Lentivirus-mediated shRNA interference targeting STAT3 inhibits human pancreatic cancer cell invasion

Figure 1 Structure of vectors. A: Lentivirus vector pGCL-GFP containing a CMV driven GFP reporter and a U6 promoter upstream of cloning restriction sites (HpaI and XhoI) to allow the introduction of oligonucleotides encoding shRNAs. The multiple cloning site (MCS) is located between the U6 promoter and CMV. B: The pEGFP-N1 vector expressed green fluorescent protein following transfection into mammalian cells. MCS is located between the immediate early promoter of CMV and the EGFP. ¹Methylated in the DNA provided by CLONTECH.

Figure 2 Selection of the most effective STAT3 specific siRNA expression vector in 293T cells. A: Phase contrast and GFP expression under a fluorescent microscope was taken after 36-48 h in 293T cells, 1: 293T cells; 2: Transfection of pEGFP-N1 vector in 293T cells; 3: Transfection of pEGFP-N1-STAT3; 4: Co-transfected of pEGFP-N1-STAT3 and pGCL-GFP-siRNA vectors in 293T cells (original magnification × 200). B: STAT3 gene was cloned from the cDNA library with production of 2340 bp by PCR. C: pEGFP-N1-STAT3 was constructed after restricted enzyme cutting and connection with production of 300 bp by PCR. 1: pEGFP-N1 vector; 2: A special sequence was inserted in pEGFP-N1 vector; 3: Marker; 4-5: STAT3 gene over-expression vector pEGFP-N1-STAT3. D: Protein level of STAT3 in 293T cells was detected by western blotting. Lanes 1-6: 293T cells, STAT3 gene over-expression vector, non-silence control, LV-STAT3siRNA-1, -2, and-3 respectively. LV-STAT3siRNA-1 and LV-STAT3siRNA-2 can significantly knock down expression of STAT3 at the protein level.

Figure 3 Expression of STAT3 suppressed by LV-siSTAT3-2 in SW1990 cells. A: SW1990 cells were infected with LV-Con or LV-STAT3siRNA-2; The cells were infected (MOI = 40), GFP expression and the phase contrast images were taken after 72 h (original magnification × 200). B: mRNA level of STAT3 after SW1990 cells were treated with LV-STAT3siRNA-2 and LV-Con detected by real-time PCR. LV-STAT3siRNA-2 significantly inhibited expression of STAT3 mRNA in SW1990 cells, ^aP < 0.05, compared with SW1990 cells, ^cP < 0.05, compared with LV-Con group. C: Western blotting analysis showed that STAT3 protein was markedly inhabited by LV-STAT3siRNA-2 in SW1990 cells.

Figure 4 Pancreatic cancer cells growth was detected by MTT assay. SW1990 cells growing in 96-well plates were infected with LV-STAT3siRNA-2 and LV-Con 1, 2, 3, 4 d respectively, and the MTT assay revealed that the cell growth was significantly suppressed by LV-STAT3siRNA-2 in SW1990 cells compared with LV-Con group and parental SW1990 group. Data are mean ± SD.

Figure 5 The expression of MMP-2 and VEGF was analyzed by real-time PCR and Western blotting in the human pancreatic cancer cell lines. LV-STAT3siRNA-2 significantly inhibited mRNA and protein expression of MMP-2 and VEGF in SW1990 cell. A: MMP-2 mRNA expression in every group. B: VEGF mRNA expression in every group. C: P-STAT3, MMP-2 and VEGF protein expression in every group.

Figure 6 Invasion assay was performed using a specialized invasion chamber. A: The blue-stained cells are those that invaded the ECMatrix and migrated through the polycarbonate membrane to the lower surface of the membrane (original magnification × 200). B: Invasion assay indicated LV-STAT3siRNA significantly decreased the invasion ability of SW1990 cells. Bars indicate mean ± SD. ^aP < 0.05 vs SW1990 group, ^cP < 0.05 vs LV-Con group.