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.     

Construction of pETNF-P16 plasmid and its expression properties in EC9706 cell line induced by X-ray irradiation

AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: Recombined plasmid pETNF-P16 was constructed and transfected into EC9706 cells with lipofectamine. ELISA,Western blot, and immunocytochemistry were performedto determine the expression properties of pETNF-P16 in EC9706 after transfection induced by X-ray irradiation. RESULTS: Eukaryotic expression vector pETNF-P16 was successfully constructed and transfected into EC9706 cells. TNFα expressions were significantly increased in the transfected cells after different doses of X-ray irradiation than in those after 0Gy irradiation (1 192.330-2 026.518 pg/mL,P&lt;0.05-0.01), and the TNFα expressions and P16 were significantly higher 6-48 h after 2 Gy X-ray irradiation (358.963-585.571 pg/mL, P&lt;0.05-0.001). No P16 expression was detected in normal EC9706 cells. However, there was strong expression in the transfected and irradiation groups. CONCLUSION: X-ray irradiation induction could significantly enhance TNFα and P16 expression in EC9706 cells transfected with pETNF-P16 plasmid. These results may provide important experimental data and therapeutic potential for gene-radiotherapy of esophageal carcinoma.     

miR-451对食管癌EC9706细胞增殖、凋亡及侵袭能力的影响

目的:探讨miR-451对食管癌EC9706细胞增殖、凋亡及侵袭能力的影响.方法:化学合成miR-451mimics,脂质体包裹转染EC9706细胞为miR-451组,同时设立无关序列(Scramble-miR)对照组、脂质体对照组和空白对照组.转染后48h,荧光定量RT-PCR检测miR-451表达量的变化,Westernblot检测Bcl-2、AKT和磷酸化AKT蛋白表达水平,流式细胞仪检测细胞凋亡情况,Transwell侵袭实验检测细胞侵袭能力的改变;MTT法检测转染后l、2、3、4、5、6d各组细胞增殖率.结果:miR-451组的miR-451表达水平显著上调(P<0.01,F=69.26),为空白对照组的15.84倍;miR-451组细胞Bcl-2、AKT和磷酸化AKT蛋白表达均显著下调(P<0.05,F=5.83);miR-451组细胞凋亡率为12.07%±1.12%,与3个对照组比较显著升高(P<0.01,F=26.72);miR-451组平均侵袭细胞数为47.4±7.4,与3个对照组比较显著降低(P<0.01,F=34.55).miR-451组细胞的生长在转染后2d出现显著抑制(P<0.05,F=5.95),并且随时间的延长而日益显著.结论:上调miR-451表达可抑制食管癌EC9706细胞增殖和侵袭,促进细胞凋亡.     

Expression properties of recombinant pEgr-P16 plasmid in esophageal squamous cell carcinoma induced by ionizing irradiation

AIM: To construct the recombinant pEgr-P16 plasmid for the investigation of its expression properties in esophageal squamous cell carcinoma induced by ionizing irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: The recombinant pEgr-P16 plasmid was constructed and transfected into EC9706 cells with lipofectamine. Western blot, quantitative RT-PCR and flow cytometry were performed to study the expression of pEgr-P16 in EC9706 cells and the biological characteristics of EC9706 cell line after transfection induced by ionizing irradiation. RESULTS: The eukaryotic expression vector pEgr-P16 was successfully constructed and transfected into EC9706 cells. The expression of P16 was significantly increased in the transfected cells after irradiation while the transfected cells were not induced by ionizing irradiation. The induction of apoptosis in transfection plus irradiation group was higher than that in plasmid alone or irradiation alone. CONCLUSION: The combination of pEgr-P16 and irradiation could significantly enhance the P16 expression property and markedly induce apoptosis in EC9706 cells. These results may lay an important experimental basis for gene radiotherapy for esophageal carcinoma.     

