -PCR中。一系列常用的的livin和 伕actin处理剂如下：livin a B upstream 5-TCCACAGTGTGCAGGAGACT-3;livi n a / B dow nstream;5/-TCCACAGTGTGCAGGAGACT-

9、3; Bactinupstream,5 /-ACGGCACAAAGACGATGGAC-3Bact in dow nstream,5 /-AGCGCAAGTACTCCGTGTG-3 zo 产品的尺寸分另为livin a是 B312/258 bp ,3-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-HCI (pH 7.5 ) , 250 mM NaCI,0.1% NP40 和口5mM EGTA包含50mM氟化钠，60mM B-丙三醇-磷酸盐,0.5mM钒酸钠,0.1mM苯 甲基磺 醯化氟10卩g/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。

10、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应（细胞信号技术、美国），最后通过增强化学荧光达到可视化（细胞信号技术、美国）。Alexesis （美国）和散塔克鲁兹生物技术（美国）购买的单克隆抗体livin （ 1:250） and actin （ 1:400）24细胞系和细胞培养我们选了一个人类胃腺癌细胞系在这一研究中。SGC-7901 （上海细胞研究所，中国上海,）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

11、（ 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37T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4C储存。2.5。 ShRNA的合成和PGPU / GFP /Neo/livin 质粒的制造通过siRNASequence-Selecto 软件设计并合成了 Livin的ShRNA序列（上海生物 技 术有限责任公司。集团公司、中国）。序列如下（表1），然后被插入pGPU/GFP/Neo （上 海 Ge nePharm 股份有限

12、公司。中国）Bbsl a nd BamH 地址产生 pGPU/GFP/Neo/liv in and pGPU/GFP/Neo/Co ntro 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（3为05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被调控子用Li-pofectAMINE 2000转染4毫克/孔空的 pGPU/GFP/Neo/vector , pGPU/GFP/Neo/livi

13、n 或 pGPU/GFP/Neo/C on trol 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 Gen eticin （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。2. 7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, F

14、L ）.。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对细胞毒性进行了测量。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。每孔加入100微升MTT溶液（1毫克/毫升），并且这些细胞放在37C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加入冰冷的70%乙醇于PBS缓冲液中储存在-4摄C暖直

15、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卩g/ml） 37下 培养细 胞并用流式细胞术（BD,美国）进行分析。2.10统计分析数据的展现要用至少三个不同实验iSD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应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

16、及 livina和livin蛋白质表达（Figs. 1 and 2 ） . olivin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） O3.2。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应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

17、选择作为扩展和另外的研究。（如图4及5）显示，livin mRNA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/livi n1转染和消极控制中没有被发现。所以SGC-7901pGPU/GFP/Neo/livi n2 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细胞数目有显著下降转染在72小时到96小时后镀（P 0.01

18、）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测定表明，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氟尿嘧啶诱导凋亡细胞数增加至约2.5 - 3倍的pGPU/G

19、FP/Neo/livin2转相比，其控制细胞（PV0.001;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，我们表明，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，也表明，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似乎是在阻断DNA损伤剂诱导细胞凋亡13

20、超过活着有效。一些组织中Livin分布研究表明，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 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。我们的第一次研究表明，活着亚型 A和B几乎都在胃癌组织（47.5%和Livin表达与 一些已知的预后因素，如分级，淋巴结转移，相关的比例计算。从文献资料表明，这两个活着亚型参与

21、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 o这些结果表明，Livin的高表达 可能对某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基础上发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 个极其有力的工具，抑制内源性基因表达24,25作出抑制Livin基 因，并试图纠

22、正胃癌细胞凋亡的不足。作者：沉默的shRNA功效的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 的研究结果还表明，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细胞凋亡反应，抑制细胞的生长，这表明，与美好生活HeLa细胞类似的结果报告了的干扰导致了对凋亡刺激的敏感性。对Crnkovic -梅坦斯18 。总之，我们的结果表明，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

