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 degrees, 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 cells were 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.
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 humancolon cancer cell line. METHODS: siRNA I, II and III were synthesized by T7 RNA polymerase-directed in vitro transcription, then transfected into humancolon cancer cells SW-480. After incubation for 6 h at 37 degrees, 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 cells were 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.
Authors: Patrick J Paddison; Amy A Caudy; Emily Bernstein; Gregory J Hannon; Douglas S Conklin Journal: Genes Dev Date: 2002-04-15 Impact factor: 11.361