N-cadherin knock-down decreases invasiveness of esophageal squamous cell carcinoma in vitro

AIM： To examine the expressions of N-cadherin and E-cadherin in specimens of 62 normal esophageal epithela, 31 adjacent atypical hyperplastic epithelia and 62 esophageal squamous cell carcinomas （ESCCs）, and to investigate the roles of N-cadherin in the invasiveness of ESCC cell line EC9706 transfected by N-cadherin shRNA.
METHODS： PV immunohistochemistry was used to detect the expression pattern of N-cadherin and E-cadherin in specimens of 62 normal esophageal epithelia, 31 adjacent atypical hyperplastic epithelia and 62 ESCCs. The invasiveness of ESCC line EC9706 was determined by transwell assay after EC9706 was transfected by N-cadherin shRNA.
RESULTS： The positive rotes of N-cadherin decreased in the carcinoma, adjacent atypical hyperplastic and normal esophageal tissues （75.8%, 61.3% and 29.0%, P 〈 0.05）, respectively, while those of E-cadherin increased （40.3%, 71.0% and 95.2%, P 〈 0.05）. The increased expression of N-cadherin and decreased expression of E-cadherin were related to invasion, differentiation, and lymph node metastasis （P 〈 0.05）. The expression level of N-cadherin decreased in the N-cadherin knocked down cells, and the invasiveness of those cells decreased significantly as well. The number of cells which crossed the basement membrane filter decreased from 123.40 ± 8.23 to 49.60 ±6.80 （P 〈 0.05）.
CONCLUSION： E-cadherin and N-cadherin expression is correlated with the invasion and aggravation of ESCC. The down-regulation of N-cadherin lowers the invasiveness of EC9706 cell line.     

转染kiss-1基因对人食管癌EC9706细胞裸鼠移植瘤的抑制作用

目的:研究转染kiss-1基因对人食管癌EC9706细胞裸鼠皮下移植瘤的作用,探讨其在食管癌基因治疗中的可行性和特异性.方法:在食管癌细胞系EC9706中转染kiss-1基因,经G418筛选,建立稳定高表达Kiss-1蛋白的细胞系.稳定表达该基因的细胞为转染kiss-1基因组,转染空质粒细胞及未处理细胞为对照组,建立裸鼠荷瘤模型;监测肿瘤生长变化,HE染色观察肿瘤病理学变化,RT-PCR、Western blot方法检测kiaa-1 mRNA和蛋白变化.结果:转染kiss-1基因组肿瘤生长受到显著抑制:HE染色显示转染kiss-1基因组及转染空质粒纽肿瘤组织内坏死均较空白对照组多;RT-PCR、Western blot结果表明转染kiss-1基因组裸鼠肿瘤组织kiss-1 mRNA和蛋白表达均显著升高,三组间比较差异具有统计学意义(F=72.685,24.807,均P<0.05).结论:转染kiss-1基因能抑制人食管癌EC9706细胞裸鼠皮下移植瘤的形成,且能有效上调kiss-1 mRNA和蛋白的表达,可为食管癌的基因治疗提供新的靶点、开辟新的思路.     

Trop-2表达下调对食管鳞癌EC9706细胞增殖和细胞迁移的影响

目的:研究肿瘤相关钙信号传导蛋白2(tumorassociated calcium signal transducer-2,Trop-2)表达下调对食管鳞癌EC9706细胞增殖和细胞迁移的影响,并探讨其可能的分子机制.方法:将Trop-2 siRNA和对照siRNA转染食管鳞癌EC9706细胞,利用实时荧光定量PCR和...     

沉默CXCR4基因对食管鳞癌EC-9706细胞中MMP-9基因表达的影响

目的:探讨在食管鳞癌EC-9706细胞中沉默CXCR4基因对MMP-9基因表达的影响,为阐明CXCR4基因在食管鳞癌侵袭转移中的作用提供实验依据.方法:化学合成2条靶向CXCR4基因的siRNA1和siRNA2,同时设立荧光标记阴性对照和空白对照.脂质体法转染入EC-9706细胞,荧光显微镜下观察转染效率.转染48h后,半定量RT-PCR检测各组细胞CXCR4和MMP-9基因mRNA表达的变化,Western blot检测各组细胞CXCR4和MMP-9基因蛋白表达的变化,侵袭小室检测各组细胞穿膜细胞数的变化,MTT检测各组细胞的A值.结果:与阴性对照和空白对照相比,转染CXCR4siRNA1和siRNA2组细胞CXCR4mRNA和蛋白的表达明显降低,差异具有统计学意义(P<0.05);同时与阴性对照和空白对照相比,转染CXCR4siRNA1和siRNA2组细胞MMP-9mRNA和蛋白的表达同样明显降低,差异具有统计学意义(P<0.05);转染CXCR4siRNA1和siRNA2组细胞穿膜细胞数与阴性对照和空白对照相比明显下降,差异具有统计学意义(P<0.05);MTT结果显示转染CXCR4siRNA1和siR...     

