Real-time RT-PCR showed that the expression of miR-146a was higher in HPDE cells compared with pancreatic cancer cells (A) and that treatment of Colo357 (B) and Panc-1 (C) cells with 25 μmol/L B-DIM or 25 μmol/L isoflavone induced the expression of miR-146a compared with vehicle control. Columns, mean from three independent experiments; bars, SEM; *, P < 0.05.

Reexpression of miR-146a by transfection resulted in the downregulation of EGFR expression and phosphorylation in Colo357 and Panc-1 cells as assessed by real-time RT-PCR (A; columns, mean from three independent experiments; bars, SEM; *, P < 0.05.) and Western blot analysis (B). The expression of IRAK-1, NF-κB (C), and MTA-2 (D) was also downregulated in pancreatic cancer cells after miR-146a transfection. However, the expressions of EGFR, p-EGFR (B); IRAK-1, NF-κB (C); and MTA-2 (D) were upregulated by anti–miR-146 transfection. C, control miRNA transfection; 146, miR-146a transfection; Anti, anti–miR-146 transfection; 3D, 5D, 6D, 9D, transfection for 3, 5, 6, or 9 days, respectively; the ratios of detected target proteins against β-actin were shown under each Western blot image.

B-DIM (25 μmol/L) or isoflavone (G2535, 25 μmol/L) treatment for 2 d decreased EGFR expression and phosphorylation in pancreatic cancer cells as assessed by real-time RT-PCR (A; columns, mean from three independent experiments; bars, SEM; *, P < 0.05.) and Western blot analysis (B). The expression of IRAK-1, NF-κB (C), and MTA-2 (D) was also downregulated in pancreatic cancer cells after B-DIM or isoflavone treatment. C, control miRNA transfection; BD, B-DIM treatment; Iso, isoflavone treatment.

miR-146a or EGFR siRNA transfection increased the level of IκBα (A) and decreased the levels of EGFR, p-IκBα, NF-κB, and MTA-2 (B) in pancreatic cancer cells. B-DIM (25 μmol/L) or isoflavone (25 μmol/L) treatment increased the level of IκBα and decreased the levels of EGFR, p-IκBα, and NF-κB in pancreatic cancer cells. EMSA (D) showed that NF-κB DNA-binding activity in pancreatic cancer cells was inhibited by the reexpression of miR-146a or the treatment of cells with 25 μmol/L B-DIM or 25 μmol/L isoflavone. C, control; 146, miR-146a transfection; siE, EGFR siRNA transfection; BD, B-DIM treatment; Iso, isoflavone treatment; 2D, treatment for 2 days; 5D, 9D, transfection for 5 or 9 days. The ratios of detected target proteins against β-actin were shown under each Western blot image.

Invasion assay showed that the reexpression of miR-146a by transfection studies or by the treatment of pancreatic cancer cells with 25 μmol/L B-DIM or 25 μmol/L isoflavone significantly inhibited the invasion of pancreatic cancer cells through the Matrigel matrix membrane. However, inhibition of miR-146 increased the invasion of pancreatic cancer cells and restored the invasive capacity of cells treated with B-DIM and G2535. A, Panc-1 cells; B, Colo357 cells; a, negative control miRNA transfection; b, transfected with anti–miR-146; c, transfected with miR-146a; d, treated with 25 μmol/L B-DIM; e, transfected with anti–miR-146 followed by B-DIM treatment; f, treated with 25 μmol/L isoflavone; g, transfected with anti–miR-146 followed by G2535 treatment (×200). The graphs showed the value of fluorescence from the invaded pancreatic cancer cells. The values indicated the comparative number of invaded pancreatic cancer cells. Columns, mean from two independent experiments; bars, SEM; P < 0.05 for each group compared with control. C, the level of miR-146 influenced the inhibitory effect of erlotinib, B-DIM, and G2535 on cell proliferation. Columns, mean from three independent experiments; bars, SEM. D, diagram showing the hypothetical cross-regulation between miR-146a, EGFR, MTA-2, IRAK-1, IκBα, and NF-κB in the inhibition of cancer cell invasion and metastasis by B-DIM and isoflavone.