siRNA干扰galectin-3表达对人胃癌细胞株SGC-7901增殖、凋亡和化疗敏感性的影响

Galectin-3属于半乳凝素家族成员,参与细胞生长和凋亡、细胞黏附、新生血管形成、肿瘤浸润和转移等多种生理和病理过程,在多种恶性肿瘤细胞中呈高表达。目的:研究siRNA干扰galectin-3表达对人胃癌细胞株SGC-7901增殖、凋亡和化疗敏感性的影响。方法:合成靶向galectin-3的siRNA并转染SGC-7901细胞,以real time PCR和蛋白质印迹法检测干扰效果。CCK-8实验检测细胞增殖,流式细胞术检测细胞凋亡。结果:Galectin-3siRNA转染24 h后转染效率为83.8%,转染后SGC-7901细胞的galectin-3表达显著受抑,mRNA和蛋白表达量分别较空白对照组降低87.8%和90.4%(P0.01)。转染后24 h、48 h和72 h,galectin-3 siRNA组SGC-7901细胞增殖抑制率分别为15.57%±1.45%、32.90%±0.76%和57.35%±1.05%,转染后72 h该组细胞凋亡率为46.17%±2.39%,均显著高于同时间点空白对照组、空脂质体组和阴性对照siRNA组(P0.01)。Galectin-3 siRNA组SGC-7901细胞由化疗药物奥沙利铂诱导的增殖抑制亦较其余三组显著增加(P0.01)。结论:以siRNA干扰galectin-3表达后,SGC-7901细胞增殖减少、凋亡增加,对化疗药物的敏感性增强,表明galectin-3有望成为胃癌基因治疗的有效靶点。     

Function of chloride intracellular channel 1 in gastric cancer cells

AIM: To investigate the effect of chloride intracellular channel 1 (CLIC1) on the cell proliferation, apoptosis, migration and invasion of gastric cancer cells.METHODS: CLIC1 expression was evaluated in human gastric cancer cell lines SGC-7901 and MGC-803 by real time polymerase chain reaction (RT-PCR). Four segments of small interference RNA (siRNA) targeting CLIC1 mRNA and a no-sense control segment were designed by bioinformatics technology. CLIC1 siRNA was selected using Lipofectamine 2000 and transfected transiently into human gastric cancer SGC-7901 and MGC-803 cells. The transfected efficiency was observed under fluorescence microscope. After transfection, mRNA expression of CLIC1 was detected with RT-PCR and Western blotting was used to detect the protein expression. Proliferation was examined by methyl thiazolyl tetrazolium and apoptosis was detected with flow cytometry. Polycarbonate membrane transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of the two cell lines.RESULTS: In gastric cancer cell lines SGC-7901 and MGC-803, CLIC1 was obviously expressed and CLIC1 siRNA could effectively suppress the expression of CLIC1 protein and mRNA. Proliferation of cells transfected with CLIC1 siRNA3 was enhanced notably, and the highest proliferation rate was 23.3% (P = 0.002) in SGC-7901 and 35.55% (P = 0.001) in MGC-803 cells at 48 h. The G2/M phase proportion increased, while G0/G1 and S phase proportions decreased. The apoptotic rate of the CLIC1 siRNA3 group obviously decreased in both SGC-7901 cells (62.24%, P = 0.000) and MGC-803 cells (52.67%, P = 0.004). Down-regulation of CLIC1 led to the inhibition of invasion and migration by 54.31% (P = 0.000) and 33.62% (P = 0.001) in SGC-7901 and 40.74% (P = 0.000) and 29.26% (P = 0.002) in MGC-803. However, there was no significant difference between the mock group cells and the negative control group cells.CONCLUSION: High CLIC1 expression can efficiently inhibit proliferation and enhance apoptosis, migration and invasion of gastric cancer cells in vitro. CLIC1 might be a promising target for the treatment of gastric cancer.     

Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity

AIM: To investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of survivin in pancreatic cancer cell line PC-2 and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity. METHODS: A siRNA plasmid expression vector against survivin was constructed and transfected into PC-2 cells with LipofectamineTM 2000. The down regulation of survivin expression was detected by semi-quantitive RT-PCR and immunohistochemical SP method and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity was detected by flow cytometry. RESULTS: The sequence-specific siRNA efficiently and specifically down-regulated the expression of survivin at both mRNA and protein levels. The expression inhibition ratio was 81.25% at mRNA level detected by semi-quantitive RT-PCR and 74.24% at protein level detected by immunohistochemical method. Forty-eight hours after transfection,apoptosis was induced in 7.03% cells by siRNA and in 14.58% cells by siRNA combined with radiation. CONCLUSION: The siRNA plasmid expression vector against survivin can inhibit the expression of survivin in PC-2 cells efficiently and specifically. Inhibiting the expression of survivin can induce ipoptosis of PC-2 cells and enhance its radiosensitivity significantly. RNAi against survivin is of potential value in gene t(?)erapy of pancreatic cancer.     

Ectopic expression of guanylyl cyclase C in gastric cancer as a potential biomarker and therapeutic target

OBJECTIVE: To investigate the expression of guanylyl cyclase C (GCC) in human gastric cancer (GC) tissues and assess the effect of GCC small interfering RNA (siRNA) on the proliferation and apoptosis of SGC‐7901. METHODS: The expression of GCC in 30 specimens and three human GC cell lines (SGC‐7901, AGS, NCI‐N87) were detected by RT‐PCR for messenger RNA (mRNA) by Western blot and immunofluorescence for proteins. Recombinant plasmids containing GCC siRNA and scrambled siRNA were constructed and transfected into SGC‐7901 cells, respectively. A cell counting kit‐8, flow cytometry (FCM) and terminal deoxynucleotidyl transferase (TDT)‐mediated dUTP‐biotin nick end‐labeling were used to evaluate cell viability, cell cycle distribution and apoptosis, followed by wound healing assay and cell adherent assay for cell motility and adherent, respectively. RESULTS: The expression of GCC was absent in paracancerous tissues, whereas the GCC mRNA and protein expressions were detected in 20/30 and 19/30 of GC specimens, respectively. Moreover, intestinal GC was statistically different from diffuse GC (P < 0.05). The proliferation of SGC‐7901 cells was markedly inhibited by GCC siRNA‐3 (P < 0.05) and cell morphological changes including volumetric reduction, karyopyknosis and karyorrhexis were observed. FCM showed that the cell count in the sub‐G0/G1 peak increased from 5.47% (48 h after transfection) to 5.63% (72 h after transfection). The wound healing assay and cell adherent assay revealed that GCC gene silencing decreased cell motility and adherent. CONCLUSION: The over‐expression of GCC has been detected in intestinal type GC. GCC siRNA can effectively inhibit the proliferation and invasion of SGC‐7901 cells and induce cell apoptosis. GCC might be a novel biomarker and therapeutic target for GC.     

Inhibitory effect of vascular endothelial growth factors-targeted small interfering RNA on proliferation of gastric cancer cells

AIM： To examine the effects of vascular endothelial growth factor （VEGF）-targeted small interfering RNA （siRNA） on proliferation of gastric cancer cellsin vitro.
METHODS： Several siRNAs were transfected into human gastric cancer cell line SGC-7901 with Lipofectamine 2000. Cells not transfected with LipofectamineTM 2000 or scrambled （SCR） siRNA served as controls. The inhibitory effect of siRNA on the expression of VEGF mRNA and protein was detected by RT-PCR and ELISA. MTT assay was used to examine the inhibition rate of cell growth.The change in cell cycling of siRNA-treated cells was detected by flow cytometry.
RESULTS： siRNA targeting human VEGF effectively inhibited the proliferation of gastric cancer cell lineSGC-7901 and the distribution of cell cycle. The percentage of G0/G1 phase was significantly higher in siRNA1- and siRNA2-transfected cells than in control cells.The expression of VEGF mRNA was significantly inhibited in siRNA1- and siRNA2-transfected cells compared with that in control cells. VEGF protein notably decreased in siRNA-transfected cells, but had no effect on SCR siRNA.
CONCLUSION： VEGF siRNA inhibits proliferation of gastric cancer cells in vitro.     

