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

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.

Figure 1

Effect of shRNA on proliferation of Colo 320 cells. A. Plasmid pGenesil-c-Myc-1,-2, -3, and -4 with pEGFP encoding green fluorescence protein were transfected to the cells. B. c-Myc-depletion inhibits colony formation of Colo 320 cells. B-1) Control Colo 320 cells. B-2) Colo 320 cells treated with pGenesil-c-Myc-1 for 24 h B-3) The percentage of colonies of pGenesil-c-Myc-1 treated cells standardized against the corresponding control. C. Cells were treated with increasing concentrations of shRNA for 48 h. C-1)Cell viability was determined by cell counts. Results are expressed as the percentage of viable cells in the treatment groups with respect to that in the corresponding control. C-2) Cell proliferation following shRNA treatments for 48 h was quantified by 3H-thymidine incorporation studies. 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).

Figure 2

Effect of shRNA on expression of c-Myc and hTERT gene in Colo 320 cells. A. c-Myc and hTERT mRNA expression of Colo 320 cells was detected by RT-PCR with different treatments. The cells were subjected to no treatment (control), pGenesil-vector group, 5, 7.5, 10, 12.5 μM shRNA for 48 h. B. c-Myc protein and hTERT expression of Colo 320 cells was detected by Western blotting with different treatments. Typical Western blot results for c-Myc and hTERT protein. Protein expression of c-Myc in Colo 320 cells was quantified by densitometric analysis. 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).

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).