肺癌沉默基因

1、Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene Report person ： 王晓磊Student id ：2014050448介导的介导的shRNA能抑制肺癌细胞中能抑制肺癌细胞中livin沉默基沉默基因的表达从而促进因的表达从而促进SGC-7901细胞凋亡细胞凋亡 Background Because of increased resistance to apoptosis in tumo

2、r cells, inhibition of specific antiapoptotic factors may provide a rational approach for the development of novel therapeutic strategies.Livin, a novel inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poorly prognostic significan

3、ce. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detected the expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene. 背景背景 由于肿瘤细胞抑制凋亡增殖,特定凋亡

4、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。1. Introduction Gastric cancer is one of the most common malignancies in the world. Most patients with this disease are diagnosed in advanced st

5、ages, 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 failure of the treatment. Among the reasons of drug resistan

6、ce, inhibited process of cell apoptosis may play an important role. 1介绍介绍 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

7、过诱导凋亡肿瘤死亡实现的4 In the present study, we investigated the expression of livin in gastric cancinomas and their adjacent tissues. The relationship between livin expression and clinical pathologic parameters was analyzed. Furthermore, we explored the feasibility of shRNA in inhibiting livin gene expressi

8、on and the apoptotic susceptibility of gastric cancer cell by shRNA-mediated silencing of the livin gene. 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. Patients and methods 2.1. Patients and tumor samples Forty sample

9、s 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 gastritis) were gained from the patients undergoing gastric endoscopic e

10、xamination (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 I to IV based on TNM classification (UICC, 2002). Tumor specime

11、ns were immediately frozen in liquid nitrogen after surgery and stored at -80 until use. Informed consent was obtained from all patients. 2患者和方法患者和方法 2.1。患者和肿瘤样本 胃癌中四十个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 ，20

12、02)。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. RT-PCR procedureTotal RNA (2 mg) extracted from frozen tissues was reverse transcribed in a final volume of 25 ml with 100 pmol of oligo(dT)15 and 200U M-MLV reverse transcriptase (promega, USA), according to the manufacturers guidelines.

13、 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 amplification was performed for 35 cycles, one cycle profile consisted of denaturationat 94 8C for 30 s, annealing at 59 8C (livin and b-a

14、ctin) for 30 s and extension at 72 8C for 30 s. A sample without RNA was included in each RTCPCR as a negative control. Sequences of livin and -actin primers used are as follows:livina/b up stream,50-TCCACAGTGTGCAGGAGACT-30;livina/downstream,50-ACGGCACAAAGACGATGGAC-30;b-actinupstream,50-AGCGCAAGTACT

15、CCGTGTG-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.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 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

16、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94。热处理在30s内59（livin and b-actin）扩大到30s内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-AGC

17、GCAAGTACTCCGTGTG-3。产品的尺寸分别为livin/是312/258 bp ，-actin 是501bp。 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 phenylmethylsu

18、lfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. 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 primar

19、y antibodies, reacted with a peroxidase-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 Alexesis Inc. (USA) and

20、Santa Cruz Biotechnology (USA). 2.3 西方吸干技术分析 病变同质性与冲力缓冲50mM Tris-HCl (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

21、(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) 2.4. Cell lines and cell culture We selected a human gastric adenocarcinoma cell lines for thisstudy. SGC-7901 (Shanghai Institute of Cell Research, Shanghai,China) is an adherent, moderately differentiated, human gastric adenocarcinoma cell

22、 line. The cell lines are gastric cancer epithelial cells and grow as adherent cells in RPMI 1640 (Hyclone Inc, USA)containing 10% FCS (Life Technologies, Inc.), 100 units/ml penicillin,and 100 mg/ml streptomycin (BioWhittaker). SGC-7901 cells were maintainedat37 8Cina humidified incubatorwithanatmo

23、sphere of 5% CO2. Cisplatin and 5-fluorouracil (Qilu pharmaceutical factory,China) were solublized in DMSO and stored at 4 8C. 2.4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 (Life Technologies, Inc.),每毫升100个单位的青霉素和毫升10

24、0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 synthesis and construction of PGPU/GFP/Neo/livin plasmidsShRNA sequences of livin were designed by software of siRNA Sequence-Selector and synthesized (Shanghai Biotech, Ltd.Corp., China). The sequen

25、ces as following (Table 1)then were inserted into BbsI and BamH sites of the pGPU/GFP/Neo(Shanghai GenePharma Co. Ltd China) to generate pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control plasmids,respectively. 2.5 ShRNA的合成和PGPU / GFP /Neo/livin质粒的制造 通过siRNASequence-Selector软件设计并合成了Livin的ShRNA序列(上海生物技术有限责任

26、公司。集团公司、中国)。序列如下(表1),然后被插入pGPU/GFP/Neo(上海GenePharma股份有限公司。中国) BbsI and BamH地址产生pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control质子。2.6. Establishment of SGC-7901 stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control For transfection experiments, SGC-7901 cells were plated into 6-well

27、 plates (3105 cells/well), 96-well plates (1104 cells/well) and 12-well plates (1.5105 cells/well) for 24 h before transfection The cells were transfected with 4 mg/well of empty pGPU/GFP/Neo/vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control plasmid using Li-pofectAMINE 2000 (Life Technologies, Inc

28、., Grand Island,NY) according to the manufacturers instructions. Forty-eight hours after transfection, the cells were passaged at 1:15 (v/v) and cultured in mediumsupplemented with Geneticin (G418) at 1000 g/ml for 4 weeks. Stably transfected clones were picked and maintained in medium containing 40

29、0 g/ml G418 for additional studies. 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

30、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。 2.7. Assay of anchorage-dependent cell growth Parent cells and cells stably expressing empty pGPU/GFP/Neo vector, pGPU/GFP/Neo/livin or pGPU/GFP/Neo/Control were seeded into 6-well plates. Cells from tr

31、iplicate wells were collected every other day. Cell numbers were determined using a Coulter counter (Coulter Electronics, Miami, FL). The number of cells per well is reported as the average SD at the indicated number of days after plating. 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

32、/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, FL).。种植一些天数后用平均SD记录每孔细胞的数量。 2.8. MTT assay Cytotoxicity was measured by MTT assay. Cells growing exponentially were plated onto 96-well plates at a density of 10000 cells/well for 24 h. The cells were then tre

33、ated with different concentrations of drugs for 48 h. One hundred microliters of MTT stock solution (1 mg/ml) were added to each well, and the cells were further incubated at 37 for 4 h. The supernatant was replaced with isopropyl alcohol to dissolve formazan production. The absorbance at wavelength

34、 595 nm was measured with a micro-ELISA reader (ClinBio-128, SLT, Austria). The ratio of the absorbance of treated cells relative to that of the control cells was calculated and expressed as a percentage of cell death. 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4

35、8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光率并用细胞死亡百分率显示出来。 2.9. Flow cytometry Cells were collected and fixed with ice-cold 70% ethanol in PBS and stored at -4 until use. After resuspension, cells were incubate

36、d with 100 ml of RNase I (1 mg/ml) and 100 ml of PI (400 mg/ml) at 37 and analyzed by flow cytometry (BD, USA). 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)进行分析。 2.10. Statistical analysis Data were expressed as the means o

37、f at least three different experiments SD. The results were analyzed by Students t-test, and P 0.05 was considered statistically significant. 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. Results 3.1. Expression of livin in gastric carcinomas In the present study, for the firs

38、t time, we evaluated by RTCPCR and westen blot the presence of livin expression in 40 gastric cancinomas, 13 para-cancerous tissues and 13 benign lesions of gastric mucosa. In para-cancerous tissues and benign lesions of gastric mucosa, no detectable levels of either mRNA isoforms were revealed, whi

39、le among tumor tissues, 19/40(47.5%) showed mRNA and protein expression of livina and livinb (Figs. 1 and 2). Livin expression correlated with some of the known prognostic variables, such as histologic grade and lymph node metastasis, but not with age, sexuality, stage and tumor infiltration extent

40、(Table 2).结果结果 3.1livin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. Characterization of stable transfectants express

41、ing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/Control We established SGC-7901 stable transfectants with either pGPU/GFP/Neo/livin, pGPU/GFP/Neo/Control plasmid, or empty pGPU/GFP/Neo/vector (Fig. 3). Some clones from each transfection were selected and analyzed by RT-PCR and Western blot to determine the

42、livin mRNA and protein expression, and others were selected for expansion and additional studies. As shown in (Figs. 4 and 5), the level of livin mRNA and protein in SGC-7901 pGPU/GFP/Neo/livin2 transfectants was reduced by more than 90%. The suppression of livin expression was not observed in pGPU/

43、GFP/Neo/livin1 transfectants and negative control. So SGC-7901 pGPU/GFP/Neo/livin2 transfectants was chosen for subsequent experiment. 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应技术选

44、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， 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/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. Inhibition of cell growth in stable transfectants The growth rate of SGC-7901 pGPU/GFP/Neo/liv

45、in2 transfectants was significantly inhibited. As shown in Fig. 6, SGC-7901 pGPU/GFP/Neo/livin2 transfectant cells number had significant decreases at 72 h and 96 h after plating (P 0.01) compared with negative control and parent cells. 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

46、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),而阴性对照和家长的细胞。 3.4. Stable transfectants were more susceptible to proapoptotic stimuli We treated SGC-7901 pGPU/GFP/Neo/livin2 transfectants and negative control cells with cytotoxic drugs (5-fluorouracil and cisplatin). MTT assay sho

47、wed that SGC-7901 pGPU/GFP/Neo/livin2 transfectants were more sensitive to cisplatin and 5-fluorouracil than negative control and parent cells (Figs. 7A, 6B). The number of apoptotic cells induced by cisplatin and 5-fluorouracil increased to about 2.5C3-fold in pGPU/GFP/Neo/livin2 transfectants comp

48、ared with their control cells (P 0.001; Fig. 7C). Furthermore, stable transfectants underwent spontaneous apoptosis more readily without proapoptotic stimuli than the control cells (P 0.05; Fig. 7C). 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/G

49、FP/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/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。4. Discussion In this study, we show

50、that livin, a new member of the IAP family, was found to be not expressed in any of the NOT cancerous gastric tissues, and expressed only in a proportion of gastric cancer patients (47.5%), and also show that suppressing livin expression or function causes spontaneous apoptosis and inhibition of SGC

51、- 7901 cells growth and make cells more susceptible to proapoptotic stimuli. It was thought that livin has two isoforms, a and b. Although both isoforms are involved in blocking apoptosis induced by TNF-a and anti-CD95 in vitro, they show some different antiapoptotic properties. livin b seems to be

52、more effective than livin a in blocking apoptosis induced by DNA damaging agents13.讨论讨论 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

53、胞凋亡13超过活着有效。Some study on tissue distribution of livin has recently shown that elevated levels of both livin isoforms a and b have been detected in heart, placenta, lung, spleen and ovary, while livin balone has been detected specifically in fetal tissues and dult kidneyand livin a alone has been de

54、tected in brain, skeletal muscle and peripheral blood lymphocytes 11-14. Furthermore, while livin expression was detected in a variety of cancerous cell lines and some tumor tissues 14-18 and anti-livin antibody was recognized in sera of gastric cancer and lung cancer patients 19,20, no data were av

55、ailable concerning the expression of livin isoforms in gastric tumor tissues. Our study for the first time demonstrates that livin isoforms a and b were almost both expressed in a proportion of gastric cancer tissues (47.5%) and livin expression correlate with some of the known prognostic variables,

56、 such as grade and lymphonode metastasis. Data from the literature have demonstrated that both livin isoforms are involved in blocking apoptosis and may give cells with livin overexpression a strong resistance to chemotherapy-induced apoptosis. Gastric cancer in general is highly resistant to chemor

57、adiotherapy and moderately resistant to apoptosis 21. These result suggested that overexpression of livin may effect the responsibility of chemotherapy on some gastric cancer patients and prognosis of patients. 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一单是在脑，骨骼肌

58、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。 The spec

59、ific interference with factors contributing to the apoptosis resistance of tumor cells may provide a novel basis for the development of rational intervention strategies in cancer therapy 22,23. Since the expression of livin could contribute to the apoptosis-resistant phenotype of cancer cells and it

60、s specific expression in tumors could make livin an interesting therapeutic target for tumor-specific intervention strategies, we chose the livin gene as a molecular target. The shRNA technology representiong an extremely powerful tool to inhibit endogenous gene expression 24,25 be made to inhibit l