23、化疗药物的结果。由于在胃癌中的表达，但活着的优惠在正常组织中，这些数据表明，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服，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办法，如高效输送到目标细胞的免疫反应或规避，一般的挑战。值得注意的是，在最近的研究表明，体内的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。这些数据表明，一个活跃的 shRNA通过血液的直接应用是主要可行的。英文翻译：对照版Expressi on of liv in in gastric can ce

24、r and in ducti on of apoptosis in SGC-7901 cells by shRNA -mediated sile nci ng of liv in geneBackgro un d-Because of in creased resista nee to apoptosis in tumor cells, i n hibiti on of specific an tiapoptotic factors may provide a ratio nal approach for the developme nt of no vel therapeutic strat

25、egies.Liv in, a no vel in hibitor of apoptosis prote in family, has bee n found to be expressed in various malig nan cies and is suggested to have poorly prog no stic sig nifica nee. However, no data are available concerning the sig nifica nee of livin in gastric cancer. In this study, we detected t

26、he expression of livin in human gastric carc ino ma a nd inv estigated the apoptotic susceptibility of SGC-7901 cell by shRNAmediated sile ncing of the liv in gene.Methods-The mRNA and protein expressi on of livi n were an alyzed by RT-PCR and wester n blot assay.The relati on ship betwee n liv in e

27、xpressi on and cli ni cal pathologic parameters was in vestigated. The small in terferi ng RNA eukaryotic expressi on vector specific to liv in was con structed by gene recomb in ati on, and the nu cleic acid was seque need. The n it was tran sfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR

28、and Western blot assay were used to validate gen e-sile ncing efficie ncy of liv in in SGC- 7901 cells. Stable clones were obta ined by G418 scree ning. The cell apoptosis was assessedby flow cytometry (FCM). Cell growth state and 50 % in hibiti on concen trati on (IC50) of 5-FU and cisplat in was d

29、eterm ined by MTT method.Results-The expressi on of liv in mRNA and protein were detected in 19 of 40 gastric carci noma cases (47.5%) and SGC-7901 cells. No expressi on of livi n was detected in tumor adjace nt tissues and benign gastric lesi on. The positive correlati on was found between livin ex

30、pression and poor differentiation of tumors as well as lymph node metastases (P 0.05). Four small in terferi ng RNA eukaryotic expressi on vector specific to liv in were con structed by gene recomb in ati on. And one of them can efficiently decrease the expression of livin, the inhibition of the gen

31、e was not less than 70% (P 0.01). The recomb in ated plasmids were extracted and tran sfected gastric can cer cells. The stable clones were obta ined by G418 scree ning, and were amplified and cultured. Whe n liv in gene was sile need, the reproductive activity of the gastric can cer cells was sig n

32、ifica ntly lower tha n the con trol groups(P 0.05). The study also showed that IC50 of 5-Fu and cisplat in on gastric can cer cells treated by shRNA was decreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplati n) (P 0.01).Con clusi ons-C Livi n is overexpressed in gas

33、tric carc inoma with a relati on ship to tumor differe n tiati on and lymph node metastases, which is suggested to be one of the molecular prog no stic factors for some cases of gastric can cer. ShRNA can in hibit livi n expressi on in SGC-7901 cells and in duce cell apoptosis.Li vin may serve as a

34、new target for apoptosis-i nduc ing therapy of gastric can cer.1. In troducti onGastric can cer is one of the most com mon malig nan cies in the world. Most patie nts with this diseaseare diag no sed i n adva n eed stages, and lose the cha nee of surgical eradicati on. Despite much progress in chemo

35、therapy,the overall survival of the patie nts with gastric cancer in advaneed stage is still poor. Resistanceof cancer cells to chemoage nts may eon tribute to failure of the treatme nt. Among the reas ons of drug resista n ee, i n hibited process of cell apoptosis may play an importa nt role.Can ce