KISS-1基因对人食管鳞癌细胞EC-9706的MMP-2表达影响

目的观察KISS-1基因对食管癌细胞EC-9706中基质金属蛋白酶2（MMP-2）表达的影响。方法将EC-9706细胞分为A、B、C组,A、B组加入pcDNA3.1-KISS-1质粒、pcDNA3.1空质粒进行转染,C组不予处理。分别采用Western blot、RT-PCR法检测细胞中的MMP-2蛋白及mRNA。结果A组KISS-1蛋白为1.143±0.118,MMP-2蛋白为0.737±0.115,B组分别为0.752±0.113、0.985±0.178,C组分别为0.761±0.127、1.038±0.233,A组与B、C组比较,P均〈0.05。A组KISS-1 mRNA为0.892±0.166,MMP-2 mRNA为0.685±0.104,B组分别为0.679±0.141、0.808±0.057,C组分别为0.686±0.148、0.815±0.073,A组与B、C组比较,P均〈0.05。结论KISS-1基因对人食管鳞癌细胞EC-9706中MMP-2的表达具有抑制作用。     

血管内皮生长因子反义寡核苷酸抑制甲状腺癌血管生成的效应

目的探讨反义寡核苷酸(ASODN)抑制甲状腺癌细胞血管内皮生长因子(VEGF)表达及内皮细胞生长的效应。方法设计合成靶向VEGF的ASODN转染人髓状甲状腺癌细胞系(TT)细胞,并制备相应条件培养基作用内皮细胞ECV304,设正义寡核苷酸(SODN)和空白对照组进行比较。观察细胞生长状态,RT PCR、免疫细胞化学法检测TT细胞VEGFmRNA和蛋白表达,四氮唑蓝法检测TT和ECV304细胞生长抑制率(IR),流式细胞仪、吖啶橙/溴化乙锭染色法检测ECV304细胞凋亡状态。结果ASODN组TT细胞VEGFmRNA和蛋白表达显著低于SODN和对照组(P<0.01),但IR差异无统计学意义(P>0.05);各转染组ECV304细胞IR差异亦无统计学意义(P>0.05);而经各ASODN组TT细胞条件培养基作用的ECV304细胞生长明显受抑,IR(分别为0.21±0.03、0.31±0.01、0.42±0.22)显著高于SODN组(0.05±0.03,P<0.01),并伴明显细胞凋亡,上述效应呈浓度依赖性。结论ASODN可通过特异性封闭甲状腺癌细胞VEGF表达,抑制内皮细胞生长,干扰肿瘤血管生成。     

Angiotensin‐I converting enzyme inhibitors suppress angiogenesis and growth of esophageal carcinoma xenografts