小干扰RNA抑制胃癌细胞环氧合酶-2的表达

目的 观察瞬时转染环氧合酶-2特异性小干扰RNA(COX-2 siRNA)对胃癌细胞增殖与凋亡的影响,探讨COX-2在胃癌发生中的作用和RNA干扰方法 对肿瘤的治疗作用.方法 以胃癌细胞系SGC7901为研究对象,瞬时转染COX-2 siRNA,转染后72 h用RT-PCR方法 分别检测COX-2siRNA组、无意义siRNA组及空白对照组的COX-2 mRNA表达;免疫组化法与Western blot检测3组细胞的COX-2蛋白质的表达;流式细胞仪检测3组的细胞周期和凋亡情况.转染后1周内每天同一时间用噻唑蓝比色分析法(MTr)检测3组癌细胞的活力并计算癌细胞的相对生存率.结果 COX-2siRNA对胃癌细胞中COX-2 mRNA及蛋白表达均有明显抑制作用,胃癌细胞增殖受到抑制,凋亡增加,但细胞周期分布无明显变化.结论 在胃癌细胞中,COX-2表达的抑制可降低胃癌细胞的增殖速度,促进肿瘤细胞凋亡,COX-2在胃癌的发生中可能具有重要作用.     

小干扰RNA抑制核因子-κB活化对HepG2细胞凋亡的影响

目的 探讨小干扰RNA(SiRNA)抑制核转录因子(NF)-κB活化对肝癌细胞凋亡的影响.方法 化学合成NF-κB siRNA和阴性对照siRNA,脂质体法转染HepG2细胞,用巢式RT-PCR和荧光定量PCR检测NF-κB mRNA表达情况;免疫组织化学法、酶联免疫吸附法、Western b10t检测NF-κB蛋白表达情况;用磷脂结合蛋白V-异硫氰酸荧光素法检测细胞凋亡,分析NF-κB表达抑制和细胞凋亡间关系.多个样本均数间的比较先行方差齐性检验,方差齐时行单因素方差分析;计数资料比较采用确切概率法分析.结果 NF-κBp65 mRNA在HepG2细胞相对表达量为1.13±0.03,在正常肝细胞L02为0.29±0.07,两者比较,t=27.02,P<0.05,差异有统计学意义.利用NF-κB siRNA干扰可下调NF-κB表达,且呈剂量、时间依赖;NF-κB siRNA转染HepG2细胞72h后,NF-κBmRNA和蛋白表达分别下降了93%和62%,抑制NF-κB表达使HepG2细胞凋亡增加85%. 结论 NF-κB在肝癌细胞中高表达,NF-κB SiRNA能特异性抑制其在肝癌细胞中活化并促进癌细胞凋亡发生.     

Effect of apoptosis on gastric adenocarcinoma cell line SGC—7901 induced by cis—9,trans—11—conjugated linoleic acid

AIM: To determine the effect of apoptosis on gastric cancer cells (SGC-7901) induced by cis-9, trans-11-conjugated linoleic acid (c9, t11-CLA) and its possible mechanism in the inhibition of cancer cells growth. METHODS: Using cell culture, flow cytometery and immunocytochemical techniques, we examined the cell growth, frequency of apoptosis and distribution of cell cycle, expression of ki67, bcl-2, Fas, and c-myc of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25,50,100 and 200 micromol x L(-1)) of c9, t11-CLA for 24 h and 48 h, with a negative control (0.1 % ethanol). RESULTS: The growth of SGC-7901 cells was inhibited by c9,t11-CLA. Eight days after treatment with various concentrations of c9,t11-CLA, as mentioned above, the inhibition rates were 5.9 %, 20.2 %,75.6 % and 82.4 %, respectively. The frequency of apoptosis on SGC-7901 cells induced by different concentrations of c9, t11-CLA (except for 25 micromol.L(-1), 24 h) was significantly greater than that in the negative control (P<0.01). To further investigate the influence of the cell cycle progression, we found that apoptosis induced by c9, t11-CLA may be involved in blocking the cell cycle of SGC-7901 cells. Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations for various time periods significantly decreased the expressions of ki67 (the expression rates were 18.70-3.20 %, at 24 h and 8.10-0.20 % at 48 h, respectively), bcl-2 (4.30-0.15 % at 24 h and 8.05 %-0 at 48 h), and c-myc (4.85-2.20 % at 24 h and 4.75-0.30 % at 48 h) as compared with those in the controls (the expressions of ki67, bcl-2, and c-myc were 15.1 % at 24 h and 13.5 % at 48 h, 6.80 % at 24 h and 8.00 % at 48 h, 5.50 % at 24 h and 5.30 % at 48 h, respectively) (P<0.01), whereas the expressions of Fas were increased (0.60-2.75 %, 24 h and 0.45-5.95 %, 48 h). CONCLUSION: The growth and proliferation of SGC-7901 cells are inhibited by c9, t11-CLA via blocking the cell cycle, pathways of bcl-2-associated mitochondria with reduced expression of bcl-2 and Fas-associated death domain protein (FADD) with enhanced expression of Fas. But expression of c-myc on SGC-7901 cells is lower than that in negative control, which needs to be studied further.     

Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer. METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting. RESULTS: The number of viable cells decreased in a dose-and time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10μmol/L for 48 h or 8μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G_1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h. CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.     

Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

AIM: To evaluate the effects of tributyrin, a pro-drug of natural butyrate and a neutral short-chain fatty acid triglyceride, on the growth inhibition of human gastric cancer SGC-7901 cell. METHODS: Human gastric cancer SGC-7901 cells were exposed to tributyrin at 0.5, 1, 2, 5, 10 and 50 mmol/L(-1) for 24-72 h. MTT assay was applied to detect the cell proliferation. [(3)H]-TdR uptake was measured to determine DNA synthesis. Apoptotic morphology was observed by electron microscopy and Hoechst-33258 staining. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect tributyrin-triggered apoptosis. The expressions of PARP, Bcl-2 and Bax were examined by Western blot assay. RESULTS: Tributyrin could initiate growth inhibition of SGC-7901 cell in a dose- and time-dependent manner. [(3)H]-TdR uptake by SGC-7901 cells was reduced to 33.6 % after 48 h treatment with 2 mmol/L(-1) tributyrin, compared with the control (P<0.05). Apoptotic morphology was detected by TUNEL assay. Flow cytometry revealed that tributyrin could induce apoptosis of SGC-7901 cells in dose-dependent manner. After 48 hours incubation with tributyrin at 2 mmol/L(-1), the level of Bcl-2 protein was lowered, and the level of Bax protein was increased in SGC-7901, accompanied by PARP cleavage. CONCLUSION: Tributyrin could inhibit the growth of gastric cancer cells effectively in vitro by inhibiting DNA synthesis and inducing apoptosis, which was associated with the down-regulated Bcl-2 expression and the up-regulated Bax expression. Therefore, tributyrin might be a promising chemopreventive and chemotherapeutic agent against human gastric carcinogenesis.     

Apoptosis of human gastric adenocarcinoma cells induced by β-ionone

AIM:To investigate the effect of β-ionone on the growth and apoptosis of gastric adenocarcinoma cell line SGC-7901.METHODS: Using M-IT, fluorescence dye (Hoechst-33258),transmission electron microscopy and the TUNEL assay,we examined growth and apoptosis of SGC-7901 cells treated with β-ionone at various concentrations (i.e. 25, 50, 100 and 200μmol/L) for 24h,48h.RESULTS:The growth of SGC-7901 cells was inhibited by β-ionone. Seven days after treatment with β-ionone at four concentrations, the inhibition rates were 12.04%, 30.59%,78.25% and 94.15%, respectively. The IC50 value of β-ionone for SGC-7901 cells was estimated to be 89μmol/L.The apoptotic morphology was demonstrated in SGC-7901 cells treated with β-ionone by Hoechst-33258 staining and electron microscopy. Apoptosis was also shown in β-iononetreated SGC-7901 cells by the TUNEL assay.CONCLUSION:β-ionone can inhibit cell proliferation and induce apoptosis of SGC-7901 cells.However, the mechanism needs to be further investigated.     