36、r cells are ofte n characterized by in creased resista nee to apoptosis 1, which mediates their in creased resista nee to various stimuli of cell apoptosis, such as DNA damage, hypoxia, nu trie nt-deprivati on 2,3. Moreover, apoptosis resista nee is con sidered to be a major cause of therapeutic fai

37、lure for tumors in cli ni cal practice, since many chemo- an d/or radiotherapeutic age nts fun cti on through the in duct ion of apoptotic tumor death 4.In hibitor of apoptosis protein (IAP s) is a no vel family of in tracellular prote ins which suppress apoptosis in duced by a variety of stimuli 5,

38、6, i nclud ing viral in fecti on, chemotherapeutic drugs, staurospori n, growth factor withdrawal, and by comp onents of the tumor n ecrosis factor-a (TNF-a)/Fas apoptotic sig nali ng pathways 7 C9. The IAPs con sists of a group of structurally related prote ins with an tiapoptotic properties 10, an

39、d may play a substa n tial role in preve nti ng tumor cell from apoptosis, and has become the focus of research in recent years. A novel member of this family is ML-IAP/livin/KIAP/BIRC7 (in the followi ng termed livi n) which has two isoforms, livi n a and liv in b 11 C14lt has bee nshow n that over

40、-expressi on of the liv in can block apoptosis in duced by a variety of proapoptotic stimuli 12. I n teresti n gly, liv in gene has bee n found to be restrictively expressed in tumor cells, but not, or to lesser amounts in most no rmal adult tissues 11 C15, an dmay con tribute to tumorige nesis by a

41、llowing malignant cell to avoid apoptotic cell death. So inhibition of livin expressi on may represe nt an in teresti ng therapeutic strategy.In the prese nt study, we in vestigated the expressi on of liv in in gastric cancino mas and their adjace nt tissues. The relati on ship betwee n liv in expre

42、ssi on and cli ni cal pathologic parameters was an alyzed. Furthermore, we explored the feasibility of shRNA in inhibiting livin gene expression and the apoptotic susceptibility of gastric cancer cell by shRNA-mediated sile ncing of the liv in gene.2. Patie nts and methods2.1. Patie nts and tumor sa

43、mplesForty samples of gastric carci noma and 13 samples of paraca ncerous tissues were collected from the patie nts who received gastrectomy (age of patie nts ranging from 29-77 years). Thirtee n samples of benign gastric lesi on (chr on ic superficial gastritis) were gained from the patie nts un de

44、rg oing gastric en doscopic exam in ati on (age of patients ranging from 33-77 years). These samples were collected from patie nts admitted to the First Affiliated Hospital of Nanjing Medical University. The patients with gastric cancer were diag no sed as being in stage I to IV based on TNM classif

45、icati on (UICC, 2002). Tumor specime ns were immediately froze n in liquid n itroge n after surgery and stored at -80Cun til use. In formed consent was obta ined from all patie n ts.2.2. RT-PCR procedureTotal RNA (2 mg) extracted from froze n tissues was reverse tran scribed in a final volume of 25

46、ml with 100 pmol of oligo(dT)15 and 200U M-MLV reverse transcriptase (promega, USA), accord ing to the manu facturer s guideli nes.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. Each amplific

47、ati on was performed for 35 cycles, one cycle profile con sisted of den aturati onat 94 8C for 30 s, ann eali ng at 59 8C (livin and b-acti n) for 30 s and exte nsio n at 72 8C for 30 s. A sample without RNA was in cluded in each RTCPCR as a n egative con trol.Seque nces of liv in and 3-actin primer

48、s used are as follows:liv ina/b up stream,50- TCCACAGTGTGCAGGAGACT- 30;liv ina/B dow nstreamACGGCACA AAGACGATGGAC-30;b-act in upstream,50-AGCGCAAGTACTCCGTGTG- 30; -acti n dow nstream, 50- AAGCAATGCTATCACCTCCC-30.The size of the amplified products were312/258 bp for livi na/b and 501 bp for b-act in

