Study On The Mechanism Of EIF4E In Chemosensitivity Of Esophageal Cancer

Backgroud and Objective:Esophageal cancer is one of the most common malignancies,which is a threat to human health worldwide.Numerous studies have found that eukaryotic translation initiation factor4E play an especially role in the development of transformation,growth and metastasis of many malignant tumors. Eukaryotic translation initiation factor4E can specifically combine mRNA mRNA m7GpppN cap structure at5’end,and it is the most effective speed regulating factor in eukaryotic cells.Once activated, eIF4E can promote carcinogenesis by rasing translation expression of many growth factors and oncogene.EIF4E was highly expressed in many human malignancies including esophageal cancer. In recent years,with the further research of the development of esophageal cancer, eIF4E has become an important gene to assess and judge the malignantance degree of esophageal cancer.Adjusting the expression levels of eIF4E,inhibiting tumor cell growth,is a new way in the field of biological therapeutics.Studies on the mechanism of eIF4E in chemosensitivity of esophageal cancer were less,this study use RNAi to regulate eIF4E expression in EC9706cells,select esophageal cancer cells with stable expression, observe the relationship between eIF4E expression and chemotherapy drug sensitivities of cisplatin,taxol and fluorouracil,giving cludes on targeting therapy of esophageal cancerMethods:1.Screening the most effective RNA interference target:293T cells were transfected with cloning plasmid-PEGFP-N1,and three kinds of different targets of RNA interference viral vector plasmid-U6-shRNA-CMV-GFP,according to lipofectamine2000instructions.24hours afer transfection, observe the expression of green fluorescence protein(GFP) in cells to evaluate the efficiency under the fluorescence microscope. Proeins were extracted36hours later,and analysed by Western Blot to detect the eIF4E expression,so that we can judge the interference effect of the five different targets and choose the strongest interference effect of RNAi viral vector plasmid in the following experiments.The grey values were analyzed by Leica Application Suite4.0.Ralative protein expression quality was caculated by the grey value ratio of eIF4E and GAPDH.2.Establish stable transfection cell lines of different eIF4E expression levels.EC9706cells were transfected respectively with plasmid-PEGFP-Nl,the strongest RNA interference viral vector plasmid-U6-shRNA-CMV-GFP-2,plasmid-PEGFP-Nl-NC, plasmid-U6-shRNA-CMV-GFP-NC with lipofectamine2000as the carrier, screened with G418to produce the stable transfection cell lines: eIF4E-OE(eIF4E overexpression cells)、eIF4E-shRNA(eIF4E silence cells)、eIF4E-OE-NC(eIF4E overexpression negative contronl cells)、 eIF4E-shRNA-NC(eIF4E silence negative contronl cells).3、Analyse stable transfection cell lines by Real Time PCR as well as Western Blot to detect the expression of eIF4E mRNA and eIF4E protein:detect the expression of eIF4E mRNA by Real Time PCR, the relative expression of eIF4E mRNA were caculated by2-△△Ct. Detect the expression of eIF4E protein by Western Blot, the grey values were analyzed by Leica Application Suite4.0,and the relative expression of eIF4E protein were caculated by the grey value ratio of eIF4E and GAPDH.3.Analyze chemotherapy drug sensitivities of stable transfection cell lines:cisplatin,fluorouracil and taxol at four different drug concentrations were added into stable transfection cell lines for respectively24h and48h.The growth inhibition ratio of eIF4E-OE(eIF4E overexpression cells)、eIF4E-shRNA(eIF4E silence cells)、 eIF4E-OE-NC(eIF4E overexpression negative contronl cells)、eIF4E-shRNA-NC(eIF4E silence negative contronl cells)、EC9706cells were evaluated by MTT method.Results:1.293T cells whichwere co-transfected plasmid-PEGFP-N1with plasmid-U6-shRNA-CMV-GFP-1,plasmid-U6-shRNA-CMV-GFP-2, plasmid-U6-shRNA-CMV-GFP-3were observed under fluorescent inverted microscope. The relative eIF4E protein expression were0.52、0.31、0.11、0.44.2. Differernt plasmids transfect EC9706cells:Green florescence was observed in eIF4E overexpression cells、eIF4E overexpression negative contronl cells、eIF4E silence cells、eIF4E silence negative contronl cells which were transfected respectively with different plasmids.3. Analysis of the expression of eIF4E mRNA by Real Time PCR: the relative expression of eIF4E mRNA in EC9706cells、eIF4E overexpression cells、eIF4E overexpression negative contronl cells、 eIF4E silence cells、eIF4E silence negative contronl cells were1.00、3.54、1.04、0.37、0.96.4.Analysis of the expression of eIF4E protein by Western Blot:the relative expression of eIF4E protein in EC9706cells、eIF4E overexpression cells、eIF4E overexpression negative contronl cells、 eIF4E silence cells、eIF4E silence negative contronl cells were0.97、1.54、1.01、0.45、1.04.5. MTT:1). at the same time point(24h48h),the inhabition rate of five cell groups increased,following the increasing concentration of three chemotherapeutic drugs,differences has statistics significance(P<0.05);2).At the same drug concentration, the inhabition rate of five cell groups increased at48h,compared with24h;3).At the same time point and the same drug concentration, the inhabition rate of five cell groups were different:the inhabition rate of eIF4E overexpression cells decreased, compared with eIF4E overexpression negative contronl cells、EC9706cells and eIF4E silence cells, differences has statistics significance (P<0.05); the inhabition rate of eIF4E silence cells increased, compared with eIF4E silence negative contronl cells、EC9706cells and eIF4Eoverexpression cells, differences has statistics significance(P<0.05).Conclusions:1. Succeeded in screening the RNA interference viral vector plasmid-U6-shRNA-CMV-GFP-2with the strongest interference effect;2. Succeeded in establishing stable transfection cell lines:eIF4E overexpression cells、eIF4E overexpression negative contronl cells、 eIF4E silence cells、eIF4E silence negative contronl cells3、The growth inhabition rates of EC9706cells、eIF4E overexpression cells、eIF4E overexpression negative contronl cells、 eIF4E silence cells、eIF4E silence negative contronl cells increased along with the drug concentrations increasement of cisplatin, fluorouracil and taxol.The inhabition rate of five cell groups increased at48h,compared with24h when reacted with cisplatin, fluorouracil and taxol at different drug concentrations. 4、Increase the expression level of eIF4E gene can reduce the sensitivities of EC9706cells to cisplatin,fluorouracil and taxol.Decrease the expression level of eIF4E gene with RNAi can promte the sensitivities of EC9706cells to cisplatin,fluorouracil and taxol.