The Influence Of Sirna On The Expression Of Eif4e And 4ebp1 In Human Esophageal Carcinoma Ec9706 Cell Line

Esophageal carcinoma is a kind of the most common malignancies in our country, which is characterized by high incidence, nonspecific appearance, easy metastasis and high death rate. The incidence of esophageal carcinoma in Henan is higher than many other provinces, but the efficacy of existing treatment is not satisfactory, so searching for new targets or new ways for biological treatment of esophageal carcinoma has become a research hotspot in recent years.Eukaryotic initiation factor 4E(eIF4E) is a 24kD protein whose gene is located in the chromosome 4q21-4q25 and is an important proto-oncogene. As one part of the eIF4F complex which is composed of eIF4E, eIF4A and eIF4G, eIF4E can specifically bind to the 5' terminal "cap" structure of eukaryotic cells' mRNA and play a role of regulation during the cap-dependent translation initiation phase. The over-expression of eIF4E can not only promote cell proliferation, but also promote malignant transformation of cells. Researches have shown that the expression levels of eIF4E were significantly increased in a number of human tumor tissues, including the head and neck squamous cell carcinoma(HNSCC), lung cancer, bladder cancer, colon cancer, breast cancer, cervical cancer, leukemia and malignant lymphoma, etc. and were also positively correlated with the progress of the disease. Some scholars believe that eIF4E is going to become the new target of gene therapy against cancer, and also the indicators for early diagnosis and assessment of prognosis. Eukaryotic initiation factor 4E-binding proteins(4EBPs) is a small protein family which contains three members: 4EBP1, 4EBP2 and 4EBP3. Among them, the study of 4EBP1 is much clearer than the others at present. 4EBP1 is composed of 118 amino acids whose encoding gene is located in chromosome 8p12, and regulates the acticity of eIF4E mainly through different phosphorylation states of its own. When the cells are in a quiescent state, 4EBP1 of low phosphorylation combines with eIF4E and forms a eIF4E-4EBPl complex, thereby preventing the cap-dependent translation initiation; after the phosphorylation of 4EBP1, eIF4E separates from it and thus lifts the inhibition of translation, the process of protein translation starts. Another study shows that the use of RNA interference inhibiting the expression of eIF4E in breast cancer cell lines can also reduce the expression of 4EBP1 simultaneously. The studies that using RNAi technology to inhibit the expression of eIF4E and 4EBP1 in esophageal carcinoma have not been reported in the literature.RNA interference(RNAi) is a widespread sequence-specific gene silencing process in the mRNA levels induced by the adoption of double-stranded RNA(dsRNA) in animals and plants. The dsRNA introduced or generated in cells is subjected to digestion with a dsRNA-specific endonuclease called Dicer and cleaved into 21bp -23bp small interfering RNAs(siRNAs), and the resultant duplexes just as siRNAs combine with a kind of poly-nuclease complex called RNA-induced silence complex(RISC). The RISC combines with mRNA which is complementary to siRNA and plays a role in digestion of mRNA as RNase. The effects of the implementation of RNAi belongs to the technology of post-transcriptional gene silencing(PTGS). At present the application of RNAi technology in cell signaling pathways, anti-virus, anti-tumor and other areas of experimental study indicates that RNAi will become an indispensable tool to study gene functions.In this study, the application of RNAi is used to inhibit the expression of eIF4E specifically in esophageal carcinoma EC9706 cells. The expression of eIF4E, 4EBP1 protein and mRNA were detected using immunocytochemistry and in situ hybridization before and after transfection; changes of cell cycle before and after the transfection were detected by flow cytometry, and provide a theoretical basis for the clinical application of RNAi technology targeting against malignancies such as esophageal carcinoma.Materials and methods1. Human esophageal carcinoma strain EC9706 is presented by the Chinese Academy of Medical Sciences Cancer Institute, State Key Laboratory of Molecular Oncology.2. Culture of the esophageal carcinoma EC9796 cells: The EC9706 cells were cultured adherently in RPMI-1640 culture medium (containing 10% fetal bovine serum, penicillin 1007ml, streptomycin 1007ml) under the condition of 37℃and 5%CO₂.3. Synthesis and identification of siRNA in vitro: The oligonucleotide templates of siRNA were designed and synthesized firstly which contained T7 RNA polymerase promoter sequence at 5' terminal that could bind to the T7 RNA polymerase to transcript the target siRNA in vitro. Two pieces of specific siRNA and a piece of nonsense siRNA were synthesized respectively, the application of 4% agarose gel electrophoresis was used to detect the length and concentration of siRNA, screening out one with better interference effect by advance experiment.4. Transfection: LipofusinX siRNA Transfection Reagent was used to transfect siRNA into EC9706 cells.5. The expression of eIF4E and 4EBP1 protein in every experimental group and control group was detected by immunocytochemistry.6. The expression of eIF4E and 4EBP1 mRNA in every experimental group and control group was detected by in situ hybridization.7. Changes of cell cycle in EC9706 cells before and after transfection were detected by the application of flow cytometry.8. Statistical analysis: The application of SPSS 13.0 statistical software was used for statistical treatment, measurement data were expressed by mean±standarddeviation( (X|-)±S); data between two groups were analyzed by t test(t-test); data morethan two groups were analyzed by the ANVOA; the level of significant difference is a =0.05. Results1. Two pieces of siRNAs were synthesized successfully in vitro and both of their length were 21bp.2. Morphological changes of EC9706 cell: After the transfection of eIF4E-specific siRNA, it was observed that the EC9706 cells became round gradually, the refractive index increased and then began to shrink.3. Immunocytochemical results: The expression of eIF4E and 4EBP1 protein in esophageal carcinoma EC9706 cells was lower in experimental groups which were transfected with specific eIF4E siRNA(150ng/μl, 200ng/μl and 250ng/μl) for 24h, 4Sh and 72h compared with cell control group(no transfection), blank control group(empty liposomes) and non-sense control group(transfected with non-sense siRNA), especially the group at the concentration of 250ng/μl for 72h showed the most obvious inhibitory effect. There was a significant difference between experimental group and control group(P<0.05). The inhibitory effect on the expression of eIF4E and 4EBP1 protein was in time-dependent manner at every dosage, but there was no significant difference in three kinds of time(P>0.05). The inhibitory effect on the expression of eIF4E and 4EBP1 protein was in dosage-ependent manner at every acting time and there was a significant difference in three kinds of dosage(P<0.05). Compared with the cell control group, no inhibitory effect on the expression of eIF4E or 4EBP1 protein was shown in both blank control group and non-sense control group, there was no significant difference among the three control groups(P>0.05).4. In situ hybridization results: The expression of eIF4E and 4EBP1 mRNA in esophageal carcinoma EC9706 cells was lower in experimental groups which were transfected with specific eIF4E siRNA(150ng/μl, 200ng/μl and 250ng/μl) for 24h, 48h and 72h compared with cell control group, blank control group and non-sense control group, especially the group at the concentration of 250ng/μl for 72h showed the most obvious inhibitory effect. There was a significant difference between experimental groups and control groups(P<0.05). The inhibitory effect on the expression of eIF4E and 4EBP1 mRNA was in time-dependent manner at every dosage, but there was no significant difference in three kinds of time(P>0.05). The inhibitory effect on the expression of eIF4E and 4EBP1 mRNA was in dosage-dependent manner at every acting time and there was a significant difference in three kinds of dosage(P<0.05). Compared with the cell control group, no inhibitory effect on the expression of eIF4E or 4EBP1 mRNA was shown in both blank control group and non-sense control group, there was no significant difference among three control groups(P>0.05).5. Cell cycle detected by FCM: Esophageal carcinoma EC9706 cells were transfected with three different dosage(150ng/μl, 200ng/μl and 250ng/μl) of eIF4E specific siRNA for 72h, there were obvious differences of percentage that the number of cells occupied in different stages compared with the cell control group and the nonsense group, the proportion of G₀/G₁ phase cells increased significantly, the proportion of S-phase cells reduced, the difference has statistical significance(P<0.05). Among the experimental groups, along with the increase of transfection dosage, the proportion of G₀/G₁ phase cells increased, the proportion of S-phase cells reduced, the difference among three transfection dosage has statistical significance(P<0.05). After the transfection of nonsense siRNA, there were no significant differences of the proportion in G₀/G₁ phase cells or S-phase cells compared with the cell control group(P>0.05) and had no significant effect on the cell cycle of esophageal carcinoma EC9706 cells.Conclusions1. eIF4E siRNA can specifically inhibit the expression of eIF4E protein and mRNA in esophageal carcinoma EC9706 cells.2. When the expression of eIF4E protein and mRNA in esophageal carcinoma EC9706 cells was depressed by specific eIF4E siRNA, the expression of 4EBP1 protein and mRNA was also reduced at the same time, which suggested that eIF4E may affect the expression of 4EBP1 through a particular mechanism or pathway.3. At the time when specific eIF4E siRNA depressed the expression of eIF4E and 4EBP1 in esophageal carcinoma EC9796 cells, the cells grew slowly and died partly, the cell cycle was arrested in G₀/G₁ phase, which indicated that eIF4E siRNA inhibited the proliferation of esophageal carcinoma EC9706 cells.