The PDAC SW1990 and CFPAC-1 cell lines were obtained from Shanghai Cell Bank (Shanghai, China). These cell lines were propagated and cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), 100 μg/ml penicillin and streptomycin, in a humidified chamber at 37°C with 5% CO₂.

Gemcitabine-resistant PDAC SW1990/GR and CFPAC-1/GR cell sublines were acquired by culturing the parental cells with gradually increasing concentrations of gemcitabine (Lilly, Neuilly-sur-Seine Cedex, France) for approximately 6 months as documented in a previous study (32). In brief, the cells were cultured in gemcitabine-conditioned medium for 3 days at the concentration of 3 μM, followed by a recovery step, and agent-free medium culturing until the cells recovered exponential growth. MTT assays were performed to evaluate the IC₅₀ of the gemcitabine-treated cells. The IC₅₀ dose was used for the gemcitabine-conditioned medium to treat the cells. By increasing the dosage of gemcitabine in the culture medium intermittently for approximately 6 months (24 weeks for SW1990 and 21 weeks for CFPAC-1 cells), stable gemcitabine-resistant cell sublines (SW1990/GR and CFPAC-1/GR) were acquired. The IC₅₀ in the SW1990/GR and CFPAC-1/GR cells was 232.2 and 314.4 μM, respectively.

miR-181b mimics, inhibitor and negative control were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). The primer sequences are shown in Table I. For gene transfection, the cells were cultured in 6-well plates to 40% confluence. miR-181b mimics, inhibitor and negative control were mixed with Lipofectamine 2000 (Invitrogen), and then added to the cell culture medium according to the manufacturer's instructions. After 24 h of transfection, total RNA and protein were prepared from the cells and subjected to qRT-PCR and western blot analyses, respectively.

A miRNA-181b lentivirus overexpression system was obtained from GenePharma. Briefly, the miRNA-181b lentiviral expression vectors, LV3-pGLV-H1-miRNA-181b-GFP-Puro and LV3-pGLV-H1-Null-GFP-Puro (null control) were constructed. The vectors were then transfected into 293-T cells. The supernatant titer was determined to be 1×10⁸ TU/ml. Subsequently, the lentivirus was used to infect PDAC cells following the manufacturer's instructions.

Total cellular RNA from the cultured cells was isolated using TRIzol reagent (Invitrogen). RNA samples (500 ng each) were then reverse-transcribed into cDNA with miRNA-181b reverse transcriptase primers (Applied Biosystems, Foster City, CA, USA) using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems). Levels of miRNA-181b and U6 expression were determined by qPCR with TaqMan MicroRNA Assays (Applied Biosystems) and an ABI 7500 machine (Applied Biosystems). The levels of mature miRNA-181b expression were then normalized to U6 and calculated as the inverse log of the ΔΔCT. All procedures were performed following the manufacturer's instructions.

Total cell protein lysates were prepared using an RIPA buffer supplemented with 1% phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined by a BCA kit (Keygen, Nanjing, China). Subsequently, 30 μg of protein sample each was separated by SDS-PAGE (10% gels) and transferred onto a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, USA) using a mini trans-blot system (Bio-Rad Laboratories, Hercules, CA, USA). Primary antibodies and corresponding secondary antibodies were then added followed by incubation. A rabbit anti-human BCL-2 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA), and a mouse anti-human GAPDH antibody and goat anti-rabbit or anti-mouse secondary antibody were obtained from Beyotime (Nantong, China). To quantify the protein expression, protein expression was normalized to GAPDH levels (Chemilmager 5500; Alpha Innotech Corp., San Leandro, CA, USA).

To detect caspase-3 activity, we first seeded the cells at a density of 1.5×10⁶ cells per well in 6-well plates. The cells were then transfected with miRNA-181b mimics, inhibitor or negative controls for 48 h. Subsequently, caspase-3 activity was analyzed using a CaspACE Assay System (Promega, Madison, Wisconsin, USA) following the manufacturer's instructions.

To detect cell viability, we first seeded 2×10³ cells in 96-well plates, and then transfected them with miR-181b mimics, inhibitor or negative controls for 72 h. Subsequently, 20 μl per well of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/ml, Sigma) was added to the cells followed by incubation at 37°C for 4 h. Then, 150 μl of dimethyl-sulphoxide (DMSO) was substituted for the supernatant, followed by oscillation for 10 min. Absorbance at 490 nm was detected using a microplate reader (Multiskan MK3; Thermo Labsystems, Franklin, MA, USA). All the results were normalized to the corresponding controls and the percentage of the control was calculated.

Cells were incubated with culture medium containing gemcitabine at a final concentration of 0.1 μM for 48 h. Cell pellets were then collected, washed with phosphate-buffered saline (PBS), resuspended in 100 μl of 1X binding buffer and stained with 5 μl phycoerythrin-Annexin V and 5 μl of 7-AAD (Becton-Dickinson, Franklin Lakes, NJ, USA) at room temperature for 15 min in the dark. A flow cytometer (Becton-Dickinson) was utilized to evaluate the apoptotic levels in each sample.

Luciferase reporter constructs carrying 60-bp-long synthetic oligonucleotides (Invitrogen, Shanghai, China) and containing wild-type putative miRNA binding sites from the human BCL-2 3′-UTR or their mutant versions (www.targetscan.org; Table II) were inserted in XbaI-FseI sites of pGL3-control vectors (Promega). The construct was confirmed by DNA sequencing. Aliquots of 1.5×10⁵ cells were seeded into 24-well plates. After 24 h, 200 ng of each independent luciferase reporter plasmid plus 80 ng of pRL-TK (Promega) plasmid as the control were co-transfected with 60 pmol of the miRNA-181b mimics, inhibitor or control. Luciferase activity was then measured 48 h after transfection using the Dual-Luciferase Reporter Assay System with a GloMax Luminometer (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected cell sample.

For in vivo chemosensitivity analyses, 2×10⁶ cells from the SW1990 and SW1990/GR cell lines transfected with LV3-pGLV-H1-miRNA-181b-GFP-Puro, LV3-pGLV-H1-Null-GFP-Puro and mock control vectors were resuspended in 25 μl of DMEM, and then injected into the pancreatic undercapsule in 4-week-old BALB/c female nude mice (6 mice in each group). BALB/c female nude mice were purchased from The Model Animal Research Center of Nanjing University, Nanjing, China. Our animal studies were approved by the Ethics Committee of Nanjing Medical University. Two weeks after cell injection, gemcitabine (150 mg/kg) was injected intraperitoneally twice weekly for 28 days. At the end of the experiments, the mice were euthanized and the tumor lesions were excised. Tumor volume was determined as V = (L × W²)/2, where L represents the length of the tumor and W represents the width of the tumor.

All in vitro experiments were performed in triplicate and repeated at least once. The Student's t-test was performed to assess statistical differences between 2 groups and the F-test was used for comparisons among 3 or more groups. The Q test was used for multiple comparisons. A P-value <0.05 was considered to indicate a statistically significant difference.

In this study, we first established gemcitabine-resistant pancreatic cancer cell sublines by cultivating SW1990 and CFPAC-1 cells with gradually increasing concentrations of gemcitabine for 6 months. At first, SW1990 and CFPAC-1 cells had partially increased viability with morphological changes, followed by permanent gemcitabine resistance with phenotypic recovery (Fig. 1A). The cell viability MTT assay confirmed the increased resistance (Fig. 1B).

To assess the role of miR-181b in PDAC cells, we first determined the miRNA-181b expression in normal and resistant cell lines. miR-181b indeed was differentially expressed between the parental and resistant cell lines, suggesting that miR-181b affects gemcitabine sensitivity in PDAC cells. In particular, miRNA-181b was expressed in the SW1990 and CFPAC-1 cells, as detected by qRT-PCR (Fig. 2A). However, the SW1990/GR and CFPAC-1/GR cells expressed significantly higher levels of miRNA-181b (Fig. 2B). SW1990, CFPAC-1, SW1990/GR and CFPAC-1/GR cells were transiently transfected with miRNA-181b mimics, inhibitor, or control, and then treated with a gemcitabine-conditioned medium (1 μM) for 72 h. The MTT assay showed that miRNA-181b mimics increased the gemcitabine sensitivity of these 4 cell lines (Table III and Fig. 2C).

To further elucidate gemcitabine sensitivity in PDAC cells, we performed a flow cytometric assay. Tumor cells were transiently transfected with miRNA-181b mimics, inhibitor or control, and then treated with gemcitabine (0.1 μM). The data showed that miRNA-181b caused the PDAC cells to undergo apoptosis (Fig. 3).

To confirm our in vitro data, we performed nude mouse xenograft assays. PDAC SW1990 and SW1990/GR cells were transfected with a lentivirus carrying miR-181b mimics or negative control sequences in vitro that were then transplanted into the pancreata of nude mice. Two weeks after tumor cell transplantation, gemcitabine was administered to the mice at 150 mg/kg twice weekly for 4 weeks. The tumor size in the mice in the miRNA-181b overexpression group was smaller than that in the mice of the null control group (Fig. 4).

To explore the underlying molecular events, we performed western blot analysis and found that miRNA-181b reduced the expression of BCL-2 protein in vitro and in tumor xenografts (Fig. 5B). We also performed ELISA to detect caspase-3 activity and found that caspase-3 activity increased with miRNA-181b overexpression, but was reduced following treatment with a miRNA-181b inhibitor (Fig. 5C).

We also performed bioinformatics analyses and found there are 2 potential miR-181b target sites in the BCL-2 3′UTR. We performed a dual-luciferase reporter assay to assess whether BCL-2 is indeed a direct downstream target of miRNA-181b. The luciferase activity was significantly restrained by miRNA-181b mimics, while the miRNA-181b inhibitor led to an enhancement of luciferase activity in normal and resistant cell lines (Fig. 5A).