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siRNA directed against c-Myc inhibits proliferation and downregulates human telomerase reverse transcriptase in human colon cancer Colo 320 cells.

Hao H, Nancai Y, Lei F, Xiong W, Wen S, Guofu H, Yanxia W, Hanju H, Qian L, Hong X - J. Exp. Clin. Cancer Res. (2008)

Bottom Line: We designed and used a short hairpin RNA to inhibit c-Myc expression in Colo 320 cells and validated its effect on cell proliferation.Upon transient transfection with plasmid encoding shRNA, it was found that expression of c-Myc and hTERT decreased in shRNA-transfected cells.In conclusion, our findings demonstrate that shRNA of c-Myc can inhibit the DNA replication in Colo 320 cells effectively and reduce telomere length and telomerase activity, therefore, it could be used as a new potential anticancer tool for therapy of human colon cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center of Experimental Medicine, Wuhan No,1 Hospital, Wuhan, 430022, PR China. hao4@163.com

ABSTRACT
The c-Myc and human telomerase reverse transcriptase gene (hTERT) gene are frequently deregulated and overexpressed in malignancy. hTERT activity is induced by c-Myc and strategies designed to inhibit c-Myc expression in cancer cells may have considerable therapeutic value. We designed and used a short hairpin RNA to inhibit c-Myc expression in Colo 320 cells and validated its effect on cell proliferation. In this study, four c-Myc-shRNA expression vectors were constructed and introduced into Colo 320 cells. The effects of c-Myc silencing on tumor cell growth was assessed by soft agar assay and DNA synthesis experiments. The expressions of c-Myc and hTERT were also assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it was found that expression of c-Myc and hTERT decreased in shRNA-transfected cells. The downregulation of c-Myc and hTERT inhibited cell growth, shortened telomere lengths, and suppressed telomerase activity. In conclusion, our findings demonstrate that shRNA of c-Myc can inhibit the DNA replication in Colo 320 cells effectively and reduce telomere length and telomerase activity, therefore, it could be used as a new potential anticancer tool for therapy of human colon cancer.

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Effects of shRNA on telomere length and telomerase activity in Colo 320 cells. A. Representative the concentration-course analysis of telomerase activity, each groups cells were mixed with 1 ml TBA solution for preparation of protein extract and 1 μg protein was subjected to TRAP assay. After hybridization and ELISA procedure, the absorbance of the samples at 450 nm was measured. The cells were subjected to no treatment (control), pGenesil-vector group, 5, 7.5, 10, 12.5 μM shRNA for 48 h. B. Mean telomere restriction fragment length was detected by RT-PCR with different treatments by Southern analysis as described under Materials and methods. Typical Southern blot results for telomere restriction fragments. Locations of the base pair markers on the DNA ladder are indicated along the left side. Significant difference was observed between the mean telomere lengths of the control and shRNA-transfected groups cells. All data were obtained from three independent experiments. Error bars represent means ± SEM. Significantly different from the corresponding control (**P < 0.01, vs control. ##P < 0.01, vs vector).
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Figure 3: Effects of shRNA on telomere length and telomerase activity in Colo 320 cells. A. Representative the concentration-course analysis of telomerase activity, each groups cells were mixed with 1 ml TBA solution for preparation of protein extract and 1 μg protein was subjected to TRAP assay. After hybridization and ELISA procedure, the absorbance of the samples at 450 nm was measured. The cells were subjected to no treatment (control), pGenesil-vector group, 5, 7.5, 10, 12.5 μM shRNA for 48 h. B. Mean telomere restriction fragment length was detected by RT-PCR with different treatments by Southern analysis as described under Materials and methods. Typical Southern blot results for telomere restriction fragments. Locations of the base pair markers on the DNA ladder are indicated along the left side. Significant difference was observed between the mean telomere lengths of the control and shRNA-transfected groups cells. All data were obtained from three independent experiments. Error bars represent means ± SEM. Significantly different from the corresponding control (**P < 0.01, vs control. ##P < 0.01, vs vector).

Mentions: We evaluated the effect of shRNA on telomerase activity. Our data suggested that shRNA could down-regulate telomerase activity in shRNA-transfected groups. Telomerase activity were shown in Figure 3A. It revealed that shRNA-transfected groups had lower telomerase activity than the control groups. shRNA at a variety of concentrations resulted in significant reduction of telomerase activity.


siRNA directed against c-Myc inhibits proliferation and downregulates human telomerase reverse transcriptase in human colon cancer Colo 320 cells.

