The Studies On The Influence Of Elemene And Eif4e Antisense Oligodeox-ynucleotide To The Expression Of Eif4e And 4ebp1 In Human Esophageal Carcinoma Ec-1 Cell Line

Esophageal carcinoma is a kind of the most common malignancies in the world. The mortality of esophageal carcinoma in my country is higher than other contries, which serious threat people's health.Esophageal carcinoma is one of the key points in the cancer research field in China.There is a multiple factor of the result of eso phageal carcinoma,polygene participates its development,and goes through multistage generation,in this progression,the overexpression of eukaryotic initiation factor 4E(eIF4E) play an important role.Eukaryotic initiation factor 4E(eIF4E) is a molecular weight of 24 KD free polypeptide and the gene of eIF4E is located in 4q21-25,which is a kind of new proto-oncogene.In eukaryotic cells,eIF4E is an important rate-limiting enzyme in the process of translation initiation.Which plays an important role in the regulation as one part of eIF4F composed by eIF4A,eIF4G,et al.The cell is under the normal physiology condition,eIF4E is low expression,which mainly promotes the translation of house-keeping gene and meet the need of cell growth and metabolism,eIF4E overexpression can promote the expression of proto-oncogene and produce a large amount of cocarcinogenic agents,which cause cells transformation and canceration.Eukaryotic initiation factor 4E-binding protein 1(4EBP1) is an important regulatory protein for eIF4E.Human 4EBP1 is composed of 118 amino acids,and the gene is located in 8p12.4EBP1 regulates the combination of eIF4E through the different phosphorylation state,thus indirectly affects the protein translation.Low phosphorylation 4EBP1 have a high affinity for eIF4E,and it can prevent the formation of translation initiation complex(eIF4F) through competitive combination of eIF4E with eIF4G.High phosphorylation 4EBP1 can't combine eIF4E,eIF4E and eIF4G,eIF4A combined to form translation initiation complex,thus start protein translation.Study shows eIF4E over-expression and 4EBP1 phosphorylation play an important role in tumor occurrence and development.People has paid attention to eIF4E as a target anti-tumor therapy.Soni et al found that inhibiting the expression of eIF4E would inhidit tumor growth,at the same time found expression of 4EBP1 was lower in breast cancer.There has been little research in the report of affecting the expression of eIF4E and 4EBP1 in esophageal cancer.Antisense oligonucleotides(ASONs) is a small section of the nucleotide sequence, which combine DNA or RNA through base pairs.ASONs can effectively block or inhibit the expression of mRNA with combination particular sequences of the target mRNA.As a result,antisense technology has become a hot spot in the field of anti-tumor research in recent years.Elemene withdraw from the Chinese herbal medicine of one kind valid composition,the chemical name is 1-methyl-1-ethylene-2,4- diisopropyl cyclohexane. It has anti-tumor function strong,poisonous side-effect small etc.Elemene has been applicated in the clinical and show a wide range of anti-tumor.However,it's anti-tumor mechanism isn't yet entirely clear.In this study,human esophageal cancer EC-1 cell as an research object,study the impact of eIF4E antisense oligonucleotide and elemene to the expression of eIF4E and 4EBP1 in human esophageal carcinoma EC-1 cell,provide a theoretical basis for clinical treatment of esophageal cancer. Materials and Methods1.Human esophageal cancer EC-1 cell is presented by Professor Cao Shihua from the University of Hong Kong.2.The culture of the esophageal carcinoma EC-1 cells:The EC-1 cells were adherent cultured with culture fluid of 10%fetal bovine serum(Penicillin, Streptomycin100 u/ml)under condition of 37℃and 5%CO₂.3.Transfection:LipofusinX was used to transfect the ASODNs into esophageal carci-noma EC-1 cells.4.Use immunocytochemistry SP method to detect experiment groups,control groups of the eIF4E and 4EBP1 protein expression.5.Use cell in situ hybridzation method to detect experiment groups,control groups of the eIF4E and 4EBP1 mRNA expression.6.Satistical analysis:All the data were analyzed by SPSS 10.0 statistical software package.The data were expressed by mean±standard deviation((?)±s).The two groups were analyzed using t test,and several groups using the ANVOA.Variance missing must be convered firstly.The level of significant difference is a=0.05.Results1.Immunocytochemistry results of elemene group:The expression quantity of eIF4E and 4EBP1 protein was lower in experimental group compared with control group after elemene(20μg/ml,40μg/ml,60μg/ml) roling in esophageal carcinoma EC-1 cells for 24h,48h,72h,especially 60μg/ml elemene at 72h.It was significantly differrent between experimental groups and blank control groups(P＜0.05).The low expression of eIF4E and 4EBP1 protein was in dosage-dependent manner at every roling time and there was significant difference in three kind of dosage(P＜0.05).The low expression of eIF4E and 4EBP1 protein was in time-dependent manner at every dosage,but there was not significant difference in three kind of time(P＞0.05).2.In situ hybridration results of elemene group:The expression quantity of eIF4E and 4EBP1 mRNA was lower in experimental group compared with control group after elemene(20μg/ml,40μg/ml,60μg/ml) roling in esophageal carcinoma