Inhibition Of Tumor Necrosis Factor Receptor 1 By SiRNA In EC109-cells Affects Cell Proliferation And Apoptosis

Objective:Tumor necrosis factor receptor 1(TNFR1) is one of the membrane receptors of tumor necrosis factor (TNF). TNFR1 distributes on the surface of human normal cell membranes generally, as well as many kinds of tumor cell membranes. After binding with its'specific ligand TNF-αor TNF-β, TNFR1 plays an important part in many aspects, such as cell activation, multiplication, apoptosis and toxicity; but the effect may be opposite in different cells. At present, the concrete mechanism is not clear. RNA interference (RNAi), a new experimental technique in recent years, is a specific post-transcriptional gene silencing progress exsisting in the organisms widely, triggered by the double-stranded RNA which is homologous with the target gene sequence. The technology is used in the experimental research more and more extensively. Applying the RNAi, we silenced the expression of TNFR1 in esophageal carcinoma cell line, and then showed the biological behavior changes between experimental group and control group.Materials and methods:1. EC109 cell line was cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Total RNA was extracted from cells by RNAiso Reagent, the genetic level of TNFR1 was detected by RT-PCR. EC109 cell line was cultivated in DMEM supplemented with 10% fetal bovine serum, then the adherent cells were collected by trypsinization, soluble subcellular protein components were extracted fractionally by ProteoExtract Subcellular Proteome Extraction Kit after PBS washing, centrifugation and spallation, membrane protein component was analysed by Western Blot detecting the protein level of TNFR1 in EC109 cell line.2. EC109 cell line was cultivated in DMEM supplemented with 10% fetal bovine serum in six pore plate preparing for transfection, after optimization of cell transfection conditions, cells were divided into three groups : blank control group(without transfection regent and siRNA), negative control group (added transfection regent and negative control-siRNA),experimental group(added transfection regent and TNFR1-siRNA). Cell total RNA was extracted by RNAiso reagent for RT-PCR detecting the effect of RNAi on genetic level. Soluble subcellular protein components were extracted fractionally by ProteoExtract Subcellular Proteome Extraction Kit after PBS washing, centrifugation and spallation, membrane protein component was analysed by Western Blot detecting the effect of RNAi on protein level.3. 24 hours after transfection, morphology investigation with inverted phase contrast microscope was adopted.4. EC109 cell line grew in 96-well plates, after optimization of cell transfection conditions, proliferation changes by methylthiazolyl tetrazolium (MTT) was adopted after transfection.5. Collecting cells in six pore plate, Flow cytometry was adopted to detect cell apoptosis changes after transfection.Results:1. It is confirmed that TNFR1 has a high expression in EC109 cell line by RT-PCR and Western Blot.2. We silenced the expression of TNFR1 successfully by transferring TNFR1-siRNA to EC109 cell line.3. After RNAi, three groups showed no diffenrences in morphology; The MTT results were similar between blank control group and negative control group within 32 hours after RNAi (P>0.05); there had a increasing proliferation in experimental group (P<0.05); The flow cytometry results were similar between blank control group and negative control group after RNAi (P>0.05); there had a decreased apoptosis in experimental group (P<0.05).Conclusions:1. TNFR1 expressed highly in EC109 cell line.2. TNFR1 gene silencing effect esophageal carcinoma cell on proliferation and apoptosis; it suggests TNFR1 plays an important role in cancer genesis and development.3. TNFR1 is inclined to be a role mediating apoptosis in esophageal carcinoma EC109 cell line.