Inhibition Of AP-2β By ShRNA Mediated RNA Interference In Colorectal Cancer Cell SW620

ObjectiveTo investigate the expression of AP-2βin SW620 cells. One siRNA specifically targeting AP-2βgene was designed and synthesized in vitro,and was transfected into the SW620 cells. The expression of AP-2βin SW620 cells transfected with siRNA is inhibited. To determine the expression of hTERT at mRNA level after transfection. The aim of the current study is to provide experimental basis for further research about the biological function of AP-2βin carcinogenesisMethods1. The expression of AP-2βat mRNA and protein levels in human colorectal cancer SW620 cells were determined by RT-PCR and Western blot assay, respectively.2. To construct shRNA mediated eukaryotic expression plasmid of targeted against AP-2βgene.3. The identified constructed plasmids were transfected into SW620 with LipofactamineTM 2000. The expression of AP-2βmRNA and protein levels were detected with RT-PCR and Western blot assay, respectively.4. To determine the expression of hTERT at mRNA level by RT-PCR after transfection.5. Fluorescence Activated Cell Sorter was applied to study cell apoptosis of SW620 cells after transfection.Results1. To construct one shRNA mediated eukaryotic expression plasmid of targeted against AP-2βgene.2. The identified constructed plasmid was transfected into SW620 with LipofactamineTM 2000. The expression of AP-2βmRNA and protein were detected with Real-time quantitative PCR and Western blot assay, respectively.3. The expression of hTERT in the transfected cells was significantly down-regulated at mRNA level (P＜0.05)4. Fluorescence Activated Cell Sorter was applied to study cell apoptosis of SW620 cells after transfection.ConclusionThe shRNA mediated eukaryotic expression vector targeting AP-2βgene was successfully constructed, which could effectively inhibite the expression of AP-2βat mRNA and protein levels. Transfection with AP-2βsiRNA expression vector could induce cell apoptosis of SW620 cells,which was probably related with down-regulating the expression of hTERT due to the decrease of its transcriptional activity. The current results may be helpful in delineating molecular mechanism of AP-2βtumorigenicity and providing a potential route for colorectal cancer gene therapy in the future.