Figure 1.
Screening colon cancer cell lines and effective hTERT-siRNA.
(A) Endogenous hTERT mRNA expression in SW480, DLD-1 and HT29 cell lines. There were significant differences among SW480, HT29 and DLD-1 cells (#P<0.01), and between SW480 and HT29 cells (*P<0.05). (B) Potency of hTERT siRNAs in SW480 cells. Levels of hTERT mRNA were significantly lower in the cells transfected with hTERT siRNAs than in cells transfected with negative control (NC) shRNA or Lipofectamine alone (Lipo). *P<0.05, #P<0.01, compared with the NC-shRNA and Lipo groups. Results are mean ± SD from three independent experiments.
Figure 2.
hTERT-shRNA inhibits hTERT expression and telomerase activity in vitro.
(A) RT-PCR analysis of hTERT mRNA expression in SW480 cultures after mock-transfection with culture medium alone (Blank), Lipofectamine alone (Lipo), negative control siRNA (NC-shRNA), or hTERT-shRNA. Levels of mRNA levels were significantly lower after 48-h transfection with hTERT-shRNA than after control transfections. ▴P<0.05, #P<0.01, compared with the Blank; *P<0.05, compared with Lipo and NC-shRNA. (B) Immunocytochemistry of hTERT protein expression. (a) Blank cells, (b) Lipo cells, (c) NC-shRNA cells, and (d) hTERT-shRNA cells. All views, ×400. Higher greyscale value indicates lower protein level. Protein levels were significantly lower after 48-h transfection with hTERT-shRNA than after control transfections. #P<0.01, compared with the other groups; *P<0.05, compared with the Blank. (C) HTERT down-regulation inhibits telomerase activity. Telomerase activity [adjusted absorbance (450 nm–630 nm)] was significantly lower in cells transfected with hTERT-shRNA than in control cells. *P<0.05, compared with the Lipo and Blank controls. Results are mean ± SD from three independent experiments.
Figure 3.
hTERT down-regulation reduces SW480 growth and proliferation and increases apoptosis at 48 h in vitro.
(A) hTERT-shRNA reduces growth and proliferation of SW480 cells. The proportion of cells in G0/G1 phase was significantly higher and the proliferation index was much lower after transfection with hTERT-shRNA than after control transfections. *P<0.05, compared with the other three groups; #P<0.05, compared with NC-shRNA. (B) hTERT-shRNA increases apoptosis of SW480 cells. Cells were transfected with (a) hTERT-shRNA or (b) NC-shRNA, stained with diaminobenzidine (brown) and counter-stained with hematoxylin (blue) to label nuclei. Images are representative results from three independent experiments. Magnification, ×400. The apoptotic index (AI) was significantly higher in cells transfected with hTERT-shRNA. #P<0.01, compared with the other three groups. (C) Transmission electron microscopy analysis of SW480 cells transfected with (a-c) hTERT-shRNA or (d) NC-shRNA. Apoptotic morphology was visible in cells transfected with hTERT-shRNA. Magnification, ×3800. (D) Rhodamine 123 fluorescence intensity analysis of SW480 cells transfected with (a) culture medium alone (Blank), (b) Lipofectamine alone (Lipo), (c) NC-shRNA or (d) hTERT-shRNA. Stained cells were analyzed by confocal scanning microscopy; higher fluorescence intensity indicates lower mitochondrial membrane potential. Magnification, ×400. Cells transfected with hTERT-shRNA showed significantly higher rhodamine 123 fluorescence than did control cells. *P<0.05, compared with NC-shRNA or Lipo; #P<0.01, compared with Blank. Results are the mean ± SD from three independent experiments.
Figure 4.
Effects of hTERT-shRNA on SW480 tumor growth, apoptosis and telomerase activity in vivo.
(A) hTERT-shRNA inhibits tumor cell growth. Tumor growth at 31 days was significantly slower in tumors treated with hTERT-shRNA than in tumors treated with saline (*P<0.05) or NC-shRNA (#P<0.01). (B) Histopathological examination of SW480 tumor tissue grafted onto nude mice and treated with hTERT-shRNA. Tumor tissue was biopsied at 31 days and stained with hematoxylin-eosin. The arrow in (a) indicates sheet necrosis; the arrow in (b) indicates inversion of the nucleoplasm to cytoplasm volume, together with mitotic pathology. Magnification, ×200. (C) RT-PCR analysis to measure hTERT mRNA expression in tumor tissue treated with saline (lanes 1–8), NC-shRNA (lanes 9–16), or hTERT-shRNA (lanes 17–24). M, 100-bp DNA ladder. The relative expression of hTERT mRNA was significantly lower in the hTERT-shRNA mice. *P<0.05, compared with the saline and NC-shRNA groups. (D) Immunocytochemistry of hTERT protein (stained brown) in tumor tissue treated with (a) saline, (b) NC-shRNA, or (c) hTERT-shRNA. Magnification, ×200. Higher greyscale values indicate lower protein levels. The relative expression of hTERT protein was significantly lower in hTERT-shRNA mice. #P<0.01, compared with the saline and NC-shRNA groups. (E) Effect of hTERT-shRNA on telomerase activity in vivo. The adjusted absorbance (450 nm - 630 nm) was significantly lower in tumors treated with hTERT-shRNA than in tumors treated with saline (*P<0.05) or NC-shRNA (#P<0.01). (F) Effects of hTERT-shRNA on apoptosis. Tumors were injected regularly with (a) saline, (b) NC-shRNA or (c) hTERT-shRNA, then biopsied and stained with diaminobenzidine (brown); nuclei were counter-stained with hematoxylin (blue). Images are representative results of three independent experiments. Magnification, ×200. The apoptotic index (AI) was significantly higher in tumors treated with hTERT-shRNA than in tumors treated with saline or NC-shRNA. #P<0.01, compared to the two control groups.