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.     

Anti-tumor effect of pEgr-IFNγ,gene-radiotherapy in B16 melanoma-bearing mice

AIM: To construct a pEgr-IFNγ Plasmid and to investigate its expression properties of interferon-γ (INF-γ) induced by irradiation and the effect of gene-radiotherapy on the growth of melanoma. METHODS: A recombined plasmid, pEgr-IFNγ, was constructed and transfected into B16 cell line with lipofectamine. The expression properties of pEgr-IFNγ were investigated by ELISA. Then, a B16 melanoma-bearing model was established in mice, and the plasmid wasinjected into the tumor tissue. The tumor received 20 Gy X-ray irradiation 36 h after injection, and IFN-γ expression was detected from the tumor tissue. A tumor growth curve at different time points was determined. RESULTS: The eukaryotic expression vector, pEgr-IFNγ, was successfully constructed and transfected into B16 cells. IFN-γ expression was significantly increased in transfected cells after X-ray irradiation in comparison with 0 Gy group (77.73-94.60 pg/mL, P<0.05-0.001), and was significantly higher at 4 h and 6 h than that of control group after 2 Gy X-ray irradiation (78.90-90.00 pg/mL, P<0.01-0.001).When the transfected cells were given 2 Gy irradiation 5 times at an interval of 24 h, IFN-y expression decreased in a time-dependent manner. From d 3 to d 15 after IFNγ generadiotherapy, the tumor growth was significantly slower than that after irradiation or gene therapy alone. CONCLUSION: The anti-tumor effect of pEgr-IFNγ generadiotherapy is better than that of genebherapy or radiotherapy alone for melanoma. These results may establish an important experimental basis for gene-radiotherapy of cancer.     

Anti-tumor effect of pEgr-IFNγgene-radiotherapy in B16 melanoma-bearing mice

AIM: To construct a pEgr-IFNγ plasmid and to investigate its expression properties of interferon-γ (INF-γ) induced by irradiation and the effect of gene-radiotherapy on the growth of melanoma.METHODS: A recombined plasmid, pEgr-IFNγ, was constructed and transfected into B16 cell line with lipofectamine. The expression properties of pEgr-IFNγwere investigated by ELISA. Then, a B16 melanoma-bearing model was established in mice, and the plasmid was injected into the tumor tissue. The tumor received 20 Gy X-ray irradiation 36 h after injection, and IFN-γ expression was detected from the tumor tissue. A tumor growth curve at different time points was determined.RESULTS: The eukaryotic expression vector, pEgr-IFNγ,was successfully constructed and transfected into B16 cells.IFN-γ expression was significantly increased in transfected cells after X-ray irradiation in comparison with 0 Gy group (77.73-94.60 pg/mL, P＜0.05-0.001), and was significantly higher at 4 h and 6 h than that of control group after 2 Gy X-ray irradiation (78.90-90.00 pg/mL, P＜0.01-0.001).When the transfected cells were given 2 Gy irradiation 5 times at an interval of 24 h, IFN-γ expression decreased in a time-dependent manner. From d 3 to d 15 after IFNγ generadiotherapy, the tumor growth was significantly slower than that after irradiation or gene therapy alone.CONCLUSION: The anti-tumor effect of pEgr-IFNγgeneradiotherapy is better than that of genetherapy or radiotherapy alone for melanoma. These results may establish an important experimental basis for gene-radiotherapy of cancer.     

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.     

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.     

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.     

Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells

AIM: To investigate the effects of anti-sense oligonudeotides (ASODNs) on mRNA expression of heparanase in human esophageal cancer EC9706 cells. METHODS: One non-sense oligonucleotide (N-ODN) and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied by in situ hybridization. RESULTS: The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs (P>0.05), but there was a significant difference between ASODNs and N-ODN or non-transfected group (ASODN1: 2.25±0.25, ASODN2: 2.21±0.23, ASODN3: 2.23±0.23, ASODN4: 2.25±0.24 vs N-ODN: 3.47±2.80 or non- transfected group: 3.51±2.93 respectively,P<0.05). CONCLUSION: The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.     

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...     

Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro

To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma.     

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.     

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。     

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...     

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.     

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. METHODS: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. RESULTS: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.     

己酮可可碱对人食管癌EC9706细胞增殖及NF—κB p65、COX-2、Ki-67蛋白表达的影响

目的探讨己酮可可碱（PTX）对肿瘤细胞的增殖抑制作用及机制。方法体外培养人食管癌EC9706细胞,用不同质量浓度的PTX进行干预,采用MTT法检测细胞增殖抑制率（IR）,免疫组化染色SP法检测核转录因子-κB（NF—κB p65）、环氧化酶-2（COX-2）、Ki-67蛋白表达。结果PTX处理后EC9706细胞IR明显升高,NF-κB P65、COX-2蛋白及Ki-67蛋白表达明显下降（P〈0．05）,且呈时间-浓度依赖性。结论PTX可抑制EC9706细胞增殖,可能机制为下调NF-κB、COX-2及Ki-67蛋白表达;此为PTX治疗食管癌提供了理论依据。     

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.     

p27kip1基因转移对食管癌细胞生存素表达和端粒酶活性的影响

背景：p27kip1是新近发现的一种抑癌基因，早期研究表明p27kip1基因转移能显著抑制食管癌细胞和人食管癌裸鼠移植瘤的生长，表明该基因疗法可能是食管癌治疗的新途径，但其抑癌机制尚未完全阐明。目的：研究p27kip1基因转移对食管癌细胞生存素（survivin）表达和端粒酶活性的影响。从而阐明p27kip1的抑癌机制，为p27kip1基因治疗食管癌提供理论依据。方法：将携带p27kip1基因的重组腺病毒（Ad—p27kip1）和LacZ重组腺病毒（Ad—LacZ）分别转染食管癌细胞系Eca9706，观察细胞形态变化。以免疫细胞化学染色和蛋白质印迹法检测p27kip1和生存素的表达．以端粒重复序列扩增程序（TRAP）聚合酶链反应（PCR）-酶联免疫吸附测定（ELISA）检测端粒酶活性。结果：经Ad—p27kip1转染后，Eta9706细胞变圆，呈葡萄串样聚集以致脱落。细胞p27kip1表达明显增强，生存素表达降低，端粒酶活性显著受抑制。结论：p27kip1基因抑制食管癌细胞生长的作用机制可能与下调生存素表达和抑制端粒酶活性有关。