Experimental Study On The Suppression Of DcR3 Gene On Cultured SW480 Cell Lines Via RNA Interference

【Objective】Decoy receptor 3, or tumor necrosis factor receptor 6B, is also called TR6 which is a newly member of tumor necrosis receptor superfamily. The dcr3 is critical intermediary for cell proliferation, differentiation and survival and anti-infection immunity by modulation of apoptosis. In mammalian cells,by the introduction of siRNAs can induce RNAi effect. This study is aimed to use this powerful tool to investigate in the human colon cancer cell line SW480, treatment with the siRNA downregulates the DCR3 levels.【Method】1. We choose chemical synthesis method to aim the target gene sequence DcR3 via online software.According to the computer aided design.The designed sequences lacked homology with other known human genes as examined by BLASTsearch of the NCBI GenBank database.High-performance purity grade, customized DcR3-siRNA RNA duplexes were purchased from Sirna (shanghai, cn). All siRNA were transfected into SW480 cells by liposome.2. The cell cycles and cell proliferations of SW480 cell were measured with MTT method after transfection.3. Extract DcR3 protein form SW480 cell aftered transfection.Study DcR3 expression by immunohistochemistry and western blot analysis to elevate the repress level of DcR3.4. The expression level of DcR3 mRNA was detected by real-time RT-PCR after they were incubated for 24h.【Results】1. The characteristic morphological changes of apoptosis were observed by reverse microscope at 24 hour post-transfection,we found SW480 cell growth has been repressed.Such as cell break into small, membrane-wrapped, fragments. The cytoplasm begins to shrink following the shrink of the nucleus. The cell shrinks, shows deformation and looses contact to its neighbouring cells.2. The MTT assay results showed that cell survival rate of SW480 was demonstrated and livingness of cells was declined3. At 24 hour post-transfection,the result of immunohistochemistry and western blot analysisthe showed the DcR3 expression level has significant degrade compared with the control,but the control compared with blank group shows no difference(P>0.05).4. Real time RT-PCR assay showed the level of DcR3 mRNA in response to DcR3-specific siRNA was decreased by 4.12 folds compared with the control. However, cellular GAPDH mRNA levels appeared unaffected.【Conclusions】1. We designed 4 group siRNA has obviously repression to the growth and proliferation of SW480 then view the apoptosis number to increase.2. It is identifided that DcR3 siRNA could inhibite proliferation and induce apoptosis in colon carcinoma cell lines SW480.3. Its inhibitory effect maybe due to specially and efficiently silence DcR3 mRNA expression, decrease the degradation of DcR3 protein.siRNA offers a useful laboratory tool in mammal cells. Our results siRNA has the inhibiting effect on DcR3 in the level of cell; DcR3 gene could be a target to treat colon carcinoma by specific gene silencing DcR3 gene. Thus, RNA interference techenology should provide a new rationale for specific gene-therapy approaches against carcinoma.