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AIM:To synthesize three small-interference RNAs (siRNAs)by T7 RNA polymerase-catalyzed reaction,and to investigatetheir efficacy on modulating the expression of serine/threoninekinase Pim-2 in human colon cancer cell line.METHODS:siRNA Ⅰ,Ⅱ and Ⅲ were synthesized by T_7 RNApolymerase-directed in vitro transcription,then transfectedinto human colon cancer cells SW-480.After incubationfor 6 h at 37℃,100 mL/L FBS in RPMI 1640 was substitutedin each well.After the transfection was repeated twice tothree times in each kind of siRNA,hPim-2 mRNA and proteinexpression were measured by RT-PCR and Westernblotting,respectively.RESULTS:Compared to the control group,after transfectedfor 48 h with hPim-2 siRNA Ⅰ,Ⅱ and Ⅲ,the relative inhibitionrates of hPim-2 mRNA expression in colon cancer cellswere 65.4% (P<0.05),46.2% (P<0.05) and 56.1% (P<0.05),respectively.The protein level of hPim-2 was decreasedat 72 h compared to the untransfected cells.The relativeinhibition percentages of hPim-2 protein by siRNA Ⅰ,Ⅱ,Ⅲwere 61.6% (P<0.05),45.8% (P<0.05) and 55.6% (P<0.05),respectively.CONCLUSION:The in vitro transcribed siRNAs can beuseful for silencing oncogene hPim-2 expression specificallyand efficiently.This may open a new path toward the useof siRNAs as a gene-specific therapeutic tool.
AIM: To synthesize three small-interference RNAs (siRNAs) by T7 RNA polymerase-catalyzed reaction, and to investigate their efficacy on modulating the expression of serine / threoninekinase Pim-2 in human colon cancer cell line. METHODS: siRNA I, II and III were synthesized by T_7 RNApolymerase-directed in vitro transcription, then transfectedinto human colon cancer cells SW-480. After incubation for 6 h at 37 ° C, 100 mL / L FBS in RPMI 1640 was substituted in each well. After the transfection was repeated twice tothree times in each kind of siRNA, hPim-2 mRNA and proteinexpression were measured by RT-PCR and Western blotting, respectively.RESULTS: Compared to the control group, after transfected for 48 h with hPim-2 siRNAs I, II and III, the relative inhibitionrates of hPim-2 mRNA expression in colon cancer cellswere 65.4% (P <0.05), 46.2% (P <0.05) and 56.1% (P <0.05), respectively.The protein level of hPim-2 was decreasedat 72 h compared to the untransfected cells.The relativeinhibition percentages of hPim- 2 protein by siRNA Ⅰ, Ⅱ and Ⅲwere 61.6% (P <0.05), 45.8% (P <0.05) and 55.6% (P <0.05) respectively.CONCLUSION: The in vitro transcribed siRNAs were be able to be used for silencing oncogene hPim-2 expression specifically and efficiently.This may open a new path toward the useof siRNAs as a gene-specific therapeutic tool.