49、respectively.2.3. Wester n Blot An alysisTissues were homoge ni zed with lysis buffer 50 mM Tris-HCI(pH 7.5), 250 mM NaCl,0.1% NP40, and 5 mM EGTA con tai ning 50 mM sodium fluoride, 60 mM b-glycerol- phosphate, 0.5 mM sodium van adate, 0.1 mM phe nyl methylsulfo nyl fluoride, 10 mg/ml aprot inin, a

50、nd 10 mg/ml leupepti n. The total prote in concen trati on was determ ined using Coomassie Brillia nt Blue. Protein samples were electrophoresed in a 10% den aturi ng SDS gel a nd tran sferred to PVDF membra ne (Roche, USA). The membra nes were in cubated with specific primary an tibodies, reacted w

51、ith a peroxidase-c on jugated sec on dary an tibody (Cell sig nali ng tech no logy,USA), and fin ally visualized by enhan ced chemilu min esce nee (Cell sig n ali ng tech no logy, USA). Monoclonal an tibodies recog nizing liv in (1:250) and act in (1:400) were purchased from Alexesis Inc. (USA) and

52、San ta Cruz Biotech nology (USA).2.4. Cell li nes and cell cultureWe selected a huma n gastric ade no carc inoma cell li nes for thisstudy. SGC-7901 (Sha n ghai In stitute of Cell Research, Sha n ghai,Chi na) is an adhere nt, moderately differe ntiated, huma n gastric ade no care inoma cell li ne. T

53、he cell li nes are gastric cancer epithelial cells and grow as adhere nt cells in RPMI 1640 (Hyclo ne Inc, USA)co n tai ning 10% FCS (Life Tech no logies, I nc.), 100 uni ts/ml pen icilli n,and 100 mg/ml streptomycin (BioWhittaker). SGC-7901 cells were maintainedat37 8Cina humidified in cubatorwitha

54、 natmosphere of 5% CO2. Cisplatin and 5-fluorouracil (Qilu pharmaceutical factory,Chi na) were solublized in DMSO and stored at 4 8C.2.5. ShRNA sy nthesis and con struction of PGPU/GFP/Neo/livin plasmidsShRNA seque nces of livi nwere desig ned by software of siRNASeque n ce-Selector and syn thesized

55、 (Sha n ghai Biotech, Ltd.Corp., Chin a). Thesequences as following (Table 1)then were inserted into Bbsl and BamH sites of the pGPU/GFP/Neo(Sha nghai Ge nePharma Co. Ltd Chi na) to gen erate pGPU/GFP/Neo/liv in and pGPU/GFP/Neo/C on trol plasmids,respectively.2.6. Establishme nt of SGC-7901 stable

56、tran sfecta nts expressi ng pGPU/GFP/Neo/livi n and pGPU/GFP/Neo/C on trolFor tran sfecti on experime nts, SGC-7901 cells were plated in to 6-well plates45(3?a 105 cells/well), 96-well plates (1 X0 cells/well) and 12-well plates (1.5 10 cells/well) for 24 h before tran sfecti onThe cells were tran s

57、fected with 4 mg/well of empty pGPU/GFP/Neo/vector, pGPU/GFP/Neo/liv in or pGPU/GFP/Neo/C on trol plasmid us ing Li-pofectAMINE 2000 (Life Tech no logies, In c., Grand Isla nd,NY) accord ing to the manu facturer?ls in struct ions. Forty-eight hours after tran sfecti on, the cells were passaged at 1:

58、15 (v/v) and cultured in mediumsuppleme nted with Gen etic in (G418) at 1000 g/ml for 4 weeks. Stably tran sfected clones were picked and mai n tai ned in medium containing 400 g/ml G418 for additi onal studies.2.7. Assay of an chorage-depe ndent cell growthPare nt cells and cells stably express ing empty pGPU/GFP/Neo vector, pGPU/GFP/Neo/liv in or pGPU/GFP/Neo/C on trol were seeded into 6-well plates. Cells from triplicate wells were c