Investigation of transcriptional gene silencing and mechanism induced by shRNAs targeted to RUNX3 in vitro

Figure 1 RUNX3 expression in the gastric cancer cells detected by Western blot. 1: Positive control: Normal gastric tissue; 2: MKN-28 cells; 3: SGC-7901 cells; 4: MGC-803 cells; 5: BGC-823 cells.

Figure 2 Identification of recombinant by restrict endonucleases digestion. M: DNA Ladder; 1: pSilencer3.1-H1/SGC7901; 2: pSilencer3.1-H1-shRNA/RUNX3/SGC-7901; 3: pSilencer3.1-H1-shRNA/RUNX3/SGC7901 cut with BamHI/HindIII.

Figure 3 RUNX3 expression in the three groups of cells detected by RT-PCR. M: DL2000 marker; 1: SGC7901 cells; 2: pSilencer3.1-H1 /SGC7901 cells cells; 3: pSilencer3.1-H1-shRNA/RUNX3/SGC7901 cells.

Figure 4 RUNX3 expression detected by Western blot. 1: SGC7901 cells; 2: pSilencer3.1-H1/c SGC7901 cells; 3: pSilencer3.1-H1-shRNA/RUNX3/SGC7901 cells.

Figure 5 RUNX3 expression in SGC7901 cells by immunohistochemistry (× 400). A: SGC7901 cells, the protein of RUNX3 expressed and located in the endochylema and the nucleus in the SGC7901 cell without transfection; B: pSilencer3.1-H1/SGC7901 cells, the protein of RUNX3 expressed and located in the endochylema and the nucleus in the SGC7901 cell with the plasmid DNA of pSilencer3.1-H1; C: pSilencer3.1-H1-shRNA/RUNX3/SGC7901 cells, the protein of RUNX3 lost expression in the SGC790 cells transfected with the recombinated plasmid-pSilencer 3.1-H1-shRNA/RUNX3.

Figure 6 The growth effect of SGC7901 cell by silencing RUNX3 (^aP < 0. 05).

Figure 7 The colony formation assay of SGC7901 in the soft agar (×100). A: SGC7901 cells, the cloning efficiency was 9.9% ± 0.3% in the SGC7901 cell without transfection; B: pSilencer3.1-H1/SGC7901 cells, the cloning efficiency was 9.7% ± 0.6% in the SGC7901 cell with the plasmid DNA of pSilencer3.1-H1; C: pSilencer3.1-H1-shRNA/RUNX3/SGC 7901 cells, the cloning efficiency was 17.4% ± 0.31% in the SGC790 cells transfected with the recombinated plasmid-pSilencer3.1-H1-shRNA/RUNX3. A significant increase of the colony formation rate in the pSilencer3.1-H1-shRNA/RUNX3/SGC790 cells was discovered compared with the controls-the SGC-79011cells and pSilencer3.1-H1/SGC7901cells (P < 0.01).

Figure 8 The cell cycle analysis by FCM. A: SGC790 cells, the cell proportions of G₀/G₁ and S stages were 43.2% ± 1.2% and 47.7% ± 1.1% in the SGC7901cell without transfection, respectively; B: pSilencer3.1-H1/SGC7901 cells, the cell proportions of G₀/G₁ and S stages were 40.3% ± 2.0% and 49.3% ± 0.9% in the SGC7901 cell with the plasmid DNA of pSilencer3.1-H1 respectively; C: pSilencer3.1-shRNA/RUNX3SGC7901 cells, the cell proportions of G₀/G₁ and S stages were 37.2% ± 1.9% and 60.5% ± 0.8% in the SGC790 cells transfected with the recombinated plasmid-pSilencer3.1-H1-shRNA/RUNX3 respectively. Compared with that of G₀/G₁ and S stages of two controls, the cell proportions of G₀/G₁ and S stages in the pSilencer3.1-H1-shRNA/RUNX3/SGC7901 cells decreased and increased obviously, respectively (P < 0.05).

Figure 9 RUNX3 expresion detected by RT-PCR in the four groups of SGC7901 cells. M: Marker; 1: Positive control, SGC7901 cells untreated with 5-Aza-CdR; 2: Negative control, pSilencer3.1-H1-shRNA/RUNX3/SGC7901cells untreated with 5-Aza-CdR; 3: Experimental group 1, pSilencer3.1-H1-shRNA/RUNX3/SGC7901cells treated with 5 × 10^-6 mol/L 5-Aza-CdR; 4: Experimental group 2, pSilencer3.1-H1-shRNA/RUNX3/SGC7901 cells treated with 1 x 10^-5mol/L 5-Aza-CdR.

Figure 10 RUNX3 protein detected by Western blotting in the four groups of SGC7901 cells. 1: Positive control, SGC7901 cells untreated with 5-Aza-CdR; 2: Negative control, pSilencer3.1-H1-shRNA/RUNX3/SGC7901cells untreated with 5-Aza-CdR; 3: Experimental group 1, pSilencer3.1-H1-shRNA/RUNX3/SGC7901cells treated with 5 × 10^-6 mol/L 5-Aza-CdR; 4: Experimental group 2, pSilencer3.1-H1-shRNA/RUNX3/SGC7901 cells treated with 1 × 10^-5 mol/L 5-Aza-CdR.