Four human pancreatic carcinoma cell lines (MIAPaCa-2, PSN-1, BxPC-3 and Panc-1) were used in the present study (8). MIAPaCa-2 and PSN-1 cell lines were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan). BxPC-3 and Panc-1 cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified incubator under 5% CO₂-95% air.

GEM was purchased from Eli Lilly and Co. (Indianapolis, IN, USA). Polyclonal rabbit anti-human DKK1 antibody (Cell Signaling Technology, Beverly, MA, USA), polyclonal rabbit anti-human sFRP2 antibody (Abcam Inc., Cambridge, MA, USA), polyclonal mouse anti-human Kremen2 antibody (Abcam Inc.) and polyclonal rabbit anti-human β-actin (Sigma-Aldrich Co., St. Louis, MO, USA) were used for western blot analysis. Recombinant human Wnt3a (R&D Systems, Minneapolis, MN, USA) was used as a Wnt/β-catenin signaling activator. In this study, Wnt3a was used at 50 ng/ml based on the protocol described in a previous study (13).

Antisense miR-29a inhibitor (anti-miR-29a), miR-29a precursor (pre-miR-29a) and their negative control oligonucleotides were obtained from Ambion Inc. (Austin, TX, USA). These were used to transfect pancreatic cancer cells by using siPORT NeoFx (Ambion Inc.) according to the instructions provided by the manufacturer. The transected cells were resuspended and cultured in regular culture medium for 24–72 h before analysis.

Total RNA and miRNA fractions were isolated from tissue samples and cell lines by TRIzol agent (Invitrogen, Carlsbad, CA, USA) and the quality of the RNA was assessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) at 260 and 280 nm (A260/280).

Reverse transcription reaction and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were performed using TaqMan human miRNA assay kit (Applied Biosystems, Foster City, CA, USA) according to the instructions supplied by the manufacturer. The expression of the target miRNA was normalized relative to that of the internal control; RNU48. Data were analyzed according to the comparative Ct method (14).

RT reaction was performed with SuperScript II (Invitrogen) based on the protocol provided by the manufacturer, followed by qRT-PCR. The expression of the target gene was normalized relative to the expression of porphobilinogen deaminase (PBGD), which was used as an internal control. The designed PCR primers were as follows; AXIN2 forward primer, 5′-GGTGTTTGAGGAGATCTGGG-3′; AXIN2 reverse primer, 5′-TGCTCACAGCCAAGACAGTT-3′; CCND1 forward primer, 5′-AAGGCCTGAACCTGAGGAG-3′; CCND1 reverse primer, 5′-CTTGACTCCAGCAGGGCTT-3′; MYC forward primer, 5′-AAGAGGACTTGTTGCGGAAA-3′; MYC reverse primer, 5′-CTCAGCCAAGGTTGTGAGGT-3′; TACSTD1 forward primer, 5′-TCCAGAAAGAAGAGAATGGCA-3′; TACSTD1 reverse primer, 5′-AAAGATGTCTTCGTCCCACG-3′; TCF3 forward primer, 5′-ATCTGTGTCCCATGTCCCAG-3′; TCF3 reverse primer, 5′-CCAGGGTAGGAGACTTGCAG-3′; and PBGD forward primer, 5′-TGTCTGGTAACGGCAATGCGGCTGCAAC-3′; PBGD reverse primer, 5′-TCAATGTTGCCACCACACTGTCCGTCT-3′.

Cells grown to semiconfluence were lysed in RIPA buffer [25 mM Tris (pH 7.5), 50 mM NaCl, 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% sodium dodecyl sulfate, 1 mM phenylmethylsulphonyl fluoride and 500 KIE/ml Trasylol, proteinase inhibitor (Bayer, Leverkusen, Germany)]. Western blot analysis was carried out as described previously (15,16).

Inhibition of cell growth in the presence of chemotherapeutic agents was assessed by the 3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich Co.) assay as described previously (15,17). Briefly, the cells were incubated for 72 h under various concentrations of GEM. After re-incubation for 4 h in MTT solution, acid-isopropanol was added to dissolve the resultant formazan crystals. The absorbance of the plate was measured in a microplate reader at a wavelength of 570 nm with a 650-nm reference and the results were expressed as the percentage of absorbance relative to untreated controls.

The binding of Annexin V was used as a sensitive method for measuring apoptosis, as described previously (15). Twenty-four hours after treatment, cells were stained with Annexin V-FITC and propidium iodide (PI) (BioVision Research Products, Mountain View, CA, USA) and analyzed on a FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). For the assessment of apoptosis, Annexin V-positive and PI-negative cells and Annexin V-positive and PI-positive cells were considered as early apoptotic cells and late apoptotic cells, respectively.

Cell cycle analysis was performed based on flow cytometric analysis, as described previously (16,17). Briefly, PI and RNase (Sigma-Aldrich Co.) were added and data were acquired on the FACSCalibur (BD Biosciences). The cell cycle was analyzed using ModFIT software (BD Biosciences).

To evaluate the activity of the Wnt/β-catenin signaling pathway, TCF/LEF transcriptional activity was examined. For the examination, the reporter assay kit (SA Biosciences, Frederick, MD, USA) was used according to the instructions provided by the manufacturer. In brief, cells were transiently transfected with the transcription factor-responsive reporter or negative control by the Lipofectamine 2000 reagent (Invitrogen). After the transfection, the cells were transfected with anti-miR-29a or its negative control oligonucleotide. After 48 h, luciferase activity was measured with the Dual-Luciferase Assay System (Promega, Madison, WI, USA) using luminometer, Lumat LB9507 (Berthold Technologies, Calmbacher, Germany). The Firefly luciferase activity, indicating TCF-dependent transcription, was normalized to the Renilla luciferase activity as an internal control to obtain the relative luciferase activity.

Data were expressed as mean ± SD. Clinicopathological parameters were compared using the χ² test and continuous variables were compared using the Student’s t-test. A p<0.05 denoted the presence of a statistically significant difference. Statistical analysis was performed using the StatView software (SAS Institute Inc., Cary, NC, USA).

All the four cell lines used in the present study expressed miR-29a though the level varied among the cells (Fig. 1). The relative expression of miR-29a was significantly lower in the transfected cells by qRT-PCR (Fig. 2A). To evaluate the effect of miR-29a on the response to GEM, we transfected anti-miR-29a in MIAPaCa-2 and PSN-1 cells, which showed higher miR-29a expression levels than the other cell lines. The MTT assay showed that cells transfected with anti-miR-29a were significantly less resistant to GEM compared to control cells (Fig. 2B). Next, we evaluated the extent of apoptosis of these cells at 24 h after treatment with GEM (MIAPaCa-2; 40 ng/ml, PSN-1; 2 ng/ml) by the Annexin V assay. The percentages of early and late apoptotic cells were significantly higher in the two cancer cell lines transfected with anti-miR-29a than in the control cells (Fig. 2C).

We also examined the influence of miR-29a on the cell cycle in MIAPaCa-2 and PSN-1. The distribution of cells in G₀/G₁ phase, S phase and G₂/M phase was similar between the miR-29a-suppressed cells and the control cells in the absence of GEM (Fig. 2D). However, 24 h after GEM treatment (MIAPaCa-2; 20 ng/ml, PSN-1; 1 ng/ml), the percentage of cells at the S phase among the miR-29a-suppressed cells was higher than the control cells (Fig. 2D).

We examined next the expression levels of these molecules in MIAPaCa-2 and PSN-1. Western blot analysis showed significantly higher protein expression levels of these molecules in the anti-miR-29a-transfected cells compared with the control cells (Fig. 3A). Furthermore, the luciferase reporter assay showed that TCF/LEF transcriptional activity, representing the activity of the Wnt/β-catenin signaling pathway, was significantly lower in the miR-29a-suppressed cells than in the control cells (Fig. 3B). We also examined the expression of five Wnt/β-catenin signaling targeted genes (AXIN2, CCND1, MYC, TACSTD1 and TCF3) in anti-miR-29a-transfected MIAPaCa-2 and PSN-1 cell lines by qRT-PCR. Suppression of miR-29a significantly reduced the mRNA expression of the targeted genes (Fig. 3C). Taken together, the results suggest that miR-29a activates the Wnt/β-catenin signaling pathway through the suppression of Dkk1, Kremen2 and sFRP2.

To further assess the effects of miR-29a, pre-miR-29a was transfected into BxPC-3 and Panc-1, which expressed lower levels of miR-29a than the other cell lines (Fig. 1). Transfection of cells with pre-miR-29a increased miR-29a level compared to the control cells (Fig. 4A). The miR-29a-overexpressing cells were significantly more resistant to GEM than the control cells, as evident by the MTT assay (Fig. 4B).

Finally, we analyzed the mechanism responsible for the miR-29a-induced resistance to GEM. We focused on the Wnt/β-catenin signaling pathway, based on the results reported by Kapinas et al (13). The addition of Wnt3a to the cultures of MIAPaCa-2 and PSN-1 resulted in the activation of Wnt/β-catenin signal in the cell lines (Fig. 5A). Furthermore, the MTT assay showed the Wnt3a-treated cells were more resistant to GEM (MIAPaCa-2; 25 ng/ml, PSN-1; 1.6 ng/ml, Fig. 5B). In addition, both the inactivated Wnt/β-catenin signal and the augmented growth-inhibitory effect by the afore-mentioned anti-miR-29a transfection were weakened after the addition of Wnt3a (Fig. 5). These findings suggest that activation of the Wnt/β-catenin signaling mediates, at least in part, the miR-29a-induced resistance to GEM.