It has recently been suggested that angiotensin‐I converting enzyme (ACE) inhibitors decrease the risk of cancer. However, studies to date have not investigated esophageal carcinoma. Therefore, we investigated the inhibitory effect of ACE inhibitors on growth of esophageal carcinoma xenografts. We used the EC9706 cell line, which expresses the highest vascular endothelial growth factor (VEGF) mRNA level, to establish xenografts in 21 BALB/c nude mice. The mice were then randomly allocated to receive normal saline, perindopril (4 mg/kg), or benazepril (6 mg/kg). Five weeks later, the nude mice were sacrificed and all tumors were dissected and weighed. The number of microvessels was counted by immunostaining endothelial cells for CD31 and the microvessel density was assessed. The EC9706 cell line showed the highest expression of VEGF mRNA of four esophageal squamous cell carcinoma lines. After treatment, the average tumor inhibitory rate in the benazepril group was 45.4%, which was significantly higher than that of the control group (P < 0.05). Similar findings were observed when we used tumor weight as an index for tumor growth inhibition (P < 0.05). By contrast, there was no significant difference between the perindopril group and the control group (P > 0.05). The benazepril group appeared to show less vascularization than the control group (P < 0.05), but we did not find a significant difference between the perindopril group and the control group (P > 0.05). The EC9706 cell line showed the highest expression of VEGF mRNA level of the four esophageal squamous cell carcinoma cell lines examined. Benazepril inhibited the growth of esophageal carcinoma in vivo. The potential mechanism of benazepril seems to involve suppression of new vessel formation. Therefore, benazepril could be used as an effective agent for the treatment of esophageal carcinoma.     

VEGF165 antisense RNA suppresses oncogenic properties of human esophageal squamous cell carcinoma

AIM: To investigate the effect of antisense RNA to vascularendothelial growth factor165 (VEGF165) on human esophagealsquamous cell carcinoma call line EC109 and the feasibilityof gene therapy for esophageal carcinoma.METHODS: Using subclone technique, the full length ofVEGF165 amino acid cDNA, which was cut from pGEM-3Zf( + ), was cloned inversely into the eukaryotic expressionvector pCEP4 . The recombinant plasmid pCEP-AVEGF165was transfected into EC109 cell with Iipofectamine. After astable transfection, dot Blot, enzyme-linked immunosorbentassay (ELISA), laser confocal imaging system analysis,transmission electron microscopy and flow cytometry wereperformed to determine the biological characteristics ofEC109 cell line before and after transfection in vitro andwhether there was a reversion in the tumorigenic propertiesof the EC109 cell in vivo.RESULTS: The eukaryotic expression vector pCEP-AVEGF165was successfully constructed and transfected into EC109cells. The expression of VEGF165 was significantly decreasedin the transfected cells while the biological characteristics ofthe cells were not influenced by the expression of antisensegene. The tumorigenic and angiogenic capabilities weregreatly reduced in nude mice, as demonstrated by reducedtumorend volume (820 ± 112.5)mm3 vs (7930 ± 1035) mn3and (7850 ± 950) mm3, P ＜ 0.01) and microvessel density(8.5 ± 1.2)mm-2 vs (44.3 ± 9.4)mm-2 and (46.4 ± 12.6)mm-2, P ＜ 0.01) in comparison between experimentalgroup, empty vector transfected group and control group.CONCLUSION: The angiogenesis and tumorigenicity ofhuman esophageal squamous cell carcinoma were effectivelyinhibited by VEGF165 antisense RNA. Antisense RNA toVEGF1, can potentially be used as an adjuvant therapy forsolid tumors.     

Effect of endothelin-1 in esophageal squamous cell carcinoma invasion and its correlation with cathepsin B

AIM： To investigate the effect of endothelin-1 in the invasion of esophageal cancer and determine whethel cathepsin B plays a role in the course.
METHODS： Western blotting was employed tc detect the expression of ET-1 protein in 75 sample., of esophageal squamous cell cancer and matched normal esophageal rnucosa. Bosentan, a dual ET （A/B）- receptor antagonist, was used to inhibit the binding of endothelin-1 and its receptors and cut down its biological role. In vitro matrigel invasion assays were made to show the invasive ability of esophageal cancer cells with and without bosentan. Subsequently, we evaluated cathepsin B activity and expression in EC9706 cell with and without bosentan.
RESULTS： We found 74.7% （56/75） tumors had an overexpression of ET-1 protein by Western blotting. Bosentan significantly inhibited matrigel invasion of cancer cells in vitro. EC9706 cells have a positive expression of cathepsin B protein, and bosentan can down-regulate its expression and activity.
CONCLUSION： Endothelin-1 may enhance the invasive ability of human esophageal cancer cells, and its role is correlated with cathepsin B.     

