Human pancreatic adenocarcinoma BxPC-3 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown in RPMI with supplements of 10% fetal bovine serum and 50 μg/ml of gentamicin at 37°C in a 5% CO₂ humidified atmosphere.

Cells were plated at a density of 1.8×10⁵ cells/ml in T75 flasks and incubated overnight at 37°C. The following day, cells were either mock-transfected or transfected with 27 nM miR-16 miRIDIAN miRNA hairpin inhibitor or miR-16 miRNA mimic and their appropriate negative controls, using the Dharmafect2 transfection reagent (Dharmacon, Lafayette, CO, USA) in accordance with the manufacturer’s protocol. miRIDIAN mimic and inhibitor oligos were prepared by dissolution in 1X small interfering RNA (siRNA) buffer. Diluted oligos were placed in Tube 1, and transfection reagent was maintained in Tube 2 in serum-free medium. Reagents were mixed gently, followed by incubation at room temperature (RT) for 5 min. The contents in Tubes 1 and 2 were then combined and incubated at RT for 30 min. Subsequently, complete medium with 10% serum was added to the mixture followed by addition to the cells. The transfected cells were allowed to grow overnight. The transfection medium was replaced after 24 h with fresh medium.

Total RNA (5 μg) was loaded and separated on a 15% denatured urea gel. After electrophoresis and transfer, the blot was hybridized with corresponding biotin pre-labeled miRNA probe (Signosis Inc., Sunnyvale, CA, USA). To normalize the hybridization, a biotin pre-labeled RNU48 probe was mixed with miRNA probe for co-hybridization. The hybridized probes were then monitored with streptavidin-HRP and chemiluminescent detection.

pGL3-BCL2 3′ UTR sense and antisense constructs were prepared as previously described (13). For luciferase reporter assay, human pancreatic cancer cells were co-transfected in a 96-well plate with 100 ng of the firefly luciferase reporter vector and 10 ng of the pEGFPC2 expression vector (transfection control; Clonetech) either alone or in combination with 50 nM miR-16 mimic oligo or 50 nM mimic negative control using the Dharmafect Duo transfection reagent. Luciferase activity was normalized to GFP expression. The relative expression was determined for the mimic negative control and miR-16-transfected samples.

Following the designated treatment, total cellular proteins were extracted. Equal protein from each sample was fractionated by SDS-PAGE and blotted onto a nitrocellulose membrane (GE Healthcare, NJ, USA). Membranes were probed with the Bcl-2 antibody (Upstate Biotechnology, Syracuse, NY, USA) followed by incubation with a HRP-conjugated secondary antibody (3,4). Finally, immunodetection was carried out by the enhanced chemiluminescence method (GE Healthcare). Immunodetection with the β-actin antibody (Sigma, St. Louis, MO, USA) served as a protein loading control.

Cells were washed 24 h post-transfection with phosphate-buffered saline and then seeded in triplicate (20,000 cells/10-cm dish). The cells were allowed to grow for an additional 2 weeks. The medium was changed every 4 days. The cells were then fixed with 4% buffered formalin (Electron Microscopy Sciences, Hatfield, PA, USA) and stained with 0.1% crystal violet for visualization and photography (19,20).

To understand whether miR-16 regulates pancreatic adenocarcinoma cell proliferation, we used a commercially available miR-16 mimic (mature). miR-16 mimics and inhibitors as well as the respective negative controls were transfected into BxPC-3 cells. The overexpression or silencing of miR-16 was verified by Northern blotting of the transfected cells (Fig. 1B). At the 48-h transfection, we achieved a significant expression of miR-16 over mock-transfected and scrambled control-containing cells (Fig. 1B). The clonogenic cell survival assay (19,20) was employed to determine the long-term survival ability of BxPC-3 cells following transfection with miR-16 and its inhibitor oligos. Notably, the overexpression of miR-16 significantly decreased the clonogenic cell survival of pancreatic tumor cells when compared to the mimic negative control (Fig. 1, panel II vs. panel I), whereas silencing of the same miR-16 had no effect on cell survival (Fig. 1A, panel III vs. panel IV). Due to the low endogenous level, silencing of miR-16 did not enhance cell survival when compared to the control.

As mentioned previously, miR-16 has the ability to down-modulate anti-apoptotic protein Bcl-2 in diverse cancer types. We attempted to ascertain whether the transfection of miR-16 mimic oligonucleotide exerts any effect on the Bcl-2 level in pancreatic cancer cells. A decrease in the Bcl-2 protein level in the BxPC-3 cells (Fig. 2, lane 2) was clearly evident due to the ectopic overexpression of miR-16. However, the level of Bcl-2 did not decrease when the hairpin inhibitor oligonucleotide of miR-16 was transfected into these cells (Fig. 2, lane 4).

The decrease in Bcl-2 protein in miR-16-overexpressed cells may be the direct effect of miRNA::mRNA complementarity or by an indirect interaction with other unknown targets. To address this issue, we used luciferase reporter constructs where the 3′ UTR sequence of the human BCL2 gene contained a presumed miR-16 complementary site that was fused into a luciferase reporter plasmid in both sense and antisense orientations (immediately downstream of the stop codon of luciferase). The resulting plasmids and control plasmid (pEGFPC2) were introduced into BxPC-3 pancreatic cancer cells together.

Notably, as shown in Fig. 3A, the co-transfection of the sense orientation constructs (pGL3 BCL2) with miR-16 mimic oligo resulted in a significant reduction in firefly luciferase reporter activity when normalized against GFP (P<0.002). In contrast, the transfection of miR-16 oligo in the presence of the luciferase vector containing BCL2 3′ UTR in the antisense orientation terminated this suppression (Fig. 3B). Moreover, the co-transfection of mimic negative control miRNA had no effect on the luciferase activity of pGL3 BCL2 and pGL3 BCL2 AS. Thus, our observation suggests that miR-16 directly regulates Bcl-2 expression in pancreatic cancer cells through the target site of 3′ UTR of BCL2 mRNA at the post-transcriptional level.