EC-1 cells for 24h,48h,72h,especially 60μg/ml elemene at 72h.It was significantly different between experimental groups and blank control groups(P＜0.05).The low expression of eIF4E and 4EBP1 mRNA was in dosage-dependent manner at every roling time and there was significant difference in three kind of dosage(P＜0.05).The low expression of eIF4E and 4EBP1 mRNA was in time-dependent manner at every dosage,but there was not significant difference in three kind of time(P＞0.05).3.Immunocytochemistry results of ASODNs group:The expression quantity of eIF4E and 4EBP1 protein was lower in ASODN₁ and ASODN₂ group compared with blank control group after two antisense oligonucleotide and one non-oligonucleotide(5μmol/L,10μmol/L,15μmol/L) transfecting esophageal carcinoma EC-1 cells for 24h,48h,72h,and there was a significant difference in them(P＜0.05).The expression of eIF4E and 4EBP1 protein was not significant lower in N-ODN group compared with blank control group,and there was not significant difference in two groups(P＞0.05).The expression of eIF4E and 4EBP1 protein was lower in ASODN₁ and ASODN₂ group,compared with N-OND group,and it was significantly different in them(P＜0.05).The expression of eIF4E and 4EBP1 protein was not significant lower in ASODN₁ group compared with ASODN₂ group,and there was not significant difference in two groups(P＞0.05).The low expression of eIF4E and 4EBP1 protein was in dosage-dependent at every transfecting time in ASODN₁ and ASODN₂ group,and there was significant different in three kind of dosage(P＜0.05).The low expression of eIF4E and 4EBP1 protein was in time-dependent manner in ASODN₁ and ASODN₂ group at every dosage,but there was not significant difference in three kind of time(P＞0.05).The expression of eIF4E and 4EBP1 protein was not significant different in different time and concentration N-OND group,and it was not significan-tly different in them(P＞0.05).4.In situ hybridration results of ASODNs group:The expression quantity of eIF4E and 4EBP1 mRNA was lower in ASODN₁ and ASODN₂ group compared with blank control group after two antisense oligonucleotide and one nonoligonucleotide (5μmol/L,10μmol/L,15μmol/L) transfecting esophageal carcinoma EC-1 cells for 24h,48h,72h,and there was a significant difference in them(P＜0.05).The expression of eIF4E and 4EBP1 mRNA was not significant lower in N-ODN group compared with blank control group,and there was not significant difference in two groups(P＞0.05).The expression of eIF4E and 4EBP1 mRNA was lower in ASODN₁ and ASODN₂ group,compared with N-OND group,and it was significantly different in them(P＜0.05).The expression of eIF4E and 4EBP1 mRNA was not significant lower in ASODN₁ group compared with ASODN₂ group,and there was not significant difference in two groups(P＞0.05).The low expression of eIF4E and 4EBP1 mRNA was in dosage-dep- endent at every transfecting time in ASODN₁ and ASODN₂ group, and there was significant different in three kind of dosage(P＜0.05).The low expression of eIF4E and 4EBP1 mRNA was in time-dependent manner in ASODN₁ and ASODN₂ group at every dosage,but there was not significant difference in three kind of time(P＞0.05).The expression of eIF4E and 4EBP1 mRNA was not significant different in different time and conce- ntration N-OND group,and it was not significantly different in them(P＞0.05).5.Immunocytochemistry results of combination group:The expression of eIF4E and 4EBP1 protein was lower in combination group compared with elemene(60μg/ml) group and ASODN₁(15μmol/L) group after elemene(60μg/ml) and ASODN₁ (15μmol/L) together roling in esophageal carcinoma EC-1 cells for 24h,48h,72h, and it was significantly different in them(P＜0.05).The low expression of eIF4E and 4EBP1 protein in time-dependent manner in combination group,and there was significant difference in three kind of time(P＜0.05).6.In situ hybridration results of combination group:The expression of eIF4E and 4E-BP1 mRNA was lower in combination group compared with elemene (60μg/ml) group and ASODN₁(15μmol/L) group after elemene(60μg/ml) and ASODN₁(15μmol/L) together roling in esophageal carcinoma EC-1 cells for 24h, 48h,72h,and it was significantly different in them(P＜0.05).The low expression of eIF4E and 4EBP1 mRNA in time-dependent manner in combination group,and there was significant difference in three kind of time(P＜0.05).Conclusions1.The expression of eIF4E protein and mRNA was lower after elemene roling in esp- hageal carcinoma EC-1 cells.Which would suggest elemene anti-cancer mechanism was related to down regulation the expression of eIF4E gene.2.The expression of eIF4E protein and mRNA was lower after different concentratei-ons of eIF4E ASODN transfecting esophageal carcinoma EC-1 cells. Which would suggest eIF4E ASODN could inhibit the expression of eIF4E gene and provide a help for esophageal carcinoma treatment.3.The expression of eIF4E protein and mRNA was lower in combination group compared with elemene and eIF4E ASODN group.Which would suggest elemene and eIF4E ASODN combination application of esophageal cancinoma EC-1 cell may be more helpful.4.The expression of eIF4E gene was lower after different concentrations of eIF4E ASODN and elemene roling in esophageal carcinoma EC-1 cells,at the same time, the expression of 4EBP1 protein and mRNA was also lower.Which would suggest eIF4E could regulate the expression of 4EBP1 gene in human esophageal cancer.