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
Background Genetic modification of dendritic cells （DCs） has been used as an effective approach to enhance anti-tumor immunity. RNA interference （RNAi）, which can cause the degradation of any RNA in a sequence-spe...Background Genetic modification of dendritic cells （DCs） has been used as an effective approach to enhance anti-tumor immunity. RNA interference （RNAi）, which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA （siRNA） specific for the li gene was transfected into DCs, and the anti-tumor immunity of li-silenced DCs was assessed. Methods The silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96 non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used. Results The li expression of DCs was significantly reduced after li siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with li siRNA plus endogenous tumor antigen （P 〈0.05）. Furthermore tumor growth was greatly inhibited when mice were immunized with DCs transfected with li siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4^= and CD8^＋ T cells were significantly activated in the li siRNA group （P 〈0.05）. Conclusion Silencing of the li gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.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.
基金This research was supported by grants from the National Natural Science Foundation of China （No. 30570828 and No. 30471961）.Acknowledgements： The authors deeply appreciate technical assistance from Q. Li and D. Q. Zhang valuble discussion with R. F Ge and help from the experimental Animal Facility technicians for animal care. We thank the International Science Editing for help in editing our manuscript.
文摘Background Genetic modification of dendritic cells （DCs） has been used as an effective approach to enhance anti-tumor immunity. RNA interference （RNAi）, which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA （siRNA） specific for the li gene was transfected into DCs, and the anti-tumor immunity of li-silenced DCs was assessed. Methods The silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96 non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used. Results The li expression of DCs was significantly reduced after li siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with li siRNA plus endogenous tumor antigen （P 〈0.05）. Furthermore tumor growth was greatly inhibited when mice were immunized with DCs transfected with li siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4^= and CD8^＋ T cells were significantly activated in the li siRNA group （P 〈0.05）. Conclusion Silencing of the li gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.
