Pre-miR-200b (cat. no. PM10492), pre-miR-301 (cat. no. PM12929), negative control (cat. no. AM17110), miR-200b inhibitor (cat. no. AM10492), miR-301 inhibitor (cat. no. AM12929) and their negative controls (cat. no. AM17010) were purchased from Ambion (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Human pancreatic carcinoma cell lines (Capan-1, Capan-2, Panc-1, MIAPaCa-2, BxPC-3 and PL45) were obtained from the American Type Culture Collection (Manassas, VA, USA). Panc-1, MIAPaCa-2 and PL45 were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.; cat. no. 10566-016) supplemented with 10% fetal bovine serum (FBS). Capan-1, Capan-2 and BxPC-3 cells were grown in RPMI-1640 (cat. no. 61870-036) with 10% FBS (both from Thermo Fisher Scientific, Inc.; cat. no. 10437028). The two media contained antibiotics (100 U/ml penicillin and 100 µg/ml strep tomycin). All cell lines were routinely passaged as monolayer cultures at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

In order to further identify the association between chemoresistance and miRNA expression, gemcitabine-resistant cells were generated. The gemcitabine-resistant cell lines (Capan-2 GEM-R and Panc-1 GEM-R) were generated in RPMI-1640 or DMEM medium with 15% FBS and continuous exposure to the respective half-maximal inhibitory concentration (IC₅₀) of gemcitabine (µM) (Tocris Bioscience, Bristol, UK) for >5 months (27). To assess the gemcitabine resistance of Capan-2 GEM-R and Panc-1 GEM-R cells, colorimetric assays were performed following treatment with gemcitabine. Secondly, in the two generated gemcitabine resistant cell lines, miR-301 was measured by reverse transcription-quantitative polymerase chain reaction.

RT-qPCR was performed as described previously (28). Total RNA was extracted from cultured cells using a standard TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) protocol. Cell pellets were suspended in an aliquot of 1 ml/well of TRIzol in a 6-well plate. Isolated RNA was reverse-transcribed using a High-Capacity cDNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was diluted and stored at −20°C prior to use. Gene expression levels were measured with a custom-designed, TaqMan qPCR (Applied Biosystems; Thermo Fisher Scientific, Inc.) containing probes for six genes: CDH1 (ID: Hs00156401_m1), ZEB1 (ID: Hs00232783_m1), ZEB2 (ID: Hs00207691_m1), Vimentin (ID: Hs00185584_m1), interleukin (IL)-6 (ID: Hs00174131_m1), miR-200b (ID: 002251) and miR-301 (ID: 002392), with GAPDH (ID: Hs99999901_s1) for mRNA or RNAU6 (ID: 001002) for miRNA as an internal control. The relative expression levels of genes, miR-200b and miR-301 relative to GAPDH or RNAU6 were calculated using the rela tive quantification ΔΔCq method (29). RT-qPCR reactions were performed using TaqMan Gene Expression Assays on an ABI prism 7900HT Sequence Detection instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec, according to the manufacturer's protocol. Each sample was assayed in triplicate.

miRNA precursor molecules corresponding to miR-200b or miR-200b inhibitor, and miR-301 inhibitor, were transfected using the RNAiMAX Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) into Capan-2 and Panc-1 cells, and the effects on the respective oligonucleotide were measured by RT-qPCR. Capan-2 and Panc-1 cells at a density of 1×10⁵ cells/well were transfected with 50 nM microRNA in a 6-well plate for RNA extraction or a 10-cm dish for gemcitabine sensitivity and proliferation assays, following the manufacturer's protocol. Cells in the 6-well plate were collected 48 h post-transfection to extract RNA and measured for miR-200b or miR-301 expression. After 12 h of transfection, transfected cells in the 10-cm dish were seeded into 96-well plates for proliferation. These transfection experiments were repeated independently three times.

The colorimetric assay was performed essentially as described in a previous method (30). Briefly, cells were seeded in a 6-cm dish at 70% confluency. After 12 h, pre-miR-200b or miR-200b/301 inhibitors and respective controls were transfected in each dish overnight. Transfected cells were seeded in 96-well plates at 4,000 cells/well in triplicate. After incubating for 12 h, cell viability was determined by treating the cells with stepwise 4-fold serial dilutions of gemcitabine (from 100 µM) and incubated at 37°C for 96 h. To evaluate cell survival, the cells were fixed with 25% glutaraldehyde for 30 min at room temperature and stained with 200 µl 0.05% methylene blue for 20 min at room temperature. The dye was eluted with 0.33 M HCl for 20 min with agitation. Absorbance was measured using a microplate reader (model no. 3550; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 598 nm. The IC₅₀ for cell growth was calculated. The morphology of Panc-1 and Capan-2 cells was assessed using light microscopy (×20 magnification).

The cell growth of pancreatic carcinoma cell lines was studied using the colorimetric methylene blue assay, as described previously (31,32). To test cell growth, cells were transfected with miR-200b or 200b inhibitor or negative control in a 10-cm dish, counting the first 12 h as Day 0. Transfected cells at 4,000 cells/well were plated in a 96-well plate for 24 h. Mean values were calculated from three different wells in triplicate for 4 days.

To evaluate whether miR-200b or acquired gemcitabine resistance contributes to a decrease in caspase-3/7, Capan-2 or Panc-1 cells were cultured for 12 h in 96-well plates in triplicate, and treated with 50 nM pre-miR-200b or miR-200b inhibitor, or their respective controls. The assay was analyzed using a caspase-3/7 assay kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol.

The invasion assay was performed in 24-well Biocoat Matrigel invasion chambers (BD Biosciences, San Jose, CA, USA), according to a previous protocol (25,33). Briefly, cells were transfected with pre-miR-200b or pre-miR-301 and the negative control in a 10-cm dish. After 12 h transfection, cells were harvested and plated in the Matrigel-coated wells (4×10⁴ cells/well) and control insert wells (4×10⁴ cells/well) using Capan-2 and Panc-1 cells, respectively. After 20 h incuba tion, seeded cells on the membrane were removed by wiping with a cotton swab, and the invasive cells through the membrane were fixed with methanol for 5 min and stained with crystal violet for 5 min. Under a light microscope (×20 magnification), invasive cells were counted in three random fields. All assays were performed in triplicate.

Immunofluorescence was performed as previously described (13). Briefly, cells were seeded into a chamber slide at 40% confluence. Following incubation overnight, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.15% Triton X-100 in PBS for 20 min. Subsequently, the cells were blocked with 5% goat serum (cat. no. ab7481; Abcam, Cambridge, MA, USA) in PBS for 1 h at room temperature. CDH1 and nuclear factor (NF)-κB protein expression was detected using anti-CDH1 (cat. no. ab15148; Abcam; 1:1,000) and anti-NF-κB (cat. no. 3033; Cell Signaling Technology Inc., Danvers, MA, USA; 1:500) antibodies at 4°C overnight, according to the manufacturer's protocol. An Alexa Fluor-conjugated antibody (cat. no. ab1500083; 1:2,000) was used as a secondary antibody at 37°C for 2 h. The cell nucleus was counterstained with DAPI for 20 min at room temperature. The coverslips were mounted on slides prior to viewing using a fluorescence microscope (magnification, ×20).

All experiments were performed in triplicate and conducted at least twice. Data are presented as the mean ± standard deviation where applicable. GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA, USA) was used for all statistical analysis. Levels of significance for comparisons between cell lines were determined by the Student's t-test distribution. To assess the correlation between miR-200b expression, CDH1 expression and IC₅₀, Pearson's correlation analysis was performed in six different pancreatic carcinoma cell lines. To analyze multiple comparisons, one-way analysis of variance and the Bonferroni test as a post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.

To examine the correlation between miR-200b and IC₅₀, the present study focused on CDH1 expression, which is known to be a target for miR-200b and the EMT markers. To identify the correlation, miR-200b and CDH1 expression was initially investigated using six different cell lines (Capan-1, Capan-2, Panc-1, MIAPaCa-2, BxPC-3 and PL45) using RT-qPCR (Fig. 2A). Next, the IC₅₀ of gemcitabine was measured using a colorimetric assay in the six pancreatic carcinoma cell lines (Fig. 2B). As expected, the results demonstrated a clear positive correlation between miR-200b and CDH1 expression according to the Pearson data (r²=0.8165; Fig. 2C). By contrast, an inverse correlation between IC₅₀ and miR-200b or CDH1 was observed (r²=0.7042 and r²=0.9030, respectively; Fig. 2C). These data indicated that miR-200b is a putative biomarker for chemosensitivity in pancreatic carcinoma.

To evaluate the functional role of miR-200b in pancreatic carcinoma, pre-miR-200b or miR-200b inhibitor were transfected into Capan-2 and Panc-1 cells using RNAiMAX Transfection Reagent. The efficacy of the transfection was confirmed using RT-qPCR. miR-200b overexpression upregulated CDH1, and suppressed ZEB1 and ZEB2 (Fig. 3A). Conversely, miR-200b inhibition reduced CDH1 expression in the two cell lines (Fig. 3B). Moreover, miR-200b overexpression improved sensitivity to gemcitabine, and miR-200b inhibitor significantly affected gemcitabine sensitivity in the two cell lines (Fig. 4). Furthermore, the capacity for cell growth was evaluated using a proliferation assay in the two cell types. Forced expression of miR-200b reduced cell growth, while miR-200b inhibition did not affect cell growth (Fig. 5A). These results were consistent across the two cell lines were in accordance with a previous study (25).

Overexpressed miR-200b enhanced apoptosis and inhibited cell invasiveness in the two cell lines (Fig. 5B and C). By contrast, downregulated miR-200b reduced apoptosis and enhanced cell invasiveness (Fig. 5B and C).

To examine the mechanisms underlying gemcitabine resistance, two sub-cell lines were generated, which were derived from Capan-2 and Panc-1 cells. These were cultured in the presence of high concentrations of gemcitabine (IC₅₀), by gradually increasing the concentration of gemcitabine over 5 months. Cells in which the IC₅₀ values were over four times higher compared with their controls were defined as Capan-2 GEM-R and Panc-1 GEM-R. The IC₅₀ of these cells was evaluated using a colorimetric assay, and they exhibited strong gemcitabine resistance compared with each control cell line (Fig. 6A). Moreover, the two GEM-R cell lines exhibited increased cell growth (Fig. 6B).

The present study also examined whether acquired gemcitabine resistance in cells was associated with miR-301 expression. Gemcitabine-resistant cells exhibited increased miR-301 and reduced miR-200b expression (Fig. 7A). No explanation became clear as to why miR-200b was reduced in gemcitabine-resistant cells. However, Wang et al (34) recently reported that gemcitabine treatment induced reduced miR-200b in pancreatic carcinoma cell lines. In addition, resistant cells exhibited an anti-apoptotic effect and increased cell invasiveness compared with control cells (Fig. 7B).

In the two gemcitabine-resistant cell lines, immunofluorescence was performed for NF-κB and CDH1. Gemcitabine-resistant cells exhibited NF-κB activation and decreased CDH1 expression, as expected (Fig. 8A). The difference was less marked in Panc-1 cells due to the lower expression levels of CHD1 and higher activation levels of NF-κB in the control cells. It was not possible within the scope of this investigation to perform western blot analysis using a fractionated sample (Cyto/Nuc) and quantification of the expression of target proteins of NF-κB. As an alternative, IL-6 expression was measured to verify the NF-κB activation in gemcitabine-resistant cells (Fig. 8B). In addition, RT-qPCR analysis demonstrated a decrease in CDH1 expression. The expression of EMT-associated genes, including vimentin, ZEB1 and ZEB2, was measured using RT-qPCR. The results did not exhibit a consistent pattern of gene expression to explain how gemcitabine resistance led to EMT in the cell lines (Fig. 9), although western blot analysis was not presented. In addition, morphology remained unaltered in gemcitabine-resistant cells (data not shown). In the same manner, miR-301 overexpression revealed decreased CDH1 expression levels, an enhanced anti-apoptotic effect, an increased IC₅₀ of gemcitabine and increased cell invasiveness, as previously reported (13). Contrary to expectations, miR-301 inhibition did not induce the remission of gemcitabine resistance (Fig. 10). These data are consistent with a previous report (13). Based on the present data, elevated miR-301 expression may affect acquired gemcitabine resistance, and an increase in miR-301 may be a predictive biomarker for acquired gemcitabine resistance in patients with pancreatic carcinoma.