Fig 1.
HNF1A expression in human pancreatic carcinoma cell lines (A and B), and human pancreatic tissues (C and D).
A, mRNA expression level in human pancreatic cancer cell lines relative to control cell line COLO357 as measured by qRT-PCR. B, protein expression in corresponding cell lines by Western blot. C, fold differences in mRNA expression levels of 27 pancreatic tumors compared to their paired adjacent non-tumor tissues. D and E, Western blots and quantitative measurement of expression of HNF1A protein in eight paired human pancreatic tumors samples (T) and their adjacent non-tumor (N) tissues. The expression levels of HNF1A are normalized to those of β-actin.
Fig 2.
Representative micrographs of HNF1A, pAKT, pmTOR in PDAC cancerous tissue or its surrounding non-tumor tissue by immunohistochemistry.
Images (A and B) show cytoplasmic and nuclear expression of HNF1A in normal pancreatic tissue. The normal pancreatic ducts are marked with single arrow. Islet cells are marked with double arrows. Pancreatic islet cells have higher level of HNF1A expression than acinar and ductal cells. Images (C and D) show the expression of HNF1A in pancreatic ductal adenocarcinoma (circled) and benign pancreatic ductal cells and islet cells adjacent to the tumor (left). Panel E-H show the representative micrographs for p-AKT (E and F) and p-mTOR (G and H) expression in normal pancreatic tissues (E and G) and pancreatic ductal adenocarcinoma (F and H). Magnifications: 100X for A, E and G; 400X for B and H, 40X for C, 200X for D and F.
Table 1.
Expression of HNF1A, pAKT, pmTOR in paired tumor and normal pancreatic tissues.
Fig 3.
HNF1A knockdown by specific siRNAs in AsPC-1 (A) and BxPC-3 (B) cells.
Cells were transfected independently with three different sequences of siRNA directed against HNF1A (siRNA1, siRNA2, siRNA3), or with a control siRNA (Control). Inhibition efficiencies were assessed by qPCR for relative levels of HNF1A mRNA at 24 h to 96 h following the transfections. Western Blot analyses of AsPC-1 and BxPC-3 cells transfected with anti-HNF1A siRNAs. Data are shown for 96 h following transfections. Expression of β-actin was used as an internal control.
Fig 4.
Effect of HNF1A knockdown on cell proliferation and cell cycle.
Pancreatic cancer cell lines were transfected with one of the two HNF1A siRNAs or a control siRNA. The proliferation rate of the siRNA-transfected cells was assessed by CellTiter-Glo assay on AsPC-1 (A) and BxPC-3 (B) cells 12 h to 72h after transfection. Cell cycle distribution was determined by Fluorescence-activated cell sorting analysis in AsPC-1(C) and BxPC-3 (D) cells. Cells were stained with PI, and analyzed for DNA content using flow cytometry 72h following transfections. The populations of cells in G1, S, and G2 phases are characterized by different intensities of PI-A fluorescence and are indicated. Data are shown relative to the control. *P<0.05 compared with each corresponding control.
Fig 5.
Effect of HNF1A knockdown on apoptosis.
72 h after transfection with anti-HNF1A siRNAs, the AsPC-1 and BxPC-3 cells were stained with FITC conjugated Annexin-V/PI. Percentage of Annexin-V/PI-stained cells was determined by flow cytometry. Dot plot was divided into a quadrant: (1) necrotic cells, (2) late apoptotic cells, (3) living cells, and (4) early apoptotic cells. Bar graph shows the percentage of apoptotic cells. Values are expressed as mean ± SD from three different experiments. *P<0.05 compared with the control.
Fig 6.
Effect of HNF1A knockdown on AKT/mTOR signaling pathway was assessed by Western Blot.
AsPC-1 and BxPC-3 cells were transfected with control siRNA and two different HNF1A siRNAs as indicated. 96h after transfection, cell lysates were immunoblotted with anti- HNF1A antibody, anti-phospho-AKT Ser473 antibody, anti-phospho-AKT Thr308 antibody, and anti-phospho-mTOR Ser2448 antibody. Membranes were stripped and re-probed with anti-AKT antibody, anti-mTOR antibody and anti-β-actin antibody. The fold differences in protein expression levels of cells transfected with HNF1A siRNA and control siRNA was presented as mean ± SD from three independent experiments.