Inhibition of adenovirus-mediated p27kip1 gene on growth of esophageal carcinoma cell strain

AIM: To investigate the inhibition of p27kip1 gene on the growth of esophageal carcinoma cell strain (EC9706). METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effect on p27kip1 expression, the growth of esophageal carcinoma cell, DNA replication, protein synthesis, cell multiplication and apoptosis were explored by means of cell growth count, 3H-TdR, 3H-Leucine incorporation, flow cytometry, DNA fragment analysis and TUNEL. RESULTS: Recombinant adenovirus Ad-p27kip1 was successfully constructed with a virus titer of 1.24 X 10(12) pfu/ml. p27kip protein expression increased markedly after EC-9706 transfection, while incorporation quantity of 3H-TdR and 3H-Leucine decreased significantly. The growth of esophageal carcinoma cell was inhibited obviously. Testing of flow cytometry displayed a typical apoptosis peak, and DNA gel electrophoresis showed a typical apoptosis ladder. TUNEL showed the apoptosis rate of Ad-p27kip1 group and control group to be 37.3% and 1.26% (P<0.001) respectively. CONCLUSION: Ad-p27kip1 can inhibit the growth and multiplication of esophageal carcinoma cells and induce apoptosis. Therefore, enhanced p27kip1 expression may be a new way to treat esophageal carcinoma.     

干扰LINC00707对食管癌细胞生物行为的影响

目的 探讨干扰LINC00707对食管癌细胞生物行为的影响及分子机制.方法 选取51例食管癌患者癌组织及癌旁正常组织,用实时荧光定量-聚合酶链反应(RT-qPCR)检测LINC00707和miR-382-5p的表达水平;将食管癌细胞EC9706随机分为对照(con)组、si-LINC00707组、si-NC组、miR-...     

Effect of miR-451 on the Biological Behavior of the Esophageal Carcinoma Cell Line EC9706

Background

MicroRNAs play important roles in coordinating a variety of cellular processes. Abnormal expression of miRNAs has been linked to several cancers. However, the functional role of miR-451 in esophageal squamous cell carcinoma remains unclear.

Aims

The present study explored the effects of miR-451 on the biological behavior of the esophageal carcinoma cell line EC9706.

Methods

Synthetic miR-451 mimics were transfected into EC9706 cells using Lipofectamine? 2000. The expression of miR-451 was analyzed by RT–PCR and the expressions of Bcl-2, AKT and phosphorylated AKT were analyzed by Western blotting. The MTT assay, soft agar colony formation assay, transwell assay and FACS were used to assess the effect of miR-451 on EC9706 cell proliferation, invasion, metastasis and apoptosis. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice.

Results

In comparison to the controls, a significant increase in the expression of miR-451 was associated with significantly decreased expressions of Bcl-2, AKT and p-AKT, and a significant increase in the apoptosis rate. The number of cell clones was significantly decreased by miR-451 expression, which also caused the inhibition of cell proliferation. The average number of cells penetrating the matrigel was significantly lower than the controls. Injection of miR-451 inhibited tumor growth in a xenograft model.

Conclusions

Upregulated expression of miR-451 induced apoptosis and suppressed cell proliferation, invasion and metastasis in the esophageal carcinoma cell line EC9706. In addition, injection of miR-451 inhibited tumor growth in a xenograft model of esophageal cancer.     

硒蛋氨酸诱导食管癌细胞株凋亡的实验研究

目的：探讨硒蛋氨酸对食管癌细胞系EC 9706凋亡的影响。方法：采用MTT比色法，细胞生长曲线描绘观察硒蛋氨酸对食管癌细胞系EC 9706增殖的影响。采用流式细胞仪观察硒蛋氨酸对EC 9706细胞诱导其凋亡的作用及对细胞周期的影响。琼脂糖凝胶电泳法检测DNA ladder。结果：硒蛋氨酸呈时间、剂量依赖性方式抑制EC 9706细胞增殖，改变细胞周期分布，增加G0／G1期细胞比例，诱导细胞凋亡。结论：硒蛋氨酸可能通过影响细胞周期分布和诱导细胞凋亡，从而抑制EC 9706细胞增殖。硒蛋氨酸可能是预防和治疗食管癌的一种新制剂。