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

Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin geneBackground-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 of apoptosis protein family, has been found to be expressed 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 expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC-7901 cell by shRNAmediated silencing of the livin gene.Methods-The mRNA and protein expression of livin were analyzed by RT-PCR and western blot assay.The relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expression vector specific to livin was constructed by gene recombination, 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 livin 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 expression of livin mRNA and protein were detected in 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin expression and poor differentiation of tumors as well as lymph node metastases (P < 0.05). Four small interfering RNA eukaryotic expression vector specific to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% (P < 0.01). The recombinated plasmids were extracted and transfected gastric cancer cells. 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 cells treated by shRNA wasdecreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P < 0.01).Conclusions-C Livin is overexpressed in gastric carcinoma with a relationship to tumor differentiation and lymph node metastases, which is suggested to be one of the molecular prognostic factors for some cases of gastric cancer. ShRNA can inhibit livin expression in SGC-7901 cells and induce cell apoptosis.Livin may serve as a new target for apoptosis-inducing therapy of gastric cancer.1. IntroductionGastric cancer is one of the most common malignancies in the world. Most 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 failure 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 damage, hypoxia, 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 (IAPs) 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 components of the tumor necrosis factor-a (TNF-a)/Fas apoptotic signaling pathways [7¨C9]. The IAPs 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 become the focus of research in recent years. A novel member of this family is ML-IAP/livin/KIAP/BIRC7 (in the following termed livin) which has two isoforms, livin a and livin b [11¨C14]. It has been shown that over-expression of the livin can block apoptosis induced by a variety of proapoptotic stimuli [12]. Interestingly, livin gene has been found to be restrictively expressed in tumor cells, but not, or to lesser amounts in most normal adult tissues [11¨C15], and may contribute to tumorigenesis by allowing malignant cell to avoid apoptotic cell death. So inhibition of livin expression may represent an interesting therapeutic strategy.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 expression and the apoptotic susceptibility of gastric cancer cell by shRNA-mediated silencing of the livin gene.2. Patients and methods2.1. Patients and tumor samplesForty 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 gastritis) were gained from the patients undergoing gastric endoscopic examination (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 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) 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 manufacturer’s 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. 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 extension at 72 8C for 30 s. A sample without RNA was included in each RT¨CPCR as a negative control.Sequences of livin and β-actin primers used are as follows:livina/b up stream,50-TCCACAGTGTGCAGGAGACT-30;livina/βdownstream,50-ACGGCACA AAGACGATGGAC-30;b-actinupstream,50-AGCGCAAGTACTCCGTGTG-30;β-acti n downstream, 50-AAGCAATGCTATCACCTCCC-30.The size of the amplified products were312/258 bp for livina/b and 501 bp for b-actin respectively.2.3. Western Blot AnalysisTissues were homogenized with lysis buffer [50 mM Tris-HCl(pH 7.5), 250 mMNaCl, 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. The total protein concentration was 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 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 Santa Cruz Biotechnology (USA).2.4. Cell lines and cell cultureWe 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 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 incubatorwithanatmosphere of 5% CO2. Cisplatin and 5-fluorouracil (Qilu pharmaceutical factory,China) were solublized in DMSO and stored at 4 8C.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 sequences 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.2.6. Establishment of SGC-7901 stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/ControlFor transfection experiments, SGC-7901 cells were plated into 6-well plates (3¡Á105 cells/well), 96-well plates (1×104cells/well) and 12-well plates (1.5×105 cells/well) for 24 h before transfectionThe 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., Grand Island,NY) according to the manufacturer¡¯s 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 400 g/ml G418 for additional studies.2.7. Assay of anchorage-dependent cell growthParent 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 triplicate 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.2.8. MTT assayCytotoxicity 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 treated 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 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.2.9. Flow cytometryCells were collected and fixed with ice-cold 70% ethanol in PBS and stored at -4℃until use. After resuspension, cells were incubated 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).2.10. Statistical analysisData were expressed as the means of at least three different experiments SD. The results were analyzed by Student’s t-test, and P < 0.05 was considered statistically significant.3. Results3.1. Expression of livin in gastric carcinomasIn the present study, for the first time, we evaluated by RT¨CPCR 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, whileamong 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 (Table 2).3.2. Characterization of stable transfectants expressing pGPU/GFP/Neo/livin and pGPU/GFP/Neo/ControlWe 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 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/GFP/Neo/livin1 transfectants and negative control. So SGC-7901 pGPU/GFP/Neo/livin2 transfectants was chosen for subsequent experiment.3.3. Inhibition of cell growth in stable transfectantsThe growth rate of SGC-7901 pGPU/GFP/Neo/livin2 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.3.4. Stable transfectants were more susceptible to proapoptotic stimuliWe treated SGC-7901 pGPU/GFP/Neo/livin2 transfectants and negative control cells with cytotoxic drugs (5-fluorouracil and cisplatin). MTT assay showed 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 2.5¨C3-fold in pGPU/GFP/Neo/livin2 transfectants compared 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).4. DiscussionIn this study, we show 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- 7901 cellsgrowth 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 more effective than livin a in blocking apoptosis induced by DNA damaging agents[13].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 kidney and livin a alone has been detected 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 available 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, 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 chemoradiotherapy 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.The specific 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 its 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 livin gene and attempt to correct the apoptosis deficiency of gastric tumor cells. The efficacy of shRNAs to silence expression of a tageted gene is different, relation with the half-life and abundance of the gene product as well as with accessibility of target mRNA [24-27]. In this study, we observed that si-livin1 was regularly more strongly silence the livin gene than si-livin2. Our study results alsoshown that silencing livin gene expression may strongly increase apoptotic response of SGC-7901 cells in the presence or absence of several proapoptotic agents and inhibit the cells growth, which indicate that the interference with livin leads to a sensitization to proapoptotic stimuli. The similar result on hela cell was reported by Crnkovic-Mertens [18].In summary, our results showed that inhibition of livin expression and function resulted in spontaneous apoptosis and inhibitor cell growth enhanced sensitivity to cytotoxic drugs in vitro. Because of the preferential expression of livin in gastric cancer but not in normal tissues, these data suggest that targeting the livin pathway alone or with cytotoxic drugs may be useful in the treatment of gastric cancer. Despite their therapeutic potential, major technical hurdles still have to be overcome, in order to apply shRNAs as drugs. Under therapeutic aspects, will have to meet the general challenges of gene therapy approaches, such as efficient delivery into the target cells or the circumvention of immune responses. Notably, recent in vivo studies showed that shRNAs could be directly applied to organs of postnatal mice by highpressure injection into the tail vein, leading to the specific inhibition of target genes [28-30]. These data show that a direct application of active shRNAs via the bloodstream is principally feasible.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。