Hao H, Nancai Y, Lei F, Xiong W, Wen S, Guofu H, Yanxia W, Hanju H, Qian L, Hong X - J. Exp. Clin. Cancer Res. (2008)

Effects of shRNA on telomere length and telomerase activity in Colo 320 cells. A. Representative the concentration-course analysis of telomerase activity, each groups cells were mixed with 1 ml TBA solution for preparation of protein extract and 1 μg protein was subjected to TRAP assay. After hybridization and ELISA procedure, the absorbance of the samples at 450 nm was measured. The cells were subjected to no treatment (control), pGenesil-vector group, 5, 7.5, 10, 12.5 μM shRNA for 48 h. B. Mean telomere restriction fragment length was detected by RT-PCR with different treatments by Southern analysis as described under Materials and methods. Typical Southern blot results for telomere restriction fragments. Locations of the base pair markers on the DNA ladder are indicated along the left side. Significant difference was observed between the mean telomere lengths of the control and shRNA-transfected groups cells. All data were obtained from three independent experiments. Error bars represent means ± SEM. Significantly different from the corresponding control (**P < 0.01, vs control. ##P < 0.01, vs vector).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2526986&req=5

Figure 3: Effects of shRNA on telomere length and telomerase activity in Colo 320 cells. A. Representative the concentration-course analysis of telomerase activity, each groups cells were mixed with 1 ml TBA solution for preparation of protein extract and 1 μg protein was subjected to TRAP assay. After hybridization and ELISA procedure, the absorbance of the samples at 450 nm was measured. The cells were subjected to no treatment (control), pGenesil-vector group, 5, 7.5, 10, 12.5 μM shRNA for 48 h. B. Mean telomere restriction fragment length was detected by RT-PCR with different treatments by Southern analysis as described under Materials and methods. Typical Southern blot results for telomere restriction fragments. Locations of the base pair markers on the DNA ladder are indicated along the left side. Significant difference was observed between the mean telomere lengths of the control and shRNA-transfected groups cells. All data were obtained from three independent experiments. Error bars represent means ± SEM. Significantly different from the corresponding control (**P < 0.01, vs control. ##P < 0.01, vs vector).
Mentions: We evaluated the effect of shRNA on telomerase activity. Our data suggested that shRNA could down-regulate telomerase activity in shRNA-transfected groups. Telomerase activity were shown in Figure 3A. It revealed that shRNA-transfected groups had lower telomerase activity than the control groups. shRNA at a variety of concentrations resulted in significant reduction of telomerase activity.

Bottom Line: We designed and used a short hairpin RNA to inhibit c-Myc expression in Colo 320 cells and validated its effect on cell proliferation.Upon transient transfection with plasmid encoding shRNA, it was found that expression of c-Myc and hTERT decreased in shRNA-transfected cells.In conclusion, our findings demonstrate that shRNA of c-Myc can inhibit the DNA replication in Colo 320 cells effectively and reduce telomere length and telomerase activity, therefore, it could be used as a new potential anticancer tool for therapy of human colon cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center of Experimental Medicine, Wuhan No,1 Hospital, Wuhan, 430022, PR China. hao4@163.com

ABSTRACT
The c-Myc and human telomerase reverse transcriptase gene (hTERT) gene are frequently deregulated and overexpressed in malignancy. hTERT activity is induced by c-Myc and strategies designed to inhibit c-Myc expression in cancer cells may have considerable therapeutic value. We designed and used a short hairpin RNA to inhibit c-Myc expression in Colo 320 cells and validated its effect on cell proliferation. In this study, four c-Myc-shRNA expression vectors were constructed and introduced into Colo 320 cells. The effects of c-Myc silencing on tumor cell growth was assessed by soft agar assay and DNA synthesis experiments. The expressions of c-Myc and hTERT were also assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it was found that expression of c-Myc and hTERT decreased in shRNA-transfected cells. The downregulation of c-Myc and hTERT inhibited cell growth, shortened telomere lengths, and suppressed telomerase activity. In conclusion, our findings demonstrate that shRNA of c-Myc can inhibit the DNA replication in Colo 320 cells effectively and reduce telomere length and telomerase activity, therefore, it could be used as a new potential anticancer tool for therapy of human colon cancer.

Show MeSH
Related in: MedlinePlus