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/threonine kinase Pim-2 in human colon cancer ...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/threonine kinase Pim-2 in human colon cancer cell line.METHODS: siRNA Ⅰ,Ⅱ and Ⅲ were synthesized by T7 RNA polymerase-directed in vitro transcription, then transfected into human colon cancer cells SW-480. After incubation for 6 h at 37℃, 100 mL/L FBS in RPMI 1640 was substituted in each well. After the transfection was repeated twice to three times in each kind of siRNA, hPim-2 mRNA and protein expression were measured by RT-PCR and Western blotting, respectively.RESULTS: Compared to the control group, after transfected for 48 h with hPim-2 siRNA Ⅰ, Ⅱ and Ⅲ, the relative inhibition rates of hPim-2 mRNA expression in colon cancer cell swere 65.4% (P<0.05), 46.2% (P<0.05) and 56.1% (P<0.05),respectively. The protein level of hPim-2 was decreased at 72 h compared to the untransfected cells. The relative inhibition percentages of hPim-2 protein by siRNA Ⅰ, Ⅱ, Ⅲ were 61.6% (P<0.05), 45.8% (P<0.05) and 55.6% (P<0.05),respectively.CONCLUSION: The in vitro transcribed siRNAs can be useful for silencing oncogene hPim-2 expression specifically and efficiently. This may open a new path toward the use of siRNAs as a gene-specific therapeutic tool.展开更多
文摘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/threonine kinase Pim-2 in human colon cancer cell line.METHODS: siRNA Ⅰ,Ⅱ and Ⅲ were synthesized by T7 RNA polymerase-directed in vitro transcription, then transfected into human colon cancer cells SW-480. After incubation for 6 h at 37℃, 100 mL/L FBS in RPMI 1640 was substituted in each well. After the transfection was repeated twice to three times in each kind of siRNA, hPim-2 mRNA and protein expression were measured by RT-PCR and Western blotting, respectively.RESULTS: Compared to the control group, after transfected for 48 h with hPim-2 siRNA Ⅰ, Ⅱ and Ⅲ, the relative inhibition rates of hPim-2 mRNA expression in colon cancer cell swere 65.4% (P<0.05), 46.2% (P<0.05) and 56.1% (P<0.05),respectively. The protein level of hPim-2 was decreased at 72 h compared to the untransfected cells. The relative inhibition percentages of hPim-2 protein by siRNA Ⅰ, Ⅱ, Ⅲ were 61.6% (P<0.05), 45.8% (P<0.05) and 55.6% (P<0.05),respectively.CONCLUSION: The in vitro transcribed siRNAs can be useful for silencing oncogene hPim-2 expression specifically and efficiently. This may open a new path toward the use of siRNAs as a gene-specific therapeutic tool.