腺病毒介导的靶向P85和Akt1短发夹RNA对人胃腺癌细胞生长抑制作用的研究

目的 构建靶向P85和蛋白激酶B1(PKB1/Akt1)的短发夹RNA(shRNA)腺病毒载体,研究其对人胃腺癌细胞SGC-7901生长的抑制效果.方法 构建腺病毒载体rAd5-P+A,体外转染SGC-7901细胞后,以实时定量PCR和Western blot分别检测P85和Akt1的mRNA和蛋白质的表达.以噻唑蓝比色分析法(MTT法)和流式细胞法评价转染后胃癌细胞的增殖活性.构建裸鼠皮下荷瘤模型进一步观察rAd5-P+A对SGC-7901细胞生长的抑制效果,并应用原位末端标记技术(TUNEL法)检测肿瘤细胞的凋亡情况.结果 成功构建的rAd5-P+A重组腺病毒载体转染SGC-7901细胞后可显著抑制p85和Akt1的mRNA表达,而P85和Aktl蛋白表达量在转染48 h、72 h后分别下调57.5%、63.7%和67.8%、75.6%,与空白对照组和通用腺病毒对照(rAd5-HK)组相比,差异具有统计学意义(P=0.005,P=0.003).与空白对照组和rAd5-HK组相比,SGC-7901细胞的增殖活性在rAd5-P+A转染后第2天明显下降(P<0.001),且rAd5-P+A转染组进入S期的细胞数减少了5.9%～7.1%,而进入G0/G1期的细胞增加了12.1%～13.7%.裸鼠皮下荷瘤模型治疗实验也显示,rAd5-P+A可抑制胃癌细胞的生长,诱导细胞的凋亡.结论 腺病毒介导的靶向P85和Akt1的shRNA可抑制人胃腺癌细胞的生长,这可能为胃腺癌靶向性联合基因治疗提供新的策略.     

腺病毒介导的靶向P85和Akt1短发夹RNA对人胃腺癌细胞生长抑制作用的研究

目的 构建靶向P85和蛋白激酶B1(PKB1/Akt1)的短发夹RNA(shRNA)腺病毒载体,研究其对人胃腺癌细胞SGC-7901生长的抑制效果.方法 构建腺病毒载体rAd5-P+A,体外转染SGC-7901细胞后,以实时定量PCR和Western blot分别检测P85和Akt1的mRNA和蛋白质的表达.以噻唑蓝比色分析法(MTT法)和流式细胞法评价转染后胃癌细胞的增殖活性.构建裸鼠皮下荷瘤模型进一步观察rAd5-P+A对SGC-7901细胞生长的抑制效果,并应用原位末端标记技术(TUNEL法)检测肿瘤细胞的凋亡情况.结果 成功构建的rAd5-P+A重组腺病毒载体转染SGC-7901细胞后可显著抑制p85和Akt1的mRNA表达,而P85和Aktl蛋白表达量在转染48 h、72 h后分别下调57.5%、63.7%和67.8%、75.6%,与空白对照组和通用腺病毒对照(rAd5-HK)组相比,差异具有统计学意义(P=0.005,P=0.003).与空白对照组和rAd5-HK组相比,SGC-7901细胞的增殖活性在rAd5-P+A转染后第2天明显下降(P<0.001),且rAd5-P+A转染组进入S期的细胞数减少了5.9%～7.1%,而进入G0/G1期的细胞增加了12.1%～13.7%.裸鼠皮下荷瘤模型治疗实验也显示,rAd5-P+A可抑制胃癌细胞的生长,诱导细胞的凋亡.结论 腺病毒介导的靶向P85和Akt1的shRNA可抑制人胃腺癌细胞的生长,这可能为胃腺癌靶向性联合基因治疗提供新的策略.     

Effect of NHE1 antisense gene transfection on the biological behavior of SGC-7901 human gastric carcinoma cells

AIM： To study the effect of type 1 Na^＋/H^＋ exchanger （NHE1 ） antisense human gene transfection on the biological behavior of gastric carcinoma cell line SGC-7901. METHODS： Antisense NHE1 eukaryotic expression on vector pcDNA3.1 was constructed by recombinant DNA technique and transfected into gastric carcinoma cell line SGC-7901 with DOTAP liposome transfection method. Morphological changes of cells were observed with optic and electron microscopes. Changes in cell proliferative capacity, apoptosis, intracellular pH （pHi）, cell cycle, clone formation in two-layer soft agar, and tumorigenicity in nude mice were examined. RESULTS： Antisense eukaryotic expressing vectors were successfully constructed and transfected into SGC-7901. The transfectant obtained named 7901 -antisense （7901-AS） stablely produced antisense NHE1. There was a significant difference between the pHi of 7901-AS cells （6.77 ± 0.05） and that of 7901-zeo cells and SGC-7901 cells （7.24 ± 0.03 and 7.26 ± 0.03, P 〈 0.01）. Compared with SGC-7901 and 7901-zeo cells, 7901-AS cells mostly showed cell proliferation inhibition, G1/G0 phase arrest, increased cell apoptotic rate, recovery of contact inhibition, and density contact. The tumorigenicity in nude mice and cloning efficiency in the two-layer soft agar were clearly inhibited. CONCLUSION： NHE1 antisense gene significantly restrains the malignant behavior of human gastric carcinoma cells, suppresses cell growth and induces cell apoptosis, and partially reverses the malignant phenotypes of SGC-7901 . These results suggest a potential role for human tumor gene therapy.     

大黄酸通过miR-29c-3p调节FSCN1的表达影响胃癌细胞的生物学行为

目的 探讨大黄酸(Rhein)对人胃癌细胞(SGC-7901)增殖、迁移、侵袭、凋亡的影响及其机制.方法 通过qRT-PCR检测大黄酸处理后细胞中miR-29c-3p的表达以及miR-29c-3p的转染效率;miR-29c-3p mimics组、NC mimics组、Rhein+miR-29c-3p inhibitor...     

Knockdown of survivin gene by vector-based short hairpin RNA technique induces apoptosis and growth inhibition in Burkitt's lymphoma Raji cell line

Survivin is the smallest member of mammalian IAP (inhibitor of apoptosis) family. It is ubiquitous during embryonic development but is not expressed in normal post-natal tissues, except the thymus, colonic epithelial cells and CD34+ hematopoietic stem cells. However, its expression is upregulated during neoplastic transformation in both solid organ and hematological malignancies, including leukemia and lymphoma. In this study, we used RNA interference with short hairpin RNA (shRNA) technique to inhibit survivin expression in a Burkitt's lymphoma cell line Raji and validated its effects on apoptosis and cell proliferation. A survivin-shRNA expression vector were constructed and introduced into Raji cells. Expression of survivin mRNA and protein was assessed by RT-PCR and western blot analysis. Apoptosis index of transfected cells was quantified by flow cytometry and cell proliferation was enumerated by trypan blue exclusion. In Raji cells treated with survivin-shRNA expression vector, survivin mRNA levels were significantly reduced by 67.14% (transient transfection) and 64.28% (stable transfection) respectively, compared with control-shRNA treated group and PBS treated group (p<0.05). The levels of survivin protein were significantly reduced by 62.50% (transient transfection) and 60.93% (stable transfection), compared with the two control groups (p<0.05). Apoptosis index was significantly increased during transient transfection and stable transfection, respectively 31.20+/-2.45% and 29.40+/-1.72% (p<0.05). Survivin-shRNA inhibited the proliferation of Raji cells of stable transfection. In conclusion, the vector-based survivin-shRNA can effectively reduce the expression of survivin gene and induce apoptosis and growth inhibition of transfected Raji cells. We suggest that survivin can be regarded as an ideal target for new anticancer intervention of NHL.     

Survivin antisense oligodeoxynucleotide inhibits growth of gastric cancer cells

AIM: To investigate the effect of transfected survivin antisense oligonucleotide (ASODN) on proliferation and apoptosis of gastric cancer cells. METHODS: The authors designed ASODNs targeting different regions of survivin mRNA, including surviving ASODN1, ASODN2 and ASODN3. ASODNs were transfected into gastric cancer cell line SGC 7901, cell growth was detected by MTT assay. Cells exposed to the potent oligonucleotide were also examined for apoptosis induction by FCM and fluorescence microscopy. Semiquantitive RT-PCR and Western blot examinations were carried for expression of survivin mRNA and protein. RESULTS: ASODN3 caused a statistically significant reduction of cell viability to 60.6% (+/-2.9%) (P<0.01), while ASODN1 and ASODN2 had no such changes (P>0.05). The cell growth was also significantly inhibited by ASODN3, compared with reversal and scrambled sequence. A significant loss of survivin mRNA was presented in ASODN3 treated cells and this was not seen in treatment with sense ODN or scramble ODN. Protein level was significantly decreased 48 h after survivin ASODN trasfected by approximately 2-fold decrease compared with untreated controls. However, ASODN3 did not induce significant apoptosis response until 48 h after transfection (P>0.05). CONCLUSION: ASODN3, which targets translation initiation part, can be identified as a most potent antisense compound. Srvivin ASODN3 may provide a novel approach to therapy of gastric cancer.