The Study About Inhibiting Specifically Expression Of Cathepsin B Gene In Esophageal Carcinoma Cells By RNAi

RNA interference (RNAi) is the process of sequence-specific posttranscriptional gene silencing (PTGS) triggered by double stranded RNA (dsRNA). The dsRNA introduced or generated in cells is subjected to digestion with a dsRNA-specific endonuclease, Dicer, into 21-25 nucleotide (nt) RNA duplexes, and the resultant duplexes, referred to as short interfering RNAs (siRNAs) duplexes, function as essential sequence specific mediators of RNAi in the RNA-induced silencing complexes (RISC). These reactions result in the cleavage of the target mRNA, he exploratory development of RNAi is manly practiced by synthesized in vitro siRNA and prodused in vivo siRNA. SiRNA can obviously inhibit expression of target gene;moreover, specificness of depressive effect is powerful. At present, experimental studies in the domain of anti-virus, anti-tumor and so on are frequently canied out bymeans of RNAi technology.CB is one kind of cysteine proteases in cytolysosome. It can directly degrade extracellular matrix(ECM) or indirectly degrade ECM by activating plasminogen activator(PA) and matrix metalloproteinase so on; thus participate invasion and matastasis process of tumor. It has been discovered that expression and activity of CB is higher than near normal tissue in many malignant tumor tissue such as gastric cancer, lung cancer, colon carcinoma, breast cancer, prostatic carcinoma, glioma, bladder carcinoma, ovarian cancer, melanoma, thyroid cancer, et al. Some scholar think that CB is likely to become a new target of tumorous gene therapy and a index of auxiliary diagnosis.Esophageal carcinoma is a kind of the most common malignancies in China, and its invasion and matastasis is the leading reason resulted in death of the sufferer. The research about the relation of the expression of CB gene and development of esophageal carcinoma is few reported. The research about inhibiting expression of correlative gene of invasion and matastasis by RNAi technology is not still reported.In this study, RNAi technology was used to inhibit cathepsin B gene in esophageal carcinoma EC9706 cells. After the treatment, we detect expression of CB gene and invasion ability of EC9706 cells. Our study would provide the laboratory evidence for the future gene therapy of esophageal carcinoma and other malignancies using RNAi.Materials and Methods1. The culture of the esophageal carcinoma EC9706 cells: The EC9706 cells were adherent-cultured with culture fluid of 10% fetal bovine serum under condition of 37°C and 5%CO₂.2. Synthesis and identification of siRNA: The DNA templates were synthesized firstly, which included T7 RNA Polymerase promoter sequence at 5' end that could bind to the T7 RNA Polymerase to transcript the target siRNA in vitro. The synthesized two siRNA were 21 nt; were specific and unspecific respectively. 4% gel electrophoresis was used to detect the length and concentration of the synthesized siRNAs.3. Transfection: Lipofectamine?2000 siRNA Transfection Reagent was used to Transfect the siRNAs into EC9706 cells. The experiment was designed three kinds of transfecting time. There were three kinds of transfecting dosage and control at every transfecting time.4. The detection after transfection: In situ hybridration and immunohistochemistry were used to detect the expression level of the target gene. Boyden chamber experiment in vitro was used to detect the invasion ability of EC9706 cells.5. Statistical analysis: The SPSS 10.0 statistical software package was used for all analyses. The date were expressed by mean±standard deviation [x|-±s] and analyzed using the ANVOA. The level of significant difference is a=0.05.Results1. SiRNAs were synthesized successfully in vitro and their lengths were 21bp.2. In situ hybridration result: The expression quantity of CB mRNA was lower in experimental groups compared control groups after transfecting for 24h, 48h, 72h, especially at 72h. It was not significantly different among different transfecting time(P>0.05). The inhibition effect was in dosage-dependent manner at every transfecting time and there was significant difference in three kinds of dosage(P <0.05). Inhibition effect didn't been observed in control groups.3. Immunohistochemistry results: The expression quantity of CB proteinum was lower in experimental groups compared with control groups after transfecting for 24h, 48h, 72h, especially at 72h. It was not significantly different among different transfecting time(P > 0.05). The inhibition effect was in dosage-dependent manner at every transfecting time and there was significant difference in three kinds of dosage(P<0.05). Inhibition effect didn't been observed in control groups.4. Boyden chamber results: Compared with control groups, the number of cellstraversed Matrigel was decreased obviously in experiment group(P<0.05).Conclusions1. SiRNA can be synthesized successfully in vitro.2. Specific siRNA of CB can inhibit the expression of CB gene and CB protein in the esophageal carcinoma EC9706 cells. It shows that RNAi can inhibit invasion and matastasis of esophageal carcinoma by inhibiting expression of CB gene.3. Boyden chamber results show that RNAi technology can suppress invasive potential of tumor cells by inhibiting expression of correlative